Glucagon receptors

The invention relates to a DNA molecule encoding a glucagon receptor or a glucagon receptor peptide, a DNA construct, a host cell comprising the construct, a glucagon receptor peptide, and a method of producing a glucagon receptor or a glucagon receptor peptide.

Glucagon is a 29 amino acid peptide hormone produced by the alpha islet cells of the pancreas. Glucagon is responsible for the maintenance of normal glucose levels in animals, including humans, by acting as a hormone opposite to insulin. In particular, while insulin is known to rapidly lower blood glucose levels, glucagon counterbalances this effect by contributing to an elevated blood glucose level.

The interaction of glucagon and insulin is very important for the maintenance of glucose levels in the body. An imbalance in glucagon or insulin is believed to play a role in several diseases, including diabetes and diabetic ketoacidosis. According to this theory, the state of hyperglycemia in diabetes may be caused not only by poor glucose utilization (caused by lowering insulin) but also by overproduction of glucose caused by elevated glucagon levels (see Unger, "Diabetes and alphacell", Diabetes 25: 136-151, 1976 ; Unger and Orci, "The essential role of glucagon in the pathogenesis of diabetes mellitus", Lancet 1: 14-16, 1975).

An important factor in the study of glucagon and its role in diseases such as diabetes is the glucagon receptor, which, when bound to glucagon, transmits a signal to the cell, thereby triggering glycogcnolysis (glycogen hydrolysis) and gluconeogenesis (glucose synthesis).

It is now believed that the effects of glucagon are mediated in part by elevated levels of intracellular cyclic adenosine monophosphate (cAMP). In particular, the binding of glucagon to its cellular receptor activates adenylate cyclase to produce cAMP, and thus increases the level of intracellular cAMP. The increase in intracellular cAMP is believed to cause glycoglycnolysis and gluconeogenesis and consequently an increase in hepatic glucose production (see Unson et al, "Biological Activities ofdes-His¹ [Glu⁹] Glucagon Amida a Glucagon Antagonist ”, Peptides 10: 11171-1777, 1989).

However, it has been suggested that there are additional metabolic pathways that stimulate glycogenolysis and gluconeogenesis. In particular, it has been reported that glucagon binds to a receptor in the hepatocyte plasma membrane coupled by the G protein to phospholipase C. Upon stimulation, this protein causes the degradation of phosphatidylinositol 4,5-biphosphate to the secondary messenger, inositol triphosphate and 1,2-diacylglycerol (Look. Wakelam et al., Activation of two signal-transduction Systems in hepatocytes by glucagon ", Nature 323: 68-71, 1986; Unson et al., Peptides 10.1171-1177, 1989; Pittneri Fain, Biochem. J. 277: 371-378, 1991). The stimulation of inositol phospholipid metabolism by glucagon may be an additional metabolic pathway by which glucagon promotes glycogenolysis and gluconeogenesis.

The present invention relates to isolated DNA molecules encoding a glucagon receptor. The term "isolated DNA molecules" as used herein refers to individual DNA molecules or sequences that are alone or separate from other components. For example, a DNA molecule is isolated when it is separated from other DNA molecules, including other chromosomal sequences with which it is naturally associated in the genome, and particularly when it is free from other structural genes. The isolated DNA molecule may contain 3 'and 5' untranslated sequences to which it is naturally linked. The glucagon receptor that is encoded by the DNA of the invention may be a rat or a human receptor. In one embodiment of the present invention, the DNA molecule encoding the glucagon receptor comprises the nucleotide sequence Sequence Identifier. No. 14 from nucleotide 145 to nucleotide 1599 In another embodiment of the present invention, the DNA molecule encodes a glucagon receptor comprising the amino acid sequence of SEQ ID NO. 15, from methionine, amino acid # 1, to threonine, amino acid # 485. In yet another embodiment, the DNA molecule comprises the nucleotide sequence of SEQ ID NO: 24, from nucleotide 53 to nucleotide 1486. In yet another embodiment, the DNA molecule encodes a glucagon receptor comprising the amino acid sequence of SEQ ID NO. No .: 25, from methionine, amino acid No. 1, to phenylalanine, amino acid No. 477. The subject of the

178 The invention also includes DNA constructs comprising a first DNA segment encoding a glucagon receptor associated with additional DNA segments needed for expression of the first DNA segment, host cells containing such DNA constructs, as well as methods for producing a glucagon receptor including the step of culturing host cells under conditions that express the segment. DNA encoding the glucagon receptor.

The invention also relates to isolated glucagon receptor peptides. In one embodiment, an isolated glucagon receptor peptide is provided comprising the sequence SEQ ID NO. No .: 15 from glutamine, amino acid # 28, to tyrosine, amino acid # 142

Figure 1 shows the structure of a representative glucagon receptor. Symbols used: EATD (N-terminal extracellular domain), surrounded by a dotted line, CM (cell membrane); ED (effector domain), surrounded by a dashed line; 1ID, first intracellular loop, 2ID, second intracellular loop; 3ID, third intracellular loop; C-ID, C-terminal intracellular domain, 1ELD, first extracellular loop; 2ELD, second extracellular loop; 3ELD, third extracellular loop, TMD1, first transmembrane domain; TMD2, second transmembrane domain; TMD3, third transmembrane domain; TMD4, fourth transmembrane domain; TMD5, fifth transmembrane domain; TMD6, the sixth transmembrane domain, TMD7, the seventh transmembrane domain.

Figure 2 graphically shows the hydrophobicity of the rat glucagon receptor.

Figure 3 shows a graph of bonding ¹²⁵I-glucagon with the glucagon receptor.

Figure 4 is a Scatchard analysis of the likely Kd for glucagon receptors.

Figure 5 is the amino acid sequence of the rat glucagon receptor with the transmembrane domains circled.

As mentioned above, the present invention provides isolated DNA molecules encoding glucagon receptors. In their native configuration, glucagon receptors are believed to exist as a membrane-bound protein consisting of the extracellular N-terminal domain as well as several smaller internal and external domains (see Figure 1). In the context of the present invention, "glucagon receptor "Refers to such protein and similar derivatives. Derivatives include allelic variants and engineered variants containing conservative amino acid substitutions and / or minor amino acid additions, substitutions, or deletions. The glucagon receptors of the present invention are capable of binding glucagon and transmitting a signal provided by glucagon to a cell. Preferably, the glucagon receptors of the present invention are capable of binding glucagon with a Kd of 100 nm or less, even more preferably 50 nm or less, and most preferably 33 nm or less. Representative assays that can be used to determine glucagon binding to the glucagon receptor are described in detail in Examples 3 and 6.

Signal transduction usually occurs when the response pathway is activated by an external stimulus, usually, but not always, directly coupled to a membrane-bound receptor. The response pathway usually triggers cellular responses such as secretion of the intracellular matrix by responsive cell lines, hormone secretion, chemotaxis, differentiation or initiation or inhibition of cell division As used herein, the association of receptors with the response pathway refers to the direct activation or signal transduction pathway. by a secondary messenger, such as a G protein, activating the cellular response pathway.

Glucagon receptors can use a variety of cellular response pathways to transmit glucagon binding signal to the cell, including, for example, the adenylate cyclase pathway and the intracellular calcium pathway. Tests for the activity of adenylate cyclase are well known to those skilled in the art, and include, e.g. described by Lin et al. (Biochemistry, 14: 1559-1563, 1975).

Glucagon receptor biological activity can be measured by an intracellular calcium concentration method (see Grynkiewicz et al., J. Biol Chem 260: 3440-3450, 1985), as well as by using a luciferase reporter system, which is detailed below. Additionally, biological responses through the inositol triphosphate pathway can be determined by measuring the metabolism of inositol triphosphate as described in the public

178 685 from Subers and Nathanson (J Mol. Cell. Cardiol., 20: 131-140, 1988) or Pittner and Fain (Blochem. J 277: 371-378, 1991). It should be noted that in the context of the present invention not all response pathways must be in place for the glucagon receptor to signal the cell. For example, some cellular responses, such as an increase in intracellular calcium levels, may be induced by the binding of glucagon to its receptor in the absence of the cAMP signal or inositol phosphate.

Isolation of glucagon receptor cDNA clones

As noted above, the present invention provides isolated DNA molecules encoding glucagon receptors. Briefly, genomic or cDNA molecules encoding glucagon receptors can be obtained from libraries that have been prepared from cells and tissues according to procedures as described below and in the examples. Cells and tissues that can be used in the context of the present invention can be obtained from a variety of mammals, for example humans, macaques, cattle, pigs, horses, dogs, rats and mice. The best cells and tissues that can be used are: adipose tissue, kidney, pancreas, heart and liver

In one aspect of the present invention, the rat glucagon receptor can be isolated and cloned using the procedures described below. Briefly, poly (A) was isolated⁺ RNA from Sprague Dawley rats, and was used as a template for cDNA synthesis as described by Houamed et al. (Science 252: 1318-1321,1991) to generate full-length cDNA. A library was created, containing approximately 1 × 10⁶ of clones, in a mammalian expression plasmid, by a cDNA targeting pathway longer than 800 bp. Plasmid DNA, prepared by pooling 5,000 clones each, was transfected into COS-7 cells, which were selected and grown on microscope slides. Transfected cells were tested 72 hours later for binding¹²⁵I-glucagon and emulsion radiography (McMachan et al., EMBOJ. 10: 2821-2832,1991). Positive pools were separated until a single clone was isolated. A plasmid obtained from a single clone, designated pLJ4, containing an approx. 2.0 kb insert that encoded a protein of 485 amino acids with a predicted molecular weight of 54,962 Da (see SEQ ID NO: 15)

In another aspect of the present invention, methods for isolating and cloning the human glucagon receptor are provided. A variety of techniques may be used, including, for example, the use of the Polymerase Chain Reaction ("PCR") to amplify glucagon receptor encoding sequences (Example 4) that can be used to identify libraries. containing sequences encoding the human glucagon receptor, followed by receptor cloning (Example 5). Particularly preferred cloning strategies for the human glucagon receptor are provided below in Examples 4 and 5. Alternatively, an expression library containing human cDNA can be prepared from suitable RNA sources as described in Example 1 and viewed as described in Example 3 by looking for clones in which are expressed for human glucagon receptor function.

Generation of recombinant glucagon receptors

The present invention provides methods for producing recombinant glucagon receptors in host cells comprising a DNA construct consisting of a first DNA segment encoding a glucagon receptor operably linked to additional DNA segments necessary for expression of the first DNA segment. As described previously, in the context of the present invention, the definition of "glucagon receptors" also includes derivatives which are substantially similar. Further, glucagon receptors can be encoded by DNA sequences that are substantially similar to the DNA sequences disclosed herein As used herein, a DNA sequence is considered "substantially similar" when: (a) the DNA sequence is obtained from the coding region of the native glucagon receptor gene (including, for example, allelic variants of the sequence disclosed below); (b) the DNA sequence is capable of hybridizing to the DNA sequences of the invention under high and low specificity conditions (see Sambrook et al., Molecular Clomng A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laborotiry Press, NY, 1989); or (c) the DNA sequence is degenerate, as a result of the genetic code, to the DNA sequence defined in (a) or (b).

178 685

Mutations in nucleotide sequences engineered to express glucagon receptor variants should preserve the reading frame of the coding sequence. Moreover, the mutations preferably should not form complementary regions that could hybridize to form secondary mRNA structures, such as loops or "hairpins", that could interfere with the translation of the mRNA for the receptor. Although the site of the mutation may be determined, it is not necessary that the nature of the mutations per se be determined. For example, in order to select the optimal characteristics of mutans at a given site, random mutagenesis of a particular codon can be performed, and the biological activity of the resulting receptor mutants tested.

Mutations can be introduced at particular loci by synthesizing oligonucleotides containing the mutant sequence flanked by restriction sites that allow ligation into fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative with the desired amino acid insertion, substitution, or deletion.

Alternatively, site-directed specific mutagenesis may be used to obtain an altered gene having a codon altered by a substitution, deletion or insertion, as appropriate. Examples of methods introducing these changes are included in the following publications: Walder et al. (Gene, 42. 133, 1986); Bauer et al. (Gene, 3773, 1985); Craik (Bio Techniąues, January 1985, 12-19); Smith et al. (Genetic Engine ering, Pnnciples and Methods Plenum Press, 1981); Sambrook et al, (ibid.).

The primary amino acid structure of the glucagon receptor can also be altered by forming covalent or aggregate conjugates with other chemical moieties such as glycosyl groups, lipids, phosphates, acetyl groups, or with other proteins and polypeptides. In a further embodiment, the glucagon receptors can be fused to other peptides to facilitate purification or identification of the glucagon receptors. For example, the glucagon receptor can be made as a fusion protein with the FLAG polypeptide sequence (see, US, US Patent No. 4,851,341; see also Hopp et al., Bio / Technology 6: 1204, 1988). The FLAG polypeptide sequence is highly antigenic and provides an epitope for binding to a specific monoclonal antibody, allowing rapid purification of the recombined protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the site following the Asp-Lys pair.

As desired, numerous DNA constructs can be made containing all or part of the nucleotide sequence of the native glucagon receptor and variants thereof, as discussed above. In the context of the present invention, a DNA construct refers to a DNA molecule or a clone of such a molecule (single or double stranded) that has been altered by human intervention such that it contains DNA segments linked or arranged in a manner not entirely found in nature. The DNA construct of the invention consists of a first DNA segment encoding a glucagon receptor operably linked to additional DNA segments necessary for expression of the first DNA segment. In the context of the present invention, additional DNA segments include transcription promoters and terminators, and may further include enhancers and other elements.

DNA constructs, also known as expression vectors, can also contain DNA segments necessary to direct the secretion of a polypeptide of interest. Such DNA segments may contain at least one secretory signal sequence. Preferred signal sequences include: glucagon signal sequence (pre-pro sequence), alpha factor signal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30: 933-943, 1982; Kurjani et al, US Patent No. 4,546,082; Brake, EP 116, 201); PHO5 signal sequence (Beck et al., WO 86/00637); BARI signal sequence (McKay et al., U S. Patent No. 4,613,572; McKay WO87 / 002670); SUC2 signal sequence (Carlson et al., Mol Cell Biol, 3: 439-447, 1983), α-1-antitrypsin signal sequence (Kurachi et al., Proc Natl Acad. Sci USA, 78: 6826-6830. 1981), signal sequence of a-2 plasmin inhibitor (Tona et al, J Blochem (Tokyo) 102: 1033-1042, 1987), signal sequence of tissue plasminogen activator (Pennica et al., Nature 301, 214-221, 1983), the PhoA signal sequence of E coli (Yuan et al., J Biol Chem, 265 13528-13552, 1990) or any bacterial signal sequence described

178 685 for example in Oliver (Ann. Rev. MicrobioL 39: 615-649, 1985). Alternatively, the secretory signal sequence may be synthesized according to the rules set out, for example, by von Heinje (Eur. J Blochem 133. 17-21, 1983; J Mol Biol 184: 99-105, 1985; Nuc Acids Res 14: 4683-4690). , 1986).

Secretory signal sequences may be used singly or in combination. For example, the first secretory signal sequence may be used in combination with a sequence encoding a third Barrier domain (described in US Patent No. 5,037,243 and incorporated herein by reference in its entirety). The sequence encoding the third Barrier domain may be positioned in the correct reading frame at the 3 'end of the DNA sequence of interest or the 5' end of the DNA segment, and in the correct reading frame with both the secretory signal sequence and the DNA segment of interest.

To allow expression, a DNA molecule encoding the glucagon receptor is inserted into a suitable DNA construct, which in turn is used to transform or transfect an expressible host cell. The host cell for use in the practice of the present invention includes mammalian, avian, plant, or insects, bacteria and fungi. Preferred eukaryotic cells include cultured mammalian cell lines (e.g. rodent or human cell lines) fungal cells, including yeast species (e.g. Saccharomyces spp. and especially S cerevisiae, Schizosaccharomyces spp. or Kluyveromyces spp.) or mold (e.g. Aspergillus spp. Neurospora spp.). Schizosaccharomyces cerevisiae strains are particularly useful. Methods for producing recombinant proteins using a variety of procanotic and eukaryotic cells are known to those skilled in the art (see "Gene Expression Technology", Methods in Enzymology, vol. 185, Goeddel (ed.) Academic Press, San Diego, Calif., 1990; see also, "Guide to Yeast Genetics and Molecular Biology", Methods in Enzymology, Gutrie and Fink (eds.) Academic Press, San Diego, Calif. , 1991). Generally, a host cell is selected on the basis of its ability to produce the protein of interest in large amounts, or its ability to carry out at least some of the processing steps necessary for the biological activity of the protein. In this way, the number of cloned DNA sequences that need to be transfected into the host cell is minimized and the total yield of biologically active protein is maximized.

Suitable yeast vectors for use in the present invention include: YRp7 (Struhl et al., Proc Natl Acad Sci USA, 76: 1035-1039, 1978), YEp 13 (Broach et al., Gene 8 '121-133, 1979), POT vectors (Kawasaki et al., US Patent No. 4,931,373, incorporated herein by reference), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108,1987), and derivatives thereof. These vectors should contain an appropriate selectable marker, which may be one of the many genes with a dominant phenotype for which suitable methods exist for recombinant selection. The most preferred selectable markers are those promoting host cell growth by providing resistance to antibiotics or allowing the cell to use specific carbon sources, and include EE772 (Broach et al., Ibid) URA 3 (Botsteini et al., Gene 8: 17,1979), HIS3 (Struhl et al., Ibid) or POT 1 (Kawasaki et al., Ibid). Another suitable marker is the CAT gene, which is responsible for resistance of yeast cells to chloramphenicol.

Preferred promoters for use in yeast include yeast glycohtic gene promoters (Hitzeman et al., J Biol Chem 255: 12073-12080, 1980; Alber and Kawasaki, J Mol Appl Genet 1: 419-434, 1982; Kawasaki, US Patent No. 4,599,311. ) or alcohol dehydrogenase genes (Young et al., Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol 101 192-201.1983) In this regard, the ΤΡΙ1 promoter (Kawasaki, US Patent No. 4,599,311,1986) and the ADH2-4 promoter are particularly preferred.^C. (Russel et al., Nature 304: 652-654; Irani and Kilgore, US Patent Application No. 07 / 784,653, incorporated herein by reference). The expression units may also include a transcription terminator. A preferred transcription terminator is the TPI1 terminator (Alber and Kawasaki, ibid)

In addition to yeast, the proteins of the invention may be produced by expression in molds, for example in Aspergillus strains (McKnight et al., US Patent No. 4,935,349, in

178 685 incorporated herein by reference). Examples of suitable promoters include. in. derived from Aspergillus nidulans glycohtic genes such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1995) and the tpiA promoter An example of a suitable terminator is the ADH3 terminator (McKnight et al, ibid, 1985). Expression units using such components are cloned into vectors capable of integrating into Aspergillus chromosomal DNA.

Techniques for transforming fungi are known in the literature, and have been described, for example, by Beggs (ibid), Hinnen et al. (Proc Natl Acad Sci USA 75: 1929-1933, 1978), Yelton et al (Proc Natl Acad Sci USA 81 : 1740-1747, 1984) and Russel (Nature 301: 167-169, 1983). The genotype of the host cells should contain the genetic defect complemented by the marker present in the expression vector. The choice of the particular host and marker is within the knowledge of one skilled in the art. In order to improve the production of a heterologous protein in yeast, for example, it is preferred that the host strain has a mutation such as e.g. a pep4 mutation (Jones, Genetics 85: 23-33, 1977) that results in reduced activity. proteolytic.

In addition to fungal cells, mammalian cells may be used in the present invention. Preferred cells for use in the present invention include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632) and 293 (ATCC No. CRL 1573 cell lines, Graham et al., J ViroL gene, 36 59-72, 1977) A preferred BHK line is the BK 570 line (deposited with the American Type Culture Collection under number CRL 10314). Additionally, a variety of mammalian cell lines may be used including rat Hepl cells (ATCC No. CRL 1600), HepII (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), human lung (ATCC No. CCL 75.1), human hepatoma (ATCC HTB052), Hep G2 (ATCC No. HB 8065), Mouse Liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2 / 0-Ag 14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777) and RINm 5AHT₂B (Orskov and Nelson, FEBS 229 (1): 175-178, 1988).

Mammalian expression vectors for use in the present invention contain a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral and cellular promoters. The viral promoters include: cytomegalovirus immediate early promoter (Boshart et al., Cell 41: 521-530, 1985) and the SV40 promoter (Subramani and ΐη., / Ο / Cell Biol 1.854-864, 1981). Cellular promoters include the mouse metallothionein 1 promoter (Palmiter et al., PatentUSANr4,579,821), the mouse V promoter_k (Bergman et al., ProcNatl AcadSci USA 81: 7041-7045, 1983; Grant et al., Nuc Acids Res 15: 5496, 1987) and the mouse V promoter_H. (Loh et al., Cell 33 85-93, 1983). Adenovirus 2 major late promoter (Kaufman and Sharp, MoI Cell Biol 2: 1304-13199,192) is particularly preferred. These vectors may contain a set of RNA storage sites downstream of the promoter and upstream of the DNA sequence encoding the peptide or protein of interest. Preferred splicing sites for RNA can be derived from adenoviruses and / or immunoglobulin genes. Also located in the expression vector is a polyadenylation signal downstream of the coding sequence. Suitable polyadenylation signals include the early and late polyadenylation signal from SV40 (Kaufman and Sharp, ibid), the polyadenylation signal from the Adenovirus 5 E1B region, and the human growth hormone gene terminator (DeNoto et al., Nuc Acids Res 9: 3719-3730,1981). The expression vector may contain a viral non-coding leader sequence, such as an adenovirus 2 three-part leader, located between the promoter and RNA splice sites. Preferred vectors may include enhancer sequences, e.g. SV40 enhancer; and mouse µ enhancer (Gillies, Cell33: 717-728, 1883). The expression vectors may also contain adenoviral VA RNA coding sequences. Suitable vectors can be obtained commercially (e.g., Stratagene, La Jolla, CA).

Cloned DNA sequences can be introduced into cultured mammalian cells by, for example, transfection using calcium phosphate (Wigier et al., Cell 14 · 725, 1978, Corsaro and Pearson, Somatic Cell Genetics 7: 609, 1981; Graham and Van der Eb, Virology 52 456, 1973), electroporation (Neumann et al., EMBO J 1: 841-845, 1982) or transfection with DEAE-dextran (Ausubel et al., (Eds), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987) which are incorporated herein by reference. In order to identify cells that have permanently integrated the cloned DNA, it is inserted together with the gene of interest or

178 685 cDNA selectable marker A preferred marker for use in cultured mammalian cells includes genes responsible for drug resistance such as neomycin, hygromycin and methotrexate. The selectable marker should be an amplifiable marker. A preferred amplifiable marker is the DHFR gene and the neomycin resistance gene. Selectable markers are described by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, incorporated herein by reference). The choice of the appropriate marker is within the knowledge of the skilled person.

The selectable markers may be introduced into the cell on a separate vector at the same time as glucagon receptor sequences, or they may be introduced on the same vector. When the same vector is used, the marker and the glucagon receptor sequence may be under the control of different promoters or the same promoter, resulting in a dicistronic transcript. Constructs of this type are known to those skilled in the art (for example, Levinson and Simonsen, US Patent No. 4,713,339). It may be advantageous to add additional DNA known as "carrier DNA" to the mixture introduced into the cell.

Transfected mammalian cells should be allowed to grow for a period of time, usually 1-2 days, in order to begin expressing DNA sequences of interest. Drug selection is made to allow continued growth of cells expressing the marker in a consistent manner. In the case of cells transfected with a selectable marker capable of amplification, the concentration of the drug may be increased stepwise to select for an increased number of copies of the cloned sequence and thus for an increased level of expression. Cells expressing the introduced sequences are selected and tested for production of the protein of interest in the desired form or level. . Cells that meet these criteria can be further cloned and grown for production.

The preferred prokaryotic hosts to be used in the practice of the present invention are Escherichia coli strains, however Bacillus and other types are also useful. Techniques for transforming these hosts and expressing foreign cloned DNA sequences are well known to those skilled in the art (see, e.g. Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, incorporated herein by reference); or Sambrook and ΐη., γ.νν.) Vectors used to express cloned DNA sequences in bacterial hosts usually contain a selectable marker such as antibiotic resistance genes and promoters active in the host cell. Suitable promoters include the trp promoter systems (Nicholas and Yanofsky, Meth. Enzymol 101: 155-164,1983), lac (Casadaban et al., J Bacteriol 143: 971-980,1980) and phagaX (Queen, J. Mol. Appl Genet 2: 1-10, 1983). Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2: 95-113, 1977), pUC plasmids (Messing, Meth Enzymol 101: 20-78, 1983; Vieira and Messing, Gene 19 '259-268, 1982). ), pCQV2 (Queen, ibid) and their derivatives. Plasmids can contain both bacterial and viral sequences.

From the guidance given herein, promoters, terminators, and methods for introducing expression vectors encoding glucagon receptors into plant, avian, and insect cells are apparent to one of skill in the art. The use of baculoviruses, for example, as vectors for the expression of heterologous DNA sequences in insect cells has been described by Atkinsoni et al. (Pestic Sci 28: 215-224,1990). Additionally, the use of Agrobacterium rhizogenes as a vector to express genes in plant cells has been described by Sinkar et al. (J Biosc (Bangalore) 11: 47-58, 1987).

Host cells containing a DNA construct of the present invention are cultured to express a DNA segment encoding a glucagon receptor. Cells are grown in accordance with standard methods in culture media containing the nutrients needed for the growth of the selected cells. Many suitable media are known to those skilled in the art and usually contain: a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, as well as other components, e.g., growth factors and serum, which may be necessary for some cells. The medium should usually select cells containing the DNA construct, for example, by selecting for drug or lack of the necessary

178 685 substances as provided by a selectable marker on the DNA construct or co-transfected with the DNA construct.

Suitable conditions for culturing yeast cells include culturing in a defined medium containing a nitrogen source which may be a non-amino acid source or yeast extract, inorganic salts, vitamins and essential amino acids at a temperature between 4 ° C and 37 ° C, and especially at 30 ° C. ° C. The pH of the medium is most preferably maintained between 2 and 8, and more preferably around a pH of 5-6. Methods of maintaining a constant pH include buffering and constant pH control. A preferred agent for controlling the pH is sodium hydroxide. Preferred buffering agents are succinic acid and BisTris (Sigma Chemical Co., St. Louis, MO). Because of the tendency of yeast to hyperglycosylate heterologous proteins, it is preferable to express the glucagon receptor of the present invention in yeast cells defective in the gene necessary for asparagine glycosylation. These cells preferably grow in a medium containing an osmotic stabilizer. The preferred osmotic stabilizer is sorbitol added to the medium at a concentration between 0.1 M and 1.5 M, and most preferably at a concentration between 0.5 and 1.0 M. Cultured mammalian cells are usually grown in commercially available serum-containing or serum-free media. The choice of a medium and conditions suitable for a particular cell line is within the knowledge of one skilled in the art.

Glucagon receptors can also be expressed in non-human transgenic animals, especially transgenic warm-blooded animals. Methods for creating transgenic animals including mice, rats, rabbits, sheep and pigs are known to those skilled in the art and are found in, for example, Hammer et al (Nature, 315: 680-683, 1985), Palmiter et al {Science, 222: 809-814 , 1983), Brister et al. (Proc Natl Acad Sci USA 82: 4438-4442), Palmiter and Brister (Cell 41: 343-345,1985) and US Patent No. 4,736,866, incorporated herein by reference. Briefly, an expression unit containing the DNA sequence to be expressed together with appropriately positioned expression control sequences is introduced into the pronuclei of fertilized oocytes. Typically, DNA is introduced by microinjection. The integration of the injected DNA is tested by analyzing the DNA from the tissue fragments, usually by a tail sample. It is usually preferable to introduce DNA into the animal's stem cell line so that it is passed on to its progeny.

In a most preferred embodiment of the present invention, a transgenic animal such as a mouse is created by introducing a mutation to avenge the glucagon receptor sequence {see, Mansour et al., "Disruption of the protooncogene int-2 in mouse embryo-derived stem cells: a general strategy fo targeting mutations. to non-selectable genes ". Nature 336: 348-352,1988). Such animals can be used as a model to study the role of the glucagon receptor in metabolism.

Glucagon receptor peptides

As mentioned above, the present invention also provides glucagon receptor peptides. In the context of the present invention, glucagon receptor peptides are understood to mean peptides containing portions of the glucagon receptor or its derivatives as described above, lacking endothelial domains, and which are at least 10 amino acids long. Briefly, the structure of the glucagon receptor as well as putative endothelial domains can be predicted from the primary translation product using, for example, a software hydrophobicity plot function such as P / C Gene or Intelligenetics Suite (Intelligenetics, Mt. View, CA), or according to the methods described in by Kyte and Doolitle {J Mol Biol 157 105-132, 1982) A plot of the hydrophobicity of the rat glucagon receptor is shown graphically in Figure 2. Aside from the graphical representation, based on this hydrophobicity analysis, it is believed that glucagon receptors have the general structure shown in Figure 1. In particular, these receptors are believed to contain an extracellular N-terminal domain, three extracellular loops and four intracellular loops, all separated by from each other with transmembrane domains

In one aspect of the present invention, there is provided an isolated glucagon receptor peptide comprising the extracellular N-terminal domain of the W glucagon receptor.

178 In a preferred embodiment, an isolated glucagon receptor peptide is provided comprising the amino acid sequence of SEQ ID NO. NO: 15 from glutamine, amino acid 28, to tyrosine, amino acid No. 142. Other isolated glucagon receptor peptides are also provided which can be selected from the extracellular and intracellular loop domains of the glucagon receptor (see Figure 11-5). In one embodiment, the glucagon receptor peptides are selected from the group consisting of 1ID (SEQ ID NO: 15, from lysine, No. 169 to histidine, No. 178), 1ELD (SEQ ID NO: 15, from tyrosine, No. 203 to Isoleucine No. 231), 2ID (SEQ ID NO: 15 from Phenylalanine No. 259 to Serine No. 266), 2ELD (SEQ ID NO: 15 from Valine No. 293 to Isoleucine No. 307 ), 3ID (SEQ ID NO: 15 from leucine No. 334 to lysine No. 345), and 3ELD (SEQ ID NO. No .: 15 aspartic acid, No. 371, to serine No. 380).

The glucagon receptor peptides of the present invention can be produced by recombinant techniques as described above or by synthetic methods, and can be purified as described below.

Purification of glucagon receptor peptides

Isolated glucagon receptor peptides can be produced, among other methods, by culturing appropriate host / vector systems to produce the recombinant translation products of the invention. Supernatants from these cell lines can be subjected to various purification procedures to isolate glucagon receptor peptides. For example, the supernatant may first be concentrated using commercially available protein concentration filters, such as Amicon or Millipore ultrafiltration units. Following concentration, the concentrate may be applied to a suitable purification substrate, for example glucagon or an anti-glucagon receptor antibody bound to a suitable substrate. Alternatively, anionic or cationic resins can be used to purify the receptor or peptide. Finally, one or more high performance liquid chromatography (RP-HPLC) steps may be used to further purify the glucagon receptor peptides.

A glucagon receptor peptide is considered "isolated" or purified, in the context of the present invention, if only one single band is visible when analyzed on SDS-polyacrylamide gel followed by staining with Coomasie blue.

Glucagon receptors, including their derivatives, as well as portions and fragments of these proteins, such as the glucagon receptor peptides described above, can be used to generate antibodies that bind specifically to the glucagon receptor. In the context of the present invention, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F (ab ') fragments₂ and Fab, as well as recombinantly produced binding partners. These binding partners include the gene variable regions derived from the gene encoding a specific monoclonal antibody. Antibodies are said to bind specifically when they bind the glucagon receptor to K._and greater than or equal to 10⁷ M.¹.

The affinity of the monoclonal antibody or single-chain antigen binding protein may be determined by any method known to those skilled in the art (see, Scatchard, Ann. NY, Acad. Sci. 51: 660-672, 1949).

Polyclonal antibodies can be raised in one of the known ways, in warm-blooded animals such as horses, cows, donkeys, chickens, mice and rats.

Briefly, the glucagon receptor is used to immunize an animal by intraperitoneal, intramuscular, intraocular, or subcutaneous injection. The immunogenicity of the glucagon receptor or glucagon receptor peptides can be enhanced by the use of an adjuvant such as Freund's complete or incomplete adjuvant. After several booster immunizations, serum samples are collected and tested for activity at the glucagon receptor. A wide variety of tests can be used to detect antibodies that bind specifically to the glucagon receptor. Exemplary tests are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Sprring Harbor Laboratory Press, 1988. Representative examples are: Countercurrent Immunoelectrophoresis (CIEP), Radioimmunoassays, Radioimmunoprecipitation, Enzymatic Immunosorbent Assays (ELISA), Dot tests

178 685

Blot, Inhibition or Competition tests, and sandwich tests (see, US Patent No. 4,376,11014,436,530; see also Antibodies A Laboratory Manual). Polyclonal sera which give a signal at least three times the background are especially preferred. Once the animal's antibody titers have reached plateau, in terms of their reactivity with the glucagon receptor, larger amounts of polyclonal serum can be obtained by either weekly exsanguination or exsanguination of the animal.

Monoclonal antibodies can be made using well known techniques (see U.S. Patent No. RE 32,001,4,902,614, 4,543,439, and 4,411,993; see also, Monoclonal Antibodies A New Dimension in Biological Analyzes, Plenum Press, Kennett, McKeam and Bechtol (eds.), 1980, and Antibodies A Laboratory Manual, Harlow and Lane (eds), Cold Spring Harbor Laboratory Press, 1988). Briefly, in one embodiment, an experimental animal such as a rat or a mouse is injected with a form of the glucagon receptor suitable to elicit an immune response against the glucagon receptor. Representative examples of suitable forms include, m. in: cells expressing the glucagon receptor, or peptides based on the sequence of the glucagon receptor. In addition, there are many known techniques to increase the resulting immune response, for example, by binding the receptor or receptor peptides to another protein such as ovalbumin or hemocyanin (KLH), or by the use of adjuvants such as Freund's complete or incomplete adjuvant. Initial immunization may be by the intraperitoneal intramuscular route of the subcutaneous intraocular route.

Between one and three weeks after the pre-immunization, the animal can be re-immunized with another boost. The animal may be bled and the serum tested for binding to the glucagon receptor using the tests described above. Additional immunizations may be performed until the animal has stabilized for reactivity to the glucagon receptor. The animal may then be given a final immunization like glucagon receptor or glucagon receptor peptide and sacrificed three to four days later. During this time, the spleen and lymph nodes can be harvested and ground into a unicellular suspension by passing the organs through a sieve or by breaking the spleen sheaths and lymph nodes surrounding the cells. In one embodiment, the red blood cells are lysed by the addition of a hypotonic solution followed by immediate isotonic equalization.

In another embodiment, suitable cells for the production of monoclonal antibodies are obtained by using an in vitro immunization technique. Briefly, the animal is killed and the spleen and lymph nodes are harvested. A unicellular suspension is prepared! places cells into a culture containing a form of the glucagon receptor suitable for eliciting an immune response, as described above. Then, lymphocytes are harvested and fused as described below.

Cells obtained by in vitro immunization, or from immunized animals as described above, can be immortalized by transfection with a suitable virus, such as Epstein-Barr virus (EBV) (see Glasky and Reading, Hybridoma 8 (4). 377-389. , 1989). Alternatively, in a preferred embodiment, the harvested spleen and / or lymph node cell suspensions are fused with appropriate myeloma cells to produce a "hybridoma" that secretes monoclonal antibodies. Suitable myeloma lines preferably are deficient in antibody construction or expression, and are further synergistic with cells of the immunized animal. Many such myeloma lines are known to those skilled in the art and can be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland, (see Catalog of Cell Lines 8c Hybndomas, edited by ATCC, 1988). Representative myeloma lines include human, UC 729-6 (ATCC No. CRL 8061), MC / CAR-Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2 / 0-Agl4 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9), and for rats Y3-Agl 2 3 (ATCC No. CRL 1631) and YB2 / 0 (ATCC No. CRL 1662). Particularly preferred lines are NS-1 (ATCC No. TIB 18) and P3X63Ag8.653 (ATCC No. CRL 1580) which can be used for fusion with mouse, rat or human cells. Fusion between a myeloma cell line and cells from an immunized animal can

178 685 can be made in a number of ways, including the use of polyethylene glycol (PEG) (see Antibodies A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988) or electrofusion (see Zimmerman and Vienken, J Membrane Biol 67). : 165-182, 1982).

After fusion, cells are plated on culture dishes containing a suitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium) (JHR Biosciences, Lenexa, Kan.). The medium may also contain additional components such as Bovine Fetal Serum (FBS, e.g. from HyClone, Logan, Utah, or JRH Biosciences), thymocytes obtained from puppies of the same species as used for immunization, or agar to harden the medium. Additionally, the medium should contain a reagent that selectively allows the growth of splenocytes fused to myeloma. The use of HAT (hypoxanthine, aminopterin and thymidine) (Sigma Chemical Co., St. Louis, Missouri) is particularly preferred. After about seven days, the resulting cells or hybridomas can be tested for the presence of antibodies that recognize the glucagon receptor. Following cloning dilution several times and retesting, a hybridoma that produces a monoclonal antibody that binds the glucagon receptor can be isolated.

Other techniques may also be used to obtain monoclonal antibodies (see William D. Huse et al., "Generation of Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lamda", Science 246: 1275-1281, December 1989, see also, L. Sastry et al, "Cloning of the Immunological Repertoire in Eschenchia coli for Generation of Monoclonal Catalytic Antibodies: Construction of Heavy Chain Variable Region - Specific cDNA Library", Proc Natl Acad Sci USA 86: 5728-5732, August 1989; see also, Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Altemative to Hybridomas", Strategies inMolecular Biology3: 1-9, January 1990; These references describe a commercially available system from Stratacyte, La Jolla, Califomia for producing antibodies by recombinant techniques.) Briefly, mRNA is isolated from a population of B lymphocytes and used to create cDNA libraries expressing heavy and light chains of immunoglobulins in the ZIMMUNOZAP (H) vectors and ZIMMUNOZAP (L). These vectors can be viewed individually or co-expressed to generate Fab fragments or antibodies (see Huse et al. in.; see also, Sastry et al., ibid.). The positive plaques can then be converted to a non-lysis-inducing plasmid that allows high-level expression of monoclonal antibody fragments in E. coli.

Similarly, binding partners can also be constructed using recombinant DNA techniques to integrate the variable regions of a gene encoding a specific binding antibody. The construction of these proteins can be made according to known methods (see James W. Larrick et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes from Single Hybridoma Cells", Biotechnology 7: 934-938, September 1989, Riechmann et al., "Reshaping Human Antibodies for Therapy", Nature 332: 323-327,1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering", Nature 328. 731 -734.1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Lysosyme Activity", Science 239: 1534-1536, 1988; Chaudhary et al., "A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin", Nature, 339: 394-397, 1989; see also, US Patent No. 5,132,405 entitled "Biosynthetic Antibody Binding Sites").

DNA segments encoding glucagon receptor-specific binding domains were amplified with hybridomas producing a specific binding monoclonal antibody, and inserted directly into the genome of a human antibody-producing cell (see Verhoeyen et al., See also Reichmann et al., Yw). This technique allows the antigen binding site of a mouse or rat specifically binding monoclonal antibody to be transferred to a human antibody. Such an antibody is preferred for human therapeutic applications because it is not as antigenic as rat or mouse antibodies.

Alternatively, the antigen binding site (variable region) can be linked to or introduced into another completely different protein (see Chaudhary et al., Supra), resulting in new

178 A protein with an antibody antigen-binding site as well as the activity of a completely different protein As is known, the antigen-binding site or the glucagon receptor binding domain is located in the variable region of an antibody. DNA sequences encoding small portions of the antibody or variable regions that bind specifically to the mammalian glucagon receptor may also be used. These portions can be readily tested for binding to the glucagon receptor using the assays described below.

The glucagon receptor of the present invention can be used to detect the presence of glucagon antagonists by a method that comprises the steps of (a) exposing the compound in the presence of glucagon agonists to a recombinant glucagon receptor associated with a response pathway under conditions and for a time sufficient for the compound to bind to the receptor and bind to it. follow the path of the answer, (b) detecting a reduction in the stimulation of the response pathway resulting from binding of the compound to the glucagon receptor relative to the stimulation of the response pathway by the glucagon agonist alone, and therefore determining the presence of a glucagon antagonist.

Glucagon agonists include molecules (including glucagon itself) that are capable of binding to the glucagon receptor and that stimulate a cellular response pathway.

A wide variety of compounds can be tested using these methods. Representative examples include the blocking antibodies described above, glucagon receptor peptides, and glucagon analogs (including both peptide and non-peptide hgands). For example, US Patent No. 07 / 741,931 provides methods for the production of a large number of glucagon analogs using pooled DNA sequences encoding these analogs. These glucagon analog-encoding pools can be generated by saturation mutagenesis of the glucagon-encoding DNA sequence (e.g., Little Gene 88 113-115; Hambers et al., Gene 88: 143-151, 1989), by segment-directed mutagenesis (e.g., Shortle et al., Proc Natl AcadSci USA 77: 5375-5379, 1980) by forced erroneous incorporation of nucleotides (e.g. Liao and Wise Gene 88: 107-111, 1990) or by using randomly mutagenized oligonucleotides (Hutchinson et al., Proc Natl AcadSci USA 83: 710-714, 1986). Single transformants expressing glucagon analogues can be cloned as described above or pooled.

Compounds are exposed to the recombinant glucagon receptor in the presence of glucagon agonists under conditions and for a time sufficient for the compound to bind and elicit a response through this pathway. Conditions and a time suitable for binding of the glucagon antagonist to the receptor may vary depending on the source of the receptor, but suitable conditions for binding typically include: a temperature between 4 ° C and 55 ° C in a buffer solution between 0 and 2 MNaCl, preferably between 0 and 0.9 MNaCl and particularly preferably in 0.1 M NaCl solution, with a pH in the range 5-9 and preferably 6.8-8. The time appropriate for binding and elicitation of a response is typically in the range of 5-15 minutes after exposure.

When a compound is exposed to the recombinant glucagon receptor in the presence of a glucagon agonist, under conditions and for a time sufficient for the compound to bind to the receptor, a reduction in stimulation of the response pathway can be detected if the compound competes with glucagon agonists for the recombinant glucagon receptor. The response pathway may be the membrane-bound adenylate cyclase response pathway, and the detection step includes measuring the reduction of cAMP through the membrane-bound adenylate cyclase pathway, relative to cyclic AMP production in the presence of glucagon alone. Preferably, the reduction in stimulation of the response pathway is equal to or greater than the reduction associated with des-His¹ -glucagon. Assays for adenylate cyclase activity can be performed, for example, using the method described by Lin et al (Biochem. 14: 1559-1563, 1975), and described in the Examples. These methods measure the level of cAMP stimulation relative to native glucagon, and typically involve exposure of the preparation of cells that express a biologically active recombinant glucagon receptor onto the mixture of glucagon and test compound in the presence of radiolabeled ATP.

Alternatively, cAMP production can be measured using methods known to those skilled in the art, including, for example, the methods described by Salomon et al. (Anal. Biochem. 58 541-548, 1976) or Krishna et al. (J Pharmacol. Exp. Ther 163: 379, 1986) or preferably at

178 685 using commercially available kits, such as Amersham Corporation's Scintillation Proximity Assay Kit. The Scintillation Proximity Assay Kit measures cAMP production by competition of iodinated cAMP with an anti-cAMP antibody. Especially preferred glucagon receptors have ED biological activity in this assay₅₀ (Effective Dose producing 50% response) less than 1 nM, more preferably ED₅₀ less than 0.7 nM, and most preferably less than 0.25 nM.

The response pathway may also include a luciferase reporter system. Briefly, luciferase is an enzyme that catalyzes photon secretion by luciferin, and can thus be detected when expressed in the presence of luciferin (Alam and Cook, Anal. Blochem. 188, 245-254, 1990). It is particularly preferred that the DNA construct comprises a cAMP response element, such as, for example, a proenkephalin cAMP response element operably linked to the luciferase cDNA. The DNA construct containing the luciferase cDNA is stably transfected into the host cell. The cell is then transfected with a second DNA construct containing a first DNA segment encoding the glucagon receptor operably linked to additional DNA segments needed for receptor expression. Upon binding of the glucagon receptor agonist, elevated levels of cAMP result in the expression of luciferase. Luciferase is exposed to luciferin and the photons released during the oxidation of luciferin by luciferase are measured.

In another embodiment, activation of the response pathway causes an increase in intracellular free calcium. Various assays can be performed to determine the concentration of free intracellular calcium, including, for example, the QuinZ fluorochrome method described by Charest et al. (J Biol Chem 259: 8769-8773, 1983) or the method based on aequorin, a light-emitting protein, described by Nakajima-Shimada {Proc Natl AcadSci USA 88: 6878-6882, 1991). A particularly preferred method is the intracellular calcium photoimaging method described in detail in Example 6. Briefly, in one embodiment, cells are transformed with a glucagon receptor expressing plasmid and grown for three days under normal culture conditions. The medium is removed and replaced with a solution containing 10 µΜ of fura-2AM {see, Grynkiewicz et al., J Biol. Chem 260: 3440-3450, 1985). Cells are incubated for 30 minutes in the dark, then washed and re-incubated for 30 to 120 minutes. Photoimaging can be performed using a Nikon Diaphot inverted fluorescence microscope equipped with a mercury lamp. Cells can first be viewed for 60 minutes to establish a zero level and then stimulated with a glucagon-containing buffer. Images are typically captured for at least three minutes post-stimulation. Software such as Inovision (Research Triangle Park, NC) can be used to process and measure the images.

The following examples are presented to illustrate the invention and not to limit it.

Example 1

CDNA synthesis and execution of cDNA libraries

A. Synthesis of rat liver cDNA

Livers were harvested from 30 g female Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) and immediately placed in liquid nitrogen. Total RNA was isolated from liver tissue using guanidine isothiocyanate (Chirwig et al., Biochemistry 18: 52-94, 1979) and CsCl gradient centrifugation. Poly (A)⁺ RNA was isolated using oligo d (T) cellulose chromatography (Aviv and Leder, Proc Natl Acad Sci USA 69: 1408-1412, 1972), cDNA was prepared on a matrix of double purified poly (T) poly (A)⁺ RNA from the liver. Ten microliters of a solution containing 10 pg of poly (A)⁺ Liver RNA was mixed with 2 μ 20 pmol / μl of ZC3747 first strand primer (SEQ ID NO: 7) and 4 μΐ of diethyl pyrocarbonate treated water. The mixture was heated at 65 ° C for 4 minutes and cooled on ice.

First mci cDNA synthesis was started by adding 8 μΐ 5 × SUPERSCRIPT buffer (GIBCO BRL, Gaithersburg, Md), 4 µl 100 mM dithiothreitol, and 2.0 µl deoxytriphosphate solution containing 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (Pharmacia LKB

178 685

Biotechnology Inc., Piscataway, NJ) into an RNA-primer mixture. The mixture was incubated at 42 ° C for 3 mm. After incubation, 6.0 μΐ 200U / pl SUPERSCRIPT reverse transcriptase (GIBCO BRL) was added. First-strand synthesis efficiency was tested in a parallel reaction by adding 10 pCi³²P-adCTP for 10 µl aliquots of the reaction mixture for labeling the reaction products. First strand synthesis reaction mixtures were incubated at 45 ° C for 45 minutes and then at 50 ° C for 15 minutes. The reaction was stopped by adding water to a final volume of 100 µl, then extracted twice with phenocchloroform (1: 1) and one extraction with chloroform / isoamyl alcohol (24: 1). Non-incorporated nucleotides were removed by two-fold cDNA precipitation in the presence of 6 µg glycogen, 2.5 M ammonium acetate and 2.5 volumes of ethanol. Unlabeled cDNA was suspended in 50 µ 50 of water and used for second strand synthesis. First strand cDNA length was determined by suspending the labeled cDNA in 20 µL of water and determining the size of the cDNA by agarose gel electrophoresis.

Second-strand synthesis was performed on a DNA-RNA hybrid from first-strand synthesis under conditions that allow the first strand to initiate second-strand synthesis by forming DNA "pins" A reaction mixture was prepared containing 20.0 µl of 5x polymerase I buffer (100 mM Tris, pH 7, 4.500 mM KCl 1.25 mM MgCl₂, 50 mM (NH₄)₂SO₄), 4.0 pl 100 mM dithiothreitol, 1.0 μΐ solution containing 10 mM of each deoxynucleotide triphosphate, 3.0 μ1 β-NAD, 15.0 pl 3 U / μΙ E coli DNA ligase (NBL Enzymez Ltd, Cramhngton, Northumbia , England), 5.0 µl 10 U / µl E coli DNA polymerase I (GIBCO BRL) and 50.0 µl unlabeled first strand DNA. Parallel reactions in which aliquots of 10 µl of the second mci synthesis were labeled by the addition of 10 µCi³²P-adCTP was used to monitor the efficiency of the second strand synthesis reaction. Reactions were incubated at room temperature for 4 minutes, and then 1.5 µ12U / µl RNase H (GIBCO BRL) was added to each of the mixtures. The reaction was incubated at 15 ° C for 2 hours and then for 15 minutes at room temperature. Reactions were stopped by the addition of 4 μ 4 500 mM EDTA followed by extraction with phenol / chloroform and chloroform / isoamyl alcohol mixtures as described above. DNA from each reaction was precipitated in the presence of ethanol and 2.5 M ammonium acetate. DNA from the unlabeled reaction was suspended in 50 µl of water. Labeled DNA was suspended and electrophoresed as described above.

Single-stranded DNA in "pins" was cut using mung bean nuclease. Reactions contained 10 μΐ 10x Mung Bean Nuclease Buffer (Stratagene Cloning Systems, La Jolla, Calif.), 4 pil of 200 mM dithiothreitol, 34 μΐ water, 50 μΐ second strand cDNA, and 2 μΐ a dilution of 110 Mung Bean Nuclease (Promega Corp, Madison , Wis.) In Stratagene MB Dilution Buffer (Stratagene Cloning Systems). The reaction was incubated at 37 ° C for 15 minutes then quenched by the addition of 20 µL Tris-HCl, pH 8.0 followed by extraction with phenol / chloroform and chloroform / isoamyl alcohol mixtures as described above. After extraction, the DNA was precipitated in ethanol and suspended in water.

Blunt ends were made in the resuspended DNA using T4 DNA polymerase. cDNA dissolved in a volume of 192 μΐ of water, mixed with 50 μΐ of 5 × T4 DNA polymerase buffer (250 mM Tris-HCl, pH 8.0, 250 mM KC1.25 mM MgCl₂), 3 μΐ 100 mM dithiothreitol, 3 μl of a solution containing 10 mM of each deoxynucleotide triphosphate and 2 μ of 6.7 U / μΙ T4 DNA polymerase (Pharmacia LKB Biotechnology Inc.). After incubation at 15 ° C for 30 minutes, the reaction was quenched by the addition of 2 µM of 500 mM EDTA, followed by serial extractions with phenol / chloroform and chloroform / isoamyl alcohol mixtures as described above. The DNA was ethanol precipitated and dissolved in 30 µ of water. Based on embedding³²P-dCTP, the cDNA yield was determined as 4 µg with 10 µg starting mRNA.

B. Preparation of a rat liver cDNA library

To facilitate cDNA cloning into a mammalian expression vector, EcoRI linkers (Invitrogen, San Diego, Calif.) Were added to the cDNA prepared as described above. 10 µl of cDNA and 800 pmoh of linker (12 µl) were mixed with 4.0 µl 10x ligase buffer (Stratagene Cloning Systems), 4.0 µl of 10 mM ATP, 6.0 µl of water and 16 units of T4 DNA ligase (4.0 µl;

178 685

Stratagene Cloning Systems) The reaction was incubated for sixteen hours with a temperature gradient from 4 ° C to 15 ° C. The reaction was stopped by the addition of 185 µ of water, 25 µ RE of REACT 2 buffer (GIBCO BRL) and then incubated for 30 to 60 minutes at 65 ° C. After incubation, the reaction mixture was extracted with phenol / chloroform and chloroform / isoamyl alcohol mixtures and ethanol precipitated as described above. After centrifugation, the DNA pellet was washed with 70% ethanol and air dried. The pellet was suspended in 18 µ of water.

To facilitate the targeted insertion of cDNA into the mammalian expression vector, the cDNA was digested with XhoI, resulting in cDNA with "sticky" ends: 5 'EcoRI and 3' XhoI. An Xho I restriction site at the 3 'end of the cDNA was introduced by primer ZC3747 (SEQ ID NO: 7). Restriction digestion was interrupted by a series of phenol / chloroform and chloroform / isoamyl alcohol extractions. The cDNA was ethanol precipitated and the resulting pellet was washed with 70% ethanol and air dried. The pellet was suspended in 1x buffer (10 mM phosphate buffer, pH 8.8, 5% glycerol, 0.125% bromophenol blue).

The resuspended cDNA was heated to 65 ° C for 10 minutes, cooled on ice, and electrophoresed on 0.9% low melting point agarose (Seaplaque GTG Low Melt Agarose, FMC Corp., Rockland, Me.) Using a BRL 1 "ladder" kb (GIBCO BRL) and 100 bp (Pharmacia LKB Biotechnology Inc.) as the size standard. Contaminating linkers and by-product fragments less than 800 bp in size were excised from the gel. The electrodes were inverted and the cDNA was electrophoresed until it was concentrated near the start of the lane. The gel fragment containing the concentrated DNA was excised, placed in a test tube and its volume determined approximately. Half the volume of the gel piece was added and the agarose was melted by heating to 65 ° C for 15 minutes. After the sample temperature was brought to 42 ° C, approx. 5 units of β-Agarase I (New England Biolabs, Beverly, Mass.) Were added. The sample was incubated for 90 minutes to digest the agarose. After incubation, 0.1 aliquot of 3 M sodium acetate was added, and the mixture was incubated for 15 minutes on ice. After incubation, the sample was centrifuged at 14,000 × g for 15 minutes at 4 ° C to remove undigested agarose. The cDNA in the supernatant was ethanol precipitated. The cDNA pellet was washed with 70% ethanol, air-dried, and suspended in 10 µL of water.

The produced cDNA was cloned into the E. coli vector pZCEP, a derivative of pCDNAl (Invitrogen), in which the origin of M1 replication and the marker enabling SopF selection were replaced with the beta lactamase cassette from pUC18. The plasmid pZCEP, linerized by digestion with EcoRI and Xhol and ligated with EcoRI cDNARI -Xhol. The produced plasmids were introduced by electroporation into E. coli cells of strain DH 10B ELECTROMAX (GIBCO BRL).

C. Human Islet Cell cDNA Synthesis

Islets from human pancreas were collected from transplant donors for which no recipient could be found. After in situ perfusion with cold UW solution (DuPont, Boston, Mass.), Each pancreas was carefully excised, the pancreatic duct was cannulated and a 4 mg / ml collagenase solution (Type V, Sigma, St. Louis, MO.) Was continuously injected. , initially at 4 ° C then 39 ° C. The gland was ground and the released fragments washed by centrifugation, passed through needles of decreasing diameter and cleared by centrifugation on a discontinuous Ficoll gradient (Warnock, 35 (Suppl. 1): 136-139, 1989). Upper interphase material was pooled and counted after islet purity determination by dithiazone staining. The islets used to create the library were more than 65% pure and those used for the Northem blot analysis were more than 40% pure. The average of the islet was 175 pm. Additionally, the isolated islets showed first and second phase of insulin secretion function after perfusion with either high glucose concentration or isobutyl methylxanthine (IBMX).

Poly (A)⁺ RNA was isolated using the FASTTRACK mRNA isolation kit (Invitrogen), according to the manufacturer's instructions. Briefly, 30,000 purified islets were lysed in lysis buffer, homogenized using tapering needles, and digested in the presence of proteinase K and RNasin followed by poly (A)⁺ RNA was purified by chromatography using oligo d (T) cellulose. The concentration and purity of the eluted fractions was determined by the optical density OD_260/280

178 685

About 2.5pg poly (A)⁺ Human islet RNA was used to make a library using the LIBRARIAN R II cDNA library construction system (Invitrogen) and ELECTROMAX E. coli DH10B cells (GIBCO BRL) according to the manufacturers' instructions. Briefly, about 2.5 pg poly (A)⁺ RNA was isolated from human islets, converted to double-stranded cDNA, and then the BstXI palindromic linker (Invitrogen) was added. The cDNA was size fractionated and the unreacted linker was removed by agarose gel electrophoresis and electroelution. Complementary DNA strands larger than 600 bp were selected.

Example 2

Isolation of rat glucagon receptor cDNA by polymerase chain amplification rat liver cDNA was used as a template to amplify glucagon receptor sequences using degenerate oligonucleotides (ZC4715 and ZC4701; SEQ ID NOS: 9 and 8) corresponding to regions of the high gene conservation family . 50 µL of the reaction mixture containing 5 ng of cDNA template (Example 1A) was set up; 100 pmoles of each of the oligonucleotides ZC4715 (SEQ ID NO: 9) and ZC4701 (SEQ ID NO: 8); 0.25 mM of each deoxynucleotide triphosphate (Cetus, Emeryville, CA); 1x 10x Promega buffer (Promega Corp.) and 12.5 units of Taq polymerase (Promega) 40 cycles of PCR (one minute at 95 ° C; one minute at 42 ° C and two minutes at 72 ° C) were performed followed by incubation for 7 minutes at 72 ° C.

A 650 bp PCR product was isolated by electrophoresis and ligated into pCRIOOO (Stratagene Clomng Systems). The resulting plasmid was used to transform E coli XL-1 cells. Plasmid DNA was isolated from the selected transformant, designated G13 / pCR10001 and sequenced (SEQ ID NO: 14). Sequence analysis of the clone revealed that the insert encodes a secretin receptor related polypeptide.

Example 3

Full-length rat glucagon receptor cDNA cloning The full-length rat glucagon receptor cDNA was obtained by viewing the library described in Example 1B in the glucagon binding assay. The library was seeded to obtain one million independent clones. Transformed colonies from each plate were scraped into LB-Amp (Sambrook et al., Yw). Cells were rinsed by centrifugation and the medium was removed. The cell pellets were resuspended in 4 ml of LB-Amp, 15% glycerol, and then stored four 1-liter aliquots at -80 ° C. The first batch in glycerol was titrated and 100 pools of 5,000 colonies were plated per plate. After colony growth, each plate was scraped into 10 ml of LB-Amp. Aliquots of cells from each pool were taken to prepare plasmid DNA. The remaining cell mixtures were brought to a final volume with 15% glycerol, aliquoted and frozen at -80 ° C. Plasmid DNA was prepared from each of the cell pools, and digested with RNase (Boeringer Mannheim, Indianapolis, Ind.) According to the manufacturer's instructions. The RNase reaction was stopped by extraction with phenol / chloroform / isoamyl alcohol (24: 24: 1), then the DNA was precipitated with ethanol

Plasmid DNA from each pool was transfected into COS-7 cells (ATCC CRL 1651), and the transfectants were tested for the presence of glucagon receptors in a binding assay. ^l25I-glucagon. Briefly, approximately 2X10 was seeded the day before transfection⁵ cells into sterile single-chamber plates (Nunc AS, Roskilde, Denmark) coated with 10 pg / ml of human fibronectin (Table 1) for 30 minutes at room temperature and washed with phosphate buffered saline (PBS, Sigma Chemical Co., St. Louis, Mo) Two micrograms of plasmid DNA from each pool were used to transfect cells in single-chamber plates using the method described by McMahan et al., (EMBO J, 10: 2821-2832, 1991; incorporated herein by reference) After transfection, cells cultured for 72 hours at 37 ° C in 5% CO₂.

178 685

Table 1

Human Fibronectin

<td>4g</td><td>human plasma fibronectin lyophilized powder (Alpha Therapeutics Corp, Los Angeles, Calif)</td>

<td>50 ml</td><td>1 mM NaPCL, pH 7.4 (a mixture of one-1 two Na), 300 mM NaCl</td>

The lyophilized powder was dissolved in the buffer solution. Ammonium sulfate was added to a concentration of 25%, and the solution was allowed to precipitate overnight at 4 ° C. Fibronectin was collected by centrifugation in a Bench Top centrifuge (Beckman Instruments, Inc., Irvine, Califf.) For fifteen minutes at 1000 rpm. The supernatant was removed and the pellet was dissolved in 10 ml of NaPO buffer solution₄ (above).

Fibronectin in a final volume of 16.9 ml was dialyzed overnight into 1 L NaPO buffer solution₄ (described above). Dialyzed material was diluted three times with 1 mM NaPO₄, pH 7.4, to obtain a solution of 1 mMNaPO₄, pH 7.4, 100 mMNaCl. Fibronectin was diluted twice with distilled water. Undissolved precipitate was removed with a glass rod.

Fibronectin was subjected to FPLC through a 50 ml DEAE FF Sepharose column (Pharmacia LKB Biotechnology Inc, Piscataway, NJ), equilibrated with three volumes of 18 mM Tris, pH 8.1, 50 mM NaCl. After washing the column with 18 mM Tris, pH 8.1.50 mM NaCl, until a blank sample was obtained, fibronectin was eluted with a salt gradient to 18 mM Tris, pH 8.1, 300 mM NaCl. Fractions were collected, and aliquots of fractions were electrophoresed on polyacrylamide gel and analyzed by Coomasie Blue staining and Western blot analysis. Peak fractions were pooled and dialyzed against 10 mM CAPS (3- (cyclohexyamino) -1 propanesulfbnic acid, Sigma), pH 11.0, 10 mM CaCl₂, 150 mM NaCl. The solution was stored at -80 ° C.

To generate transfectants for the binding assay ¹²⁵I-glucagon, the medium was aspirated from above the cells, and they were washed three times with cold (4 ° C) PBS. After the final wash, cells were coated with Binding Assay Medium containing 0.5 nM^I25I-glucagon (Amersham receptor grade, specific activity 2000 Ci / mmol; Amersham). The cells were rocked at 30 ° C for one hour. The medium was aspirated from the cells, cold (4 ° C) Glucagon-Free Binding Assay Medium was added, and the cells were incubated for 5 minutes at room temperature. The medium was aspirated from the cells and rinsed three times with cold (4 ° C) PBS. After the final wash, cells were fixed with 1 ml of 2.5% glutaraldehyde in PBS at room temperature for 20 minutes. Glutaraldehyde was removed and cells were washed three times with PBS. Slides were air dried for one hour at room temperature, dipped in a photographic liquid emulsion (Eastman Kodak Co., Rochester, Ν. Y.) according to the manufacturer's instructions, and dried at room temperature in the dark for at least 30 minutes. Slides were placed in a darkened box for 72 hours at 4 ° C. Cells capable of binding to glucagon were detected at 2.5X magnification in the dark field. In one pool, # 57, cells capable of binding glucagon were found.

<td colspan="2">Table 2 Medium for the Binding Test</td>

<td>RPMI</td><td>1640 (Sigma) containing 20pg / ml bacitracin (Sigma) 25 mM HEPES buffer, pH 7.4 1% penicillin / streptomycin solution (Sigma) 2 mM glutamine 50 U / ml aprotymny (Sigma) 1% Bovine Serum Albumin, Fraction V (Sigma)</td>

178 685

1M sodium bicarbonate

8.4 g of solid NaCO₃

Sodium bicarbonate was poured into a 100 mL measuring vessel, and 80 cm were added³ distilled water. The solution was stirred until completely dissolved. Distilled water was added to 100 ml. The solution was mixed again and stored at 4 ° C in a sealed vessel.

Medium

One milliliter of IM sodium bicarbonate was added to 1 liter of distilled water. Four liters were prepared with the solution cooled overnight, or prepared with cold distilled water.

69% sucrose solution with g of sucrose

The sucrose was dissolved in 31 ml of distilled water by heating. The concentration was tested using a refractometer. Depending on the needs, either sucrose or distilled water was added to bring the concentration to 69% ± 0.5%.

42.3% sucrose solution with g sucrose

Sucrose was dissolved in 57 ml of distilled water by heating. The concentration was measured with a refractometer. As needed, 69% sucrose solution or water was added, bringing the concentration to 42.3 ± 1%

BufferX2 for the binding assay

100 mM HEPES, pH 7.3

300 mM NaCl mM EDTA

2% bovine serum albumin

1.6 mg / ml bacitracin

Imaging Buffer

140 mM NaCl 10 mM HEPES 5.6 mM glucose 5 mM KC1 mM MgSO₄ mM CaCl₂

Solution Fura-2 AM mg fura-2 AM (Molecular Probes) ml DMSO ml Imaging Buffer

Fifty milligrams of fura-2 AM was dissolved in 50 mL of DMSO. After dissolution, the solution was mixed with 5 ml of Imaging Buffer.

An aliquot of plasmid DNA from pool # 57 was subjected to PCR amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID NOS: 8 and 9). A 50 µl reaction mixture was prepared containing between 200 ng and 400 ng of plasmid DNA from pool # 57; 100 pmoles of each of the oligonucleotides ZC4701 and ZC4715 (SEQ ID NOS: 8 and 9); 50 mM KCl; 10 mM Tns-HCl, pH 9.0 (at 20 ° C); 1.5 mM MgCl₂; 0.01% gelatin; 0.1% Triton X-100; 0.2 mM of each deoxynucleotide triphosphate (PharmaciaLKB Biotechnology Inc) and 1 Unit of Taq polymerase (Promega). 30 cycles of PCR reactions were performed (two minutes at 95 ° C, two minutes at 45 ° C, and two minutes at 72 ° C) followed by a 7-minute incubation at 72 ° C. The reaction mixture was stored at 4 ° C. Analysis of the PCR product by gel electrophoresis showed the presence of a 700 bp band which was almost the same size as the product described in Example 2.

The pool # 57 glycerol sample was titrated and 20 plates of 500 colonies each were plated.

178 The glucagon binding as described above. In one pool, # 57-18, cells capable of binding glucagon were found.

Pool plasmid DNA sample # 57-18 was subjected to PCR amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID NOS. 8 and 9) as described above. Analysis of the PCR product by gel electrophoresis showed the presence of a 700 bp band, confirming the presence of the glucagon receptor DNA sequence.

The glycerol sample of pool # 57-18 was titrated and 6 plates of 50 colonies and 47 plates of 20 colonies were plated. Colonies were pooled and glycerol and plasmid DNA samples were made as described above. Aliquots from each of the pools were transfected into COS-7 cells and the transfectants were tested in a glucagon binding assay as described above. In addition, an aliquot of plasmid DNA from each pool was amplified using oligonucleotides ZC4701 and ZC4715 (SEQ ID NOS: 8 and 9) as described above. Four pools (# 57-18-16, # 57-18-18, # 57-18-36 and # 57-18-48) containing glucagon-binding cells were detected, which could be detected by PCR amplification confirming the 700 bp band.

For isolation, cDNA was plated on each of two 150 mm plates of 2,500 colonies of pool # 57-18. Filter impressions were obtained according to the method described by Hanahan and Meselson (Gene 10: 63,1980) and Sambrook et al. (Ibid), incorporated herein by reference. A hybridization probe was obtained by PCR amplification of plasmid DNA from pools # 57-18 using the oligonucleotides and methods described above. The PCR product was purified from a low melting point agarose gel and started using the MEGAPRIME kit (Amersham, Arlington Heights, III) according to the manufacturer's instructions. The filters were hybridized in a solution containing 6XSSC, 5X Denhardt's solution, 5% SDS, 200 pg / ml of sonicated salmon sperm DNA and a PCR fragment labeled with³²P o specific activity 2x 10³ cpm / ml. Filters were hybridized overnight at 65 ° C. Excess label was removed by three washes with 2XSSC, 1% SDS at 65 ° C. The film was exposed to the filters for four hours at -80 ° C. Positive clones containing the pLJ4 plasmid were identified and sequenced. Plasmid pLJ4 is deposited with the American Type Culture Collection (12301 Parklawn Dr., Rockville, MD 20852) as K coli transformant, under number 69056 August 21, 1992.

Restriction site analysis and sequence analysis showed that pLJ4 contains an insert of approximately 2 kb, encoding a 485 amino acid protein with a predicted molecular weight of 54,962 Da. The nucleic acid sequence and amino acid sequence are shown in Sequence Identifiers. No.14 and 15. Hydropathy analysis using the method of Kyte and Doolitlle (J Mol Biol 157: 105-132, 1982; incorporated herein by reference) revealed eight hydrophobic amino acid intervals corresponding to the N-terminal signal sequence and seven transmembrane domains (Figure 2). In addition, analysis of the predicted amino acid sequence revealed the presence of four potential N-linked glycosylation sites located in extensive hydrophilic sequences and the presence of six cysteines in the same region.

Example 4

Isolation of human glucagon receptor cDNA by amplification by the human islet cDNA polymerase chain reaction (Example 1C) was used as a template to amplify human glucagon receptor sequences using degenerate oligonucleotides ZC4715 and ZC4701 (SEQ ID NOS: 9 and 8). A 50 µl reaction mixture containing 5 ng of template cDNA (Example 1C), 100 pmoh of each of the oligonucleotides ZC4715 (SEQ ID NO: 9) and ZC4701 (SEQ ID NO: 9) was set up. No .: 8); 0.25 mM of each deoxynucleotide triphosphate (Cetus Emeryville, Calif.,); IX Buffer 10Χ Promega (Promega). 40 cycles of PCR reactions (one minute at 95 ° C, one minute at 45 ° C and two minutes at 72 ° C) were run, followed by an incubation of 7 minutes at 72 ° C.

178 685

A PCR product of approximately 750 bp in length was isolated by gel electrophoresis. One tenth of the isolated PCR product was used as a template for an additional PCR reaction using the oligonucleotides ZC4758 and ZC 4778 (SEQ ID No. 10111), which were designed to introduce a BamHI restriction site at the 3-end 'and an EcoRI restriction site at the 5' end of the PCR product for cloning purposes. A 50 µl reaction was set up as described above. 40 cycles of PCR reactions (one minute at 95 ° C, one minute at 50 ° C, and half a minute at 72 ° C) were run, followed by a 7 minute incubation at 72 ° C.

To test transformants for the presence of human glucagon receptor sequences, the insert present in each of the transformants was amplified using oligonucleotides ZC447 and ZC967 (SEQ ID NOS: 1 and 2) which were designed as universal primers to sequence the pUC plasmid and complementary to the pUC sequences flanking the insert PCR product. Forty-eight transformants were loaded into a 25 µL reaction mixture containing 20 pmol of each of the oligonucleotides; 0.125 mM of each of the oligonucleotide triphosphates (Cetus, Emeryville, Calif.,); IX buffer 10Χ Promega (Promega) and 1.25 units of Taq polymerase (Promega). 30 cycles of PCR reactions were performed (one minute at 95 ° C, one minute at 45 ° C, and a half minute at 72 ° C) followed by a 7 minute incubation at 72 ° C.

The PCR products were then tested by Southern hybridization (Southern, J. Mel. Biol. 98: 503.1975; and Sambrooki et al., Ibid., Which are hereby referenced) using the 1.9 kb EcoRI-Xhol fragment of pLJ4, labeled random primers using the MEGAPRIME kit (Amersham) as a probe. One of the clones, G30, hybridized to the full-length rat DNA probe. The nucleotide sequence of G30 is shown in SEQ ID NO. No .: 16.

Example 5

Human glucagon full-length cDNA cloning

To identify a library containing the human glucagon receptor encoding sequences, a series of libraries were tested by PCR using the ZC5433 and ZC5432 oligonucleotide primers (SEQ ID NOS: 13 and 12) designed to include the sequence from the G30 clone described above. Acquired and made human genomic and cDNA libraries from human liver, cells, islets, brain and placenta were studied (Table 3). Separate 50 μΐ reactions were set up with DNA from various libraries in the volumes listed in Tables 4.20 pmol / plkazdegoz ZC5433 iZC5432 (Sequence Identifiers 13 and 12), 0.25 mM of each deoxynucleotide triphosphate, 5 μΐ 10X Taq I buffer (Promega), 15 mM MgCl_?, 19.5 μΐ distilled water and 0.5 μΐ 5 U / μΙ Taq I (Promega) polymerase. Additionally, reactions containing pLJ4 were set up as a positive control and without DNA as a negative control.

Table 3 Library and DNA sources

<td>Library / DNA</td><td>Source</td>

<td>human liver cDNA, NIH</td><td>R. Bertolloti (NIH, Bethesda, Md)</td>

<td>human genomic # 946203</td><td>Stratagene</td>

<td>human genomic # 944201</td><td>Stratagene</td>

<td>human islet cell cDNA</td><td>Example 1C</td>

<td>Agtl 1 HEPG2</td><td>Prepared as described by Hagen im, (US Patent 4,784,950, incorporated herein by reference in its entirety)</td>

<td>human cDNA first strand of the brain</td><td>Clontech</td>

<td>human cDNA first strand placenta</td><td>Clontech</td>

<td>human cDNA first strand liver</td><td>Clontech</td>

178 685

Table 4 Volumes

<td>Library / DNA</td><td>Volume (diluted with water up to 15 μΐ)</td>

<td>human liver cDNA, NIH</td><td>1 μΐ from a 400 ng / μΐ solution</td>

<td>human genomic # 946203</td><td>15 μΐ phage solution</td>

<td>human genomic # 944201</td><td>15 μΐ phage solution</td>

<td>human islet cell cDNA</td><td>1 μΐ of solution 1 2 μg / μl dil. 1 3</td>

<td>Agtl 1 HEPG2</td><td>15 μΐ phage solution</td>

<td>human cDNA first strand of the brain</td><td>1 μΐ of a 10 ng / μΐ solution</td>

<td>human cDNA first strand placenta</td><td>1 μΐ of a 10 ng / μΐ solution</td>

<td>human cDNA first strand liver</td><td>1 μΐ of a 10 ng / μΐ solution</td>

30 cycles of PCR reactions were performed (one minute at 94 ° C, one minute at 50 ° C, and half a minute at 72 ° C) followed by a 10 minute incubation at 72 ° C. The PCR product was then analyzed by agarose gel electrophoresis. Only the human liver library, derived from NIH, made it possible to obtain a band of 320-410 bp, similar to that observed with pLJ4 as a positive control.

Human liver libraries cloned into pcD2 plasmid (Chen and Okayama, Mol Cell Biol 7: 2745-2752, 1987) obtained from Dr. Roger Bertolloti (National Institutes of Health, Bethesda, Md) were used to obtain a full length cDNA clone encoding human glucagon receptor. A library was seeded to obtain one million individual clones. Colonies transformed from each of the plates were scraped into 10 ml LB-Amp (Sambrook et al., Ibid). Cells were washed by centrifugation and the medium was removed. The cell pellet was suspended in 4 ml of LB-Amp, 15% glycerol and divided into four one-ml aliquots which were stored at -80 ° C. One of the glycerol samples was titrated and plated as 100 pools of 5,000 colonies each. After colony growth, each plate was scraped into 10 ml of LB-Amp. Aliquots of cells from each of the pools were taken to make plasmid DNA. The remaining cell mixtures were brought to a final volume with 15% glycerol, aliquoted and frozen at -80 ° C. Plasmid DNA was prepared from each cell pool, and DNA was digested with RNase (Boeringer Mannheim, Indianapolis, Ind.) According to the manufacturer's instructions. The RNase reactions were terminated by extraction with phenol / chloroform / isoamyl alcohol (24: 24: 1) and the DNA was then precipitated with ethanol.

Aliquots of the dissolved plasmid DNA from each pool were pooled into groups of 10 (i.e. 1-10, 11-20, 21-30, 31-40, etc.). Plasmid DNA was diluted 1-20 and 1 µl of DNA from each of the pools was used to make a PCR reaction mix identical to the mix described above. The reaction mixture was amplified under the conditions described above. Analysis of the PCR product by agarose gel electrophoresis indicated that pools 31-40 contained a band size 320-410 identical in size to the positive control.

Plasmid DNA prepared from the original pools 31 to 40 was diluted 1:20 and 1 µΐ of each pool was used to make a reaction mixture identical to that described above. The PCR amplification of the reaction mixtures was performed under the conditions described above. Analysis of the PCR product by agarose gel electrophoresis showed that pool # 40 gave a band of approx. 310-420 bp.

The plasmid DNA concentration was estimated by electrophoresis of a 1 µl dilution of a 10% pool # 40 on an agarose gel at 70 ng / µΐ. Seventy nanograms of pool # 40 plasmid DNA were electroporated into © coli cells of strain DH1 OB at 2.3 kV, 400Ω. 25 pF and resuspended in 1 ml SOC (Sambrook et al., Ibid). Three 10 'dilutions of cells were prepared²,10'³ and 10 '⁴. One hundred microliters of each dilution is in

178 685 for four plates. The colony count was approximately 10,000 colonies per plate for four plates containing cells from a 10 'dilution² and approximately 1000 colonies per plate for four plates containing cells from a 10 'dilution³. Duplicate filter prints were prepared from each of the four 10 'plates³ and one 10 '², marked as pools # 1 to # 5. One of the filters from each replicate was placed on the solid medium and colonies were allowed to form. Colonies were scraped off and used to make plasmid DNA for PCR amplification. Additionally, the three remaining 10 'tiles were scraped off² and plasmid DNA was prepared from them.

The remaining filters were pre-rinsed with 3XSSC with 0.5% SDS at 65 ° C with shaking for 12 hours to remove bacterial sediment. Filters were prehybridized in Ullrich buffer (Ullnch, EMBOJ 3: 361-364, 1984) with 50% formamide, 1% SDS overnight at 37 ° C. G30 clone DNA, labeled with the Amersham MEGAPRIME kit (Amersham) according to the manufacturer's instructions, boiled and added to the hybridization solution (Ullrich buffer + 5-% formamide) at a final concentration of 6X10⁵ cpm / ml. Filters were incubated overnight at 37 ° C with shaking. After overnight incubation, the probe solution was removed and the filters were washed for five minutes at 65 ° C in 2XSSC + 0.1% SDS. Following the first wash, the filters were washed in the same solution for fifteen minutes at 65 ° C and then for five minutes at room temperature with shaking. The last bath was repeated twice more. Filters were exposed to the film overnight at room temperature. A single colony on plate # 2, which corresponded to pool # 2, was consumed after hybridization with G30. This colony was picked and dispersed to obtain individual colonies. Plasmid DNA was prepared from several single colonies and then subjected to DNA sequence analysis. One of the clones, 40-2-2, was further sequence analyzed and deposited with the American Type Culture Collection (12301 Parklawn Dr., Rockville, MD 20852) as transformant E coli under number 69055 on August 21, 1992. The partial DNA sequence of clone 40-2-2 and the predicted amino acid sequence is shown as SEQ ID NO. No: 17 and 18.

To confirm the presence of glucagon receptor sequences on the hybridization filters, PCR amplification was performed using each of the DNA preparations (plasmid DNA made from each of the replicate filters and plasmid DNA made from each of the three 10 'plates).²) The pooled plasmid DNA was diluted 1:20 with water, and 1 μΐ of each DNA was used as a template to set up the PCR reaction and performed as described above. Agarose gel electrophoresis of the PCR product showed that pool # 2 and three of the four 10,000 pots contained the PCR band between 310 and 420 bp. The presence of the PCR-generated band in pool # 2, which corresponded to plate # 2, confirmed the presence of glucagon receptor DNA sequences.

F. Strategy for the cloning of the 5 'human glucagon receptor sequence

Analysis of a portion of the clone 40-2-2 cDNA sequence and the full-length rat glucagon receptor cDNA sequence revealed that clone 40-2-2 was missing the N-terminal sequence of approximately 25 amino acids. The 5 'end cDNA sequence of the human glucagon receptor was obtained using an adaptation of the method described by Frohman et al {Proc Natl AcadSci USA 85: 8998-9002, 1988). Briefly, an oligonucleotide primer was designed with a sequence hybridizing to the sequence near the 5 'end of the coding sequence of clone 40-2-2. The primer was hybridized to a G-tagged template from the first strand of human liver cDNA and the primer was extended 5' with Taq I polymerase. A second poly d (C) primer was "glued" to the G-labeled cDNA template to allow for polymerase chain amplification followed by cloning, sequencing and assembly with the coding region present in clone 40-2-2.

Three cDNA templates were prepared. The first matrix was the commercially available first strand human liver cDNA. Second and third cDNA templates were prepared by synthesizing the first strand cDNA from commercially available human liver mRNA (Clontech) using an ohgonucleotide containing a sequence specific for the human glucagon receptor or using the traditional oligo d (T) primer

178 685

A second cDNA template was prepared by synthesis from human liver mRNA using oligonucleotide ZC5433 (SEQ ID NO: 13), which is specific for the human glucagon receptor coding sequence. Reaction mixture containing 2 μΐ 1 pg / pl human liver mRNA, 8 μ1 20 pmol / µM ZC5433 (SEQ ID NO: 13) and 0.5 µM 10 mM Tris, pH7.4, 0.1 mM EDTA were incubated for 7 minutes at 68 ° C and then two minutes on ice. After incubation, 4 µM of 5X SUPERSCRIPT buffer (GIBCOBRL), 1 µM was added to the reaction mixture. 0.25 miCi / μΙ cc³²P-dCTP and 5 µΐ SUPERSCRIPT reverse transcriptase. The reaction was incubated for one hour at 45 ° C. The reaction was stopped by the addition of 80 µΐ TE. RNA was hydrolyzed by the addition of 1 µΐ 0.5% EDTA and 1 µΐ KOH. The hydrolysis reaction was incubated at 65 ° C for 5 minutes. After incubation, the sample was diluted to 1 ml with 50 mM KOH, 0.1 mMEDTA and passed through a CENTRICON 100 concentrator (Amicon, Danvers, Mass.). The column was washed with 1 ml of 50 mM KOH, 0.1 mM EDTA. The concentrated cDNA was harvested and neutralized with half the volume of 100 mM HCl. The neutralized cDNA sample was ethanol precipitated and then dissolved in 26 µL of distilled water.

A third cDNA template was prepared by synthesis from human liver mRNA using the purchased oligo d (T) primer. A reaction mixture was prepared containing 2 µg lpg / µm human liver mRNA, 1 µl 1 µg / µl oligo d (T) 18 (NewEngland Biolabs, Beverly, Mass) and 5 µl 10 mM TRIS, pH 7.4, 0.1 mM EDTA. The cDNA synthesis was performed under the conditions specified for the synthesis described above.

First strand cDNA was labeled with G. Set up tubes containing 4 µΐ first strand human liver cDNA (QUICK CLONE; Clontech, Palo Alto, Califf), 4 µΐ first strand cDNA from ZC5433 primer (SEQ ID NO. No .: 13), or 4 μΐ of the first strand of cDNA from oligo d (T) primer 22 μ 22 of water, 8 μΐ of 5X buffer (Promega), 4 μ of 10 mM dGTP and 2 μΐ 15 U / μΙ of terminal transferase were added to each reaction mixture, then the reaction was incubated for 30 minutes at 37 ° C and then for 10 minutes at 65 ° C. The reactions were diluted to 90 µΐ with 10 mM Tris, pH 7.4, 1 mM EDTA and ethanol precipitated.

Second strand synthesis of all labeled G cDNAs was performed identically. Each cDNA was first dissolved in 50 µ 50 of distilled water. To each of the cDNAs was added 100 µmol of ZC4814 (SEQ ID NO: 20) in 5 µL. The cDNA was glued to the primer by heating the mixtures to 68 ° C for five minutes followed by a two-minute incubation on ice. After gluing the cDNA with the primer, 20 μΐ of polymerase I X5 buffer (example 1), 1 μΐ 100 mM dithiothreitol, 2 μΐ of a solution containing 10 mM of each dNTP, 2 μΐ 0.5 miCi / μΙa were added to each of the mixtures.³²P-dCTP, 1 μΐ 3 U / μΙ E. coli DNA Ligase (New England Biolabs), 5 μΐ 7 U / μΙ E. coli DNA Polymerase I (Amersnam). The reactions were incubated for five minutes at 22 ° C and then 1.5 μΐ 2U / pl RNase H (GIBCO-BRL) were added and further incubated at 16 ° C for two hours The reaction was quenched by the addition of 200 μΐ 10 mM Tris (pH 8 0), 1 mM EDTA followed by 150 μΐ water-saturated phenol and 150 μΐ chloroform. The mixture was stirred vigorously and centrifuged for three minutes at 22 ° C to phase separation. The aqueous phase was taken and extracted again with phenol and chloroform as described above. After the second extraction with phenol-chloroform mixture, the aqueous layer was extracted with chloroform. The aqueous phase cDNA was precipitated by the addition of 5 µg muscle glycogen, 100 µM 8 M ammonium acetate and 300 µM isopropanol to selectively precipitate large nucleic acid particles and leave unincorporated oligonucleotide primers in the supernatant. The cDNA was centrifuged and the pellet was washed with 70% ethanol and then air dried. The pellet was dissolved in 15 μΐ of redistilled water.

Double-stranded cDNA was amplified in five parallel reactions using oligonucleotide primers homologous to the 5 'end of ZC4814 (Seq. No .: 20) and containing restriction sites to improve cloning, and an oligonucleotide primer specific for the 5 'extreme end of the coding sequence present in clone 40-2-2. Each of the reaction mixtures contained 5 μΐ 10X Taq buffer 1.3 μΐ 25 mM MgC12, 5 μΐ of a solution containing 2.5 mM of each of dNTPs, 1 μΐ of double-stranded cDNA, and 0.5 μΐ of Taq I polymerase (Promega). Distilled water was added to a final volume of 50 µL. The reaction was denatured for five

178 685 minutes at 98 ° C before the addition of 1 μΐ each of 10 pmol / μΐ ZC5624 (SEQ ID NO: 21) and 20 pmol / μΐ ZC4812 (SEQ ID NO: 19) Each sample was coated with 70 μΐ of mineral oil kept at 90 ° C Reactions were amplified with thirty cycles (95 ° C for 60 seconds, 57 ° C for 40 seconds and 72 ° C for 60 seconds) followed by incubation for 7 minutes at 72 ° C.

Each of the PCR products was electrophoresed on an agarose gel, and the amplified fragment was cut and cloned into pCRIOOO using the TA clonig kit (Invitrogen). Three clones from each ligation were selected and tested for insert using ZC5624 oligonucleotide primers (Sequence ID No.: 21) and ZC4812 (SEQ ID NO: 19) in independent PCR reaction mixtures each containing a sample from a clone as the source of the template DNA. PCR reactions were performed as described above, except that only 30 reaction cycles were performed. Single clones containing an insert from the original PCR reactions were subjected to DNA sequence analysis. Of the two clones that were shown to contain an error free sequence encoding the 5 'end of the human glucagon receptor, one was selected to provide the sequence encoding the 5' end of the human glucagon receptor. Clone 9A was digested with EcoRI and KpnI to obtain a 551 bp fragment containing the coding sequence for the 5 'end of the human glucagon receptor. Sequence encoding the 3' end of the glucagon receptor was obtained as a 561 bp KpnI-BamHI fragment from clone 40-2-2. The EcoRI-KpnI fragment and the 561 bp KpnI-BamHI fragment were ligated in the presence of EcoRI and BamHI to prevent concatamerization. The ligation product, a 1112 bp fragment, was gel purified and named 9A1. For convenience, fragment 9A1 was ligated into the EcoRI-BamHI digested plasmid pUC18. The ligation mixture was transformed into E. coli strain DH10b cells, and selected clones were tested for the presence of the insert. One of the clones had an insert

The glucagon receptor coding sequence was introduced into a mammalian expression vector using plasmid p9A11 and clone 40-2-2 to obtain the complete glucagon receptor coding sequence. The plasmid p9Al1 was digested with pVuI and BamHI to isolate a 967 bp fragment. Clone 40-2-2 was digested with BamHI and SacI to isolate the 828 bp fragment containing the 3 'end of the human glucagon receptor coding sequence. The mammalian expression vector pHZl was linearized by digestion with EcoRI and the ends were filled with T4 DNA polymerase. The linearized "blunt ended" vector was digested with SacI. Pvull-BamHI fragments, 967 bp in size, and BamHI-Sacl, 828 bp in size, were ligated into the SacI digested pHZ 1 vector.

The pHZ1 plasmid is an expression vector that can be used to express the protein in mammalian cells or a frog oocyte-based translation system with in vitro transcribed mRNA. The pHZ1 expression unit consists of the murine metallothionein-1 promoter, the T7 bacteriophage promoter, flanked by multiple cloning sites containing unique restriction sites for insertion of coding sequences, a human growth hormone terminator, and a T7 bacteriophage terminator. In addition, pHZ1 contains an E coli origin of replication, a bacterial beta lactamase gene, a selectable marker expression unit containing the SV40 promoter and origin, a neomycin resistance gene, and the SV40 transcription terminator.

The plasmid pHZ1 containing the glucagon receptor cDNA sequence in the correct orientation to the promoter is designated pLJ6 '. The insert and its links to the vector have been sequenced to confirm the presence of the correct sequence. The plasmid pLJ6 'was deposited with the American Type Culture Collection (12301 Parklawn Cr, Rockville, MD 20852) under number 69183 on January 15, 1993. The DNA sequence and the predicted amino acid sequence of the glucagon receptor cDNA present in pLJ6 'are shown in SEQ ID NOS, respectively. No. 24 and 25.

Example 6

Cloning of Glucagon receptor cDNA from human islet cells

In addition to cloning the glucagon receptor from human liver cells, glucagon receptor cDNA was obtained from a human islet cell library. A portion of the lu cDNA library

178 685 islet cells (Example 1C) were subjected to PCR amplification using oligonucleotide ZC5763 (SEQ ID NO: 22) which is an antisense nucleotide containing an XhoI site flanked by sequences from the 3 'untranslated end of the human glucagon receptor cDNA. The presence of restriction sites in the oHgonucleotide primers aids the targeted cloning of the resulting PCR product into an appropriate plasmid vector. A PCR reaction mixture was set containing 4 μ islet cell cDNA library (Example 1C), 8 μΐΐ10 Promega PCR buffer (Promega), 20 pmoles ZC5763 (SEQ ID NO: 22), 20 pmoli ZC5849 (SEQ ID NO: 23), 1 μΐ of a solution containing 20 mM each of the deoxyribonucleotides and 46.5 μΐ of water. The mixture was heated to 95 ° C for three minutes, then the temperature was reduced to 80 ° C for three minutes. The mixture was kept at 80 ° C until use. To start the reaction, 20 µ of enzyme mixture containing 2 µΐ of 10Χ Promega PCR buffer (Promega), 2 µl of 5U / µl Taq I polymerase (Cetus) and 16 µΐ of water were added. The reaction was covered with 50 μΐ of mineral oil (Sigma) and subjected to thirty cycles (95 ° C for a minute, 55 ° C for a minute, and 72 ° C for two minutes and fifteen seconds, with incubation at 72 ° C extended by three seconds each cycle) followed by incubation for ten minutes at 72 ° C. After the final incubation at 72 ° C, the reaction was held at 4 ° C. Agarose gel electrophoresis analysis of 10 µL aliquots of the PCR product showed the presence of a fragment of approximately 18 to 1.9 kb. Based on human glucagon receptor cDNA present in pLJ6 '; 1846 bp fragment was expected.

The PCR reaction was subjected to chloroform extraction followed by two phenol: chloroform extractions followed by a final chloroform extraction. After the final extraction, 5 µL of 4 µg / µl glycogen support (Boehringer Mannheim Corporation) was added and the mixture was precipitated in the presence of ammonium acetate and ethanol. The DNA was collected by centrifugation and the pellet was washed with 70% ethanol. The DNA pellet was dissolved in water and digested with Xhol and EcoRI. DNA was gel purified and cloned into the pBLUESCRIPT SK plasmid⁺ (Stratagene Cloning Systems) linearized by Xhol and EcoRI. The ligation mixture was transformed into E. coli cells of strain DH10B (GIBCO-BRL). Plasmid DNA was prepared from selected transformants, and subjected to restriction digestion and Southern blot analysis. Clones were compared to the human glucagon receptor cDNA insert present in pLJ6 '. Based on the diagnostic restriction enzyme digestion, selected clones were subjected to sequence analysis. It showed that one of the pSLIGR-1 clones contained the glucagon receptor coding sequence. The pSLIGR-1 coding region contained three nucleotide changes compared to the liver cDNA present in pLJ6 '. One of the changes was a silent mutation, the other two caused amino acid changes as shown in Table 5. Analysis of the changes indicates that they may be the result of polymorphs representing allelic variants.

Table 5

<td>Amino acid number</td><td>Amino acid according to the Liver cDNA sequence</td><td>Amino acid according to the cDNA sequence of Islet Cells</td>

<td> 184 265</td><td>Phe Cheese</td><td>Leu Gly</td>

Example 7

Cloning of the human glucagon receptor gene

In order to obtain a genomic clone of the human glucagon receptor, two libraries were screened using the human glucagon receptor cDNA as a probe. The amplified human lung genomic library of Caucasian male lambdaFIX II and the amplified human lung genomic library lambdaFIX (both from Stratagene Cloning Systems catalog numbers 946203 and 944201) were searched for the human glucagon receptor gene.

178 685

The amplified human lung genomic library was titrated and plated at approximately 4X10⁴ colony forming units (pfu) with E. coli strain LE392 cells (Stratagene Clomng Systems) on each of thirty 150 mm diameter plates. An additional ten plates of 6X10 were sown⁴ E. coli strain LE392 cells. Plates were incubated overnight at 37 ° C. Thirty plates were selected for viewing.

Filters were made in duplicate for each of the thirty plates. Each filter was made by covering the plate with HYBOND nylon membrane (Amersham) according to the manufacturer's instructions. The filters were removed from the plate and cells were lysed in 1.5M NaCl and 0.5M NaOH for five minutes at room temperature. Filters were neutralized for five minutes in 1M Tris-HCl (pH 7 5), 1.5 M NaCl and fixed with 1200 µL UV energy in a STRATALINKER (Stratagene Cloning Systems). After fixation, the filters were washed three times in 0.25XSSC, 0.25% SDS, 1 mMEDTA at 65 ° C. After washing, the filters were split into six lots of 10 filters and prehybridized in a prehybridization solution (5XSSC, 5X Denhardt's solution, 0.2% SDS, 1mM EDTA) filtered through a 0.45 µm filter followed by the addition of 100 µg / ml denatured immediately prior to use. hot salmon sperm DNA Filters were prehybridized overnight at 65 ° C.

Human glucagon receptor cDNA from clone p40-2-2 was randomly primed with the MEGAPRIME kit (Amersham) using the manufacturer's protocol. Prehybridization solution from each batch of filters was removed and replaced with fresh prehybridization solution supplemented with 28 5Χ10⁶ cpm of the probe. Filters were hybridized at 65 ° C for twenty hours. After hybridization, the hybridization solution was removed, the filters were washed four or five times with a washing solution containing 0.25XSSC, 0.2% SDS, 1mM EDTA at room temperature. After rinsing, the filters were washed in eight consecutive baths at 65 ° C, followed by a final rinse at 70 ° C. After a 70 ° C bath, the films were autoradiographed on film (XAR-5, Eastman Kodak Co., Rochester, NY) for four days at -70 ° C using an intensifying screen.

Examination of the autoradiographs revealed the presence of four hybridization regions with the radiolabeled probe. An agar fragment from each of the four regions was taken for purification. Each agar fragment was soaked overnight in 1 ml of SM (Maniatis et al., Molecular Cloning · A Laboratory Manual, Cold Spring Harbor, NY, 1982; incorporated herein by reference), 1% chloroform. After overnight incubation, phage from each agar fragment were diluted with 1 · 1000 SM. Aliquots of 5.25 and 50 µL were plated with E. coli strain LE392 cells. Plates were incubated and single blots were prepared from the plates plated with 5 and 25 µL. Filters were made, prehybridized and hybridized as described above. Filters were exposed to an autoradiographic film.

Examination of the autoradiographs revealed positively tagged regions from each of the four clones. Ten agar fragments were taken from positively labeled regions representing at least two positive regions for each of the original clones. Agar fragments were treated as described above. Phage from each agar fragment was diluted 1: 10000 in SM. Aliquots of 2.5 μΐ and 10 μΐ were seeded with E. coli strain LE392 cells. The plates were incubated and single prints were prepared from each of the plates with suitably isolated colonies. Filters were prepared and prehybridized as described above. The autoradiograms from the filters showed the presence of exposed areas corresponding to individual colonies. Twelve positive colonies representing at least one clone from each of the four original positives were picked. One colony from each plate was further analyzed.

Agar fragments from phage clones 2-2-1,3-1-1 and 14-2-1 and 11-2-1 tested as described above. The phage were diluted 1: 100 in SM and plated on E. coli strain LE392 cells. Double-stranded DNA was prepared as described in Grossberger (Nuc Acids Res 15: 6737, 1987; incorporated herein by reference). Double-stranded DNA was digested with Xbal to release the genomic insert. Agarose gel electrophoresis showed that clones 2-2-1 and 11-2-1 contained an approximately 1.9 kb Xbal insert, clone 14-2-1 contained a 15 kb insert, and clone 3 -3-1 in

178 685 ran in the 13 kb insert. Southern blot analysis of the XbaI and Xbal-BamHI digested clones showed that the human glucagon receptor cDNA is shown in Figure 6.

Table a 6

<td>Number New Maple</td><td>Original Clone number</td><td>Hybridization with the Xbal fragment</td><td>Hybridization with the Xbal-BamHI fragment</td>

<td>clone 1</td><td> 2-2-1</td><td>~ 9kb</td><td>4 2 kb, 1 9 kb</td>

<td>clone 6</td><td> 11-2-1</td><td>~ 9kb</td><td>4 2 kb, 1 9 kb</td>

<td>clone 2</td><td> 14-2-1</td><td>~ 15 kb</td><td>4 2 kb, 1 9 kb</td>

<td>maple 5</td><td> 3-3-1</td><td>~ 13 kb</td><td>1 6 kb</td>

Clones 11-2-1 and 14-2-1 were further analyzed. Clone 2-2-1 appeared to be identical to clone 11-2-1 hence its further analysis was abandoned. For convenience, the names of the clones were changed as described in Table 6. Double-stranded DNA was prepared from each of the phage colonies for cloning into a plasmid vector according to the method described by Maniatis. The DNA was digested with XbaI, gel purified, and cloned into pBLUESCRIPT SK plasmid (Stratagene Clomng Systems) linearized with Xbal digestion and treated with calf alkaline phosphatase to prevent recirculation. The ligation mixtures were electroporated into DH1 OB ELECTROMAX cells (GIBCO-BRL) in a GENEPULSER machine (Bio-rad Laboratories; Richmond; CA) at 400 ohms, 25 mifarads and 2.3 kV. Plasmid DNA was prepared from selected transformants. Clones containing the genomic insert from clones 6 and 2 were named pSLHGR6 and pSHLGR2. The clones of pSLHGR6 and pSHLGR2 were sequenced. Sequence analysis and comparison with the coding region of the human glucagon receptor revealed the presence of 12 exons spanning the coding region. Chromosome analysis, performed on the smears of metaphasic chromosomes described by Dumam et al (Mol Cell Biol 8: 1863-1867, 1988; provided herein as reference) using a biotinylated glucagon receptor gene probe and a probe specific for the centromeric chromosome 17 region. Chromosome denaturation, hybridization and single color detection were performed as described by Pinkel et al. (Proc Natl Acad SCI, 83: 2934-2938, 1986, incorporated herein by reference) and modifications by Kievits et al. (Cell Cytogenet. Genet. 53: 134-136, 1990, incorporated herein by reference), except that hybridization was performed in a 65% (v / v) mixture of formamide / dextran sulfate / 2XSSC and washing after hybridization was 65% mixture of formamide / 2XSSC at 42 ° C followed by a rinse in 0.1XSSC at 55C as described by Palmiter et al. (Proc Natl Acad Sci USA, 89: 6333-6337,1992, incorporated herein by reference). The translocation in the q position was confirmed²⁵ by DAPI staining of some of the metaphasic smears as described by TestA et al. (Cytogenet Cell Genet 60: 247-249, 1992; incorporated herein by reference). DAPI staining resulted in a pattern resembling Q-fringes.

The amplified human placenta library (Stratagene) was screened unsuccessfully for the human glucagon receptor gene using the method described above. The library was titrated and plated at approximately 5X10⁴ pfu with E. coli strain LE392 cells (Stratagene Cloning Systems) on each of thirty 150 mm diameter plates. An additional eleven plates of 105 pfu and E. coli strain LE392 cells were seeded. Plates were incubated overnight at 37 ° C. Thirty-eight plates were selected for viewing.

The nylon filters were blotted, rinsed and prehybridized as described above. The glucagon receptor cDNA fragment of human G30 islet cells (Example 4) was randomized with primers of the MEGAPRIME kit (Amersham) using the manufacturer's protocol. The prehybridization solution from each batch of filters was removed and replaced with fresh prehybridization solution supplemented with 1 1Χ10⁶ cpm probe Filters were hybridized at 65 ° C overnight. After hybridization, the hybridization solution was removed, the filters were washed four or five times with a washing solution containing 0.25XSSC, 0 2% SDS,

178 685 mM EDTA at room temperature After rinsing, the filters were washed in eight consecutive baths at 65 ° C followed by a final rinse at 70 ° C. After a bath at 70 ° C, the films were autoradiographed on film (XAR-5, Eastman Kodak Co.) for four days at -70 ° C using the intensifier screen.

Examination of the autoradiographs showed no convincing positive signals; however, seven areas of poor marking were selected for further analysis. Clones were re-screened as described above, but the re-performed autoradiographs showed no labeling at all. So these clones were not searched for.

Example 8

Expression of glucagon receptor cDNA in mammalian cells

A. Expression of Rat Glucagon Receptor in BHK570 Cells

Plasmid pLJ4 was co-transfected with plasmid pLJ 1 into BHK570 cells (deposited with the American Type Cell Collection under number 10314) using the calcium phosphate method described by Graham and Van der Eb (Virology 52: 456, 1973, incorporated herein by reference). P lazmid obtained from PLJ P416 plasmid containing the Adenovirus 5 ori, SV40 enhancer, adenovirus major late promoter 2 triple leader of adenovirus 5, splice site 3 'and 5' cDNA DHFR^r, SV40 polyadenylation signal and pML-1 vector sequences (Lusky and Botchan, Nature 293: 79-81, 1981) The EcoRI-Xbal DNFR expression unit from plasmid p416 was ligated into pUC 18 linearized by digestion with EcoRI and Xbal to generate pL J1. Transfected cells were grown in medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 1XPSN antibiotic mix (GIBCO-BRL 600-5640), and 20mM L-Glutamine). After a few days in non-selective medium, the medium was replaced with the selection medium (medium containing 250 mM methotrexate (ΜΤΧ)). Cells were split and diluted 1:20 and 1:50 in selection medium in 10 cm plates. After 7-10 days of selection at 250 mM, colonies were picked using cloning cylinders in 24-well plates. The resulting clones were tested for glucagon binding as described above (Example 3). Binding of glucagon was also performed with all cells by seeding 2X10⁵ cells of each clone into the well of a 24-well plate. The plates were incubated for 72 hours at 37 ° C and 5% CO₂. Glucagon binding was performed as described in Example 3, except that after the last wash in PB S, cells were removed from the well by trypsinization into tubes. Tubes were counted in a gamma counter. The cells with the highest counts, and hence capable of binding the highest amount of glucagon, were selected for further study. Selected transformants were also tested for glucagon-dependent cAMP response as described in Example 8D and for glucagon-dependent intracellular calcium response as described in Example 8E. The glucagon binding assay was also performed on cell membrane preparations from the transfectants described above.

B. Glucagon binding by membrane preparation

Membranes of pLJ4 transfected BHK cells were compared with rat liver membrane preparations for their binding capacity ¹²⁵I-glucagon membranes were prepared as described by Rodbell et al. (J Biol Chem 246: 1861-1871, 1971, incorporated herein by reference). Two batches of liver membranes were prepared from approximately 80 g of livers from 12 to 16 decapitated young female rats. Livers were quickly removed and placed in an ice-cooled vessel. Livers were divided into 10 gram portions. The portions were crushed with scissors and connective tissue was removed. The aliquots were individually homogenized in a Dounce homogenizer. 25 ml of Mediumk (Table 2) was added to each batch crushed in the homogenizer, and each batch was homogenized on ice with eight strong strokes of the homogenizer pestle. After homogenization, the homogenates were collected in 450 ml of cold Medium (Table 2). The pooled homogenates were mixed for three minutes and filtered through two layers of cloth followed by four layers of cloth. The homogenates were centrifuged for 30 minutes at 1500Xg at 4 ° C. Supernatants were removed and the pellets were pooled in a clean Dounce homogenizer. The pellets were suspended with three gentle movements of the pestle. The suspended pellets were precipitated in a measuring cup with a capacity of

178 685

250 ml containing 62 ml of 69% Sugar Solution (Table 2). Distilled water was added to a final volume of 110 ml, stirred vigorously and kept cool. The concentration of the solution was adjusted to 44% +/- 0.1% using 69% sucrose or water as measured by a refractometer (Bausch &amp; Lomb, Rochester, NY). The sucrose suspension was divided equally into 25X89mm ultracentrifuge tubes. Each slurry was carefully covered with 20 ml of 42.3% Sugar Solution (Table 2), and the slurries were centrifuged for 150 minutes at 24,000 rpm in a SW28 rotor (Sorvall, DuPont Company, Wilmington, Del.) At 4 ° C.

After centrifugation, floating material from each tube was removed by aspirating into a 10 ml syringe through an 18G needle. The material from each tube was pooled and suspended in approx. 10 ml of Medium (Table 2) by drawing and ejecting the mixture through the needle into the centrifuge tube. The tube was filled with Medium (Table 2) and centrifuged for 15 minutes at 15,000 rpm in an SS-34 rotor (Sorvall). The supernatant was carefully collected and discarded. The pellet was suspended in Medium (Table 2) and diluted 1: 1000 with distilled water. The absorbance was read in 1 cm cuvettes to determine the protein content. Protein concentration was determined using the formula:

A x A ^ 224nm ^Λ^ 236ηπ r + · - r ii / ₇

------------- x fold - dilutions - mg - proteins / ml 6 45 ⁶

The membrane preparation was aliquoted, frozen in a dry ice / methanol bath, and stored at -80 ° C.

Membranes were prepared from transfected BHK cells growing basins in a 150 mm plate in selection medium. Two plates of transfectants growing from the basin were rinsed twice with cold phosphate buffered saline (PBS; Sigma Chemical Co., St. Louis, Mo.) and 10 ml of PBS containing 1 mM PMSF was added to each plate. Cells from each plate were scraped into PBS solution, and transferred to fresh tubes. Each plate was washed with 5 ml of PBS containing 1 mM PMSF, and the rinsed fluid was added to the appropriate cells. The cells were centrifuged at 2000 rpm in a table top centrifuge at 4 ° C. Supernatants were removed and cells were resuspended in 30 ml of 5 mM Hepes, 1 mM PMSF, pH 7.5. Cells were incubated on ice for 15 minutes and then centrifuged at 47800Xg at 4 ° C. Each pellet was suspended in PBS solution containing 1 M PMSF, aliquoted and frozen at -80 ° C.

A competition study using a glucagon binding assay was performed on rat liver membrane preparations and transformed BHK cells. Briefly, the tubes containing 20 μΐ glucagon from 10 '¹¹ M to ΙΟ '⁶ M diluted in 10 mM HO Ac or 20 μΐ BSA. 100 µ prob of Binding Buffer, 20 µΐ was added to each tube¹²⁵I-glucagon (Amersham); 20 μ / 1 mM NaHCO₃; 20 μΐ 10 mMHOAc, 0.5% BSA (Novo Nordisk N / A, Bagsvaerd, Demnark) and 40 μΐ distilled water. The binding reaction was initiated by adding 20 µ of the membrane preparation to each of the tubes. The reaction was incubated for 30 minutes at 30 ° C. Membranes were collected by centrifugation in a microcentrifuge at high speed for 10 minutes at 4 ° C. Supernatants from each sample were aspirated, and pellet pulses were counted. Competition with unlabeled glucagon resulted in nearly identical sigmoidal curves for glucagon binding (Figure 3). According to Scatchard analysis (Scatchard, Ann. N. K Acad Sci 51: 660-672, 1949; incorporated herein by reference), the K d was 50 nM for the cloned receptor and 49 nM of rat liver membranes (figure 4).

The specificity of the receptor encoded by pLJ4 was investigated by its ability to compete for the binding of related peptide hormones to ¹²⁵I-glucagon. Micromolar amounts of glucagon and related peptides were added to the membrane preparations of pLJ4 transfected cells (Table 7), along with¹²⁵I-glucagon in the binding assay described above. Only native glucagon 1 antagonist, des-His ^ Glu amide⁹ ] -glucagon were able to compete with ¹²⁵I-glucagon for membrane binding.

178 685

Table 7

<td>1 μΜ</td><td>human glucagon (Sigma)</td>

<td>1 μΜ</td><td>1 9 des-His amide [Glu] glucagon (synthesized on 431 A peptide synthesizer A Applied Biosystems using RTNK AMIDĘ MBHA resin (Bachem Bioscience Inc, Philadelphia, Penn))</td>

<td>200 nM</td><td>human glucagon-like peptide (GLP) (Sigma)</td>

<td>20 nm</td><td>porcine intestinal vasoactive peptide (VIP) (Sigma)</td>

<td>1 μΜ</td><td>salmon calcitonin (Sigma)</td>

<td>1 μΜ</td><td>swine secretin (Sigma)</td>

<td>1 μΜ</td><td>bovine parathyroid hormone (PTH) (Sigma)</td>

C. Expression of Rat Glucagon Receptor in COS-7 Cells

The ability of pLJ4 transfected COS-7 cells to be stimulated by glucagon to increase cAMP levels was tested using Amersham SPA (Amersham) as described below (Example 8D). The test showed that glucagon-stimulated pLJ4 transfectants accumulate approximately five times more cAMP compared to with control COS-7 cells transfected with vector alone. Related peptides such as secretin, VIP, PTH, GLP and calcitonin were added to the transfectants at concentrations from 100 nM to 1000 nM and tested for their ability to induce elevated cAMP levels. The test results showed that none of the related peptides is capable of significantly elevating cAMP levels.

D. Whole Cell Luciferase and Adenyl Cyclase Studies, Rat glucagon receptor cDNA was expressed in the BHK570 cell line, stably transfected with ZK6, an expression unit containing a promoter containing at least one cAMP response element, luciferase DNA and an hGH terminator. This cell line measures luciferase activity, adenylate cyclase activity, and intracellular calcium levels in response to glucagon binding to its receptor.

The proenkephalin (CRE) cAMP response element in plasmid ZK6 was obtained from Zem233. Zem233 was obtained from the Zem67 and Zem106 plasmids. The Zem 106 plasmid was constructed from the Zem93 precursor. To construct Zem93, the KpnI-BamHI fragment containing the MT-1 promoter was isolated fromMThGHIII (Palmiter et al., Science 222: 809-814, 1983) and introduced into pUC18. approximately 600 bp of the 5 'sequence from the MT-1 promoter was eliminated.

CRE proenkephalin was introduced into the 5 'end of the SV40 Zem 109 promoter by first digesting Zemloo with EcoRI and Sstl to isolate the vector containing fragment. The oligonucleotides ZC982 and ZC983 (SEQ ID NOs: 3 and 4) were designed to encode, when paired, the CRE proenkephalin from nucleotide-71 to -133 (Comb. and m., Nature 323: 353-356, 1986) flanked at the 5 'end by an EcoRI site and at the 3' end by an SstI site. Oligonucleotides ZC982 and ZC983 (Sequence Identifiers Nos. of the Zem224 plasmid.

The Zam67 plasmid was obtained by digesting pIC19R (Marsh et al., Gene 32: 481-486, 1984) with Smal and HindIII digest. The SV40 ori region at position 270 (Pvull) was transferred by ligating at position 5171 (HindIII) into linearized pICI9R to form Zem67. The HindIII-BamHI fragment containing the neomycin resistance gene and the S V40 terminator from plasmid pSV2-neo (America Type Culture Collection No. 37149) was introduced into HindIII-BglII digested Zem67 to create Zem220.

An expression unit containing the SV40 promoter-neomycin resistance gene-SV40 terminator as an EcoRI fragment was isolated. The Zem224 plasmid was digested with EcoRI and treated with calf alkaline phosphatase to prevent recirculation. The neomycin expression unit was ligated and linearized with Zem224. The plasmid containing the SV40 proximal CRE promoter was designated Zem233

178 685

The Zem233 plasmid was modified by introducing an additional CRE sequence, a TATA box and a LacZ coding portion and poly (A) sequences immediately 3 'from the proenkephalin CRE sequence such that the resulting expression unit was in the reverse orientation to the neomycin resistance unit present in Zem233. The Zem233 plasmid was linearized by digestion with BamHI and SstL. Oligonucleotides ZC3509 and ZC3510 (SEQ ID NOS. Nos .: 5 and 6) was designed so that when paired, the resulting duplex encoded the alpha glycoprotein CRE (Delegeane et al, Cell Biol 7: 3994-4002,1987) with a 5'Sstl sticky end and a 3'EcoRI sticky end. Oligonucleotides were paired according to standard methods. The TATA box was obtained as an EcoRI-PstI fragment spanning nucleotides -79 to +18 of the thymidine kinase gene (McKnight, Cell 31: 355-366, 1982). The 3 'sequence of the LacZ gene and its associated poly (A) sequence was obtained as a PstI-BamHI fragment from plasmid pLacF (obtained from Jaques Peschon, Immunex Corp., Seattle, Wash,) containing the LacZ coding region and the mouse protamine terminator cloned into pUC 18 Zem233 was linearized with SstI-BamHI and the sequences were ligated: the SstI-EcoRI fragment of the ZC3509 / ZC3510 adapter, the EcoRI-PstI fragment containing the TATA box and the LacZ PstI-BamHI sequence. A plasmid containing an expression unit in the correct orientation with respect to the Zem233 neomycin resistance gene expression unit was designated KZ5.

The luciferase gene and human growth hormone (hGH) terminator sequences were used to alter the LacZ coding sequence and the poly (A) coding sequences present in KZ5. The luciferase gene was originally derived from plasmid alfa-1681uc (Delcgeano et al., Mol Cell Biol., 7: 3994-4002, 1987) and deWet et al., Mol Cell Biol, 7 725-737, 1987) as a fragment of Xhol-Xbal 1.7 kb in length. The hGH terminator was obtained as the Xbal-SalI fragment from Zem219b (deposited as an E coli transformant with the American Typc Culture Collection (Rockville, Md) under ATCC No. 68979). The luciferase gene and hGH terminator sequences were cloned for convenience into Xhol-Sall linearized pICI9H (Marsh et al., Ibid). The resulting plasmid KZ8 was digested with Xho I and SalI to isolate the luciferase-hGH terminator sequence. The KZ5 plasmid was digested with Sal to isolate the vector containing fragment and treated with calf alkaline phosphatase to prevent recirculation. The Xhol-Sall fragment containing the luciferase hGH terminator was ligated into the digested Sal KZ5. The plasmid containing the luciferase-hGH terminator in the correct orientation to the promoter was designated KZ6.

The KZ6 plasmid was transfected into BHK570 cells (deposited with the American Type Culture Collection under number 10314) using the calcium phosphate precipitation method described by Graham and Van der Eb (Eirology 52: 456, 1973, incorporated herein by reference). Transfected cells were cultured in medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 1XPSN antibiotic mixture (GIBCO-BRL) and 2.0mM L-glutamine). After a few days in non-aggregating medium, the medium was changed to G418 selection medium (medium containing 500 pg / ml G418). Cells were allowed to grow to confluence, then trypsinized and seeded in marginal dilutions into the wells of 96-well plates. Cells were allowed to grow for one to two weeks in G418 selection medium. Clones from single colony containing wells were tested for their ability to respond to forskolin in the luciferase assay described below. Forskolin raises cellular cAMP levels and activates cAMP-independent biological response pathways in a receptor-independent manner. The clone capable of responding to forskolin was designated BHK / KZ6-19-46.

BHK / KZ6-19-46 cells were co-transfected with pLJ4 and pLJ1 or pZCEP and pLJ1 (pZCEP transfectants were used as negative control) as described above using calcium phosphate transfection. Transfectants were selected in 250 nM methotrexate as previously described.

The transfectants were tested in triplicate for the induction of CREluciferase responses by selected agonists. Six randomly selected pLJ4 transfectants and random pZCEP transfectants (negative control) were tested. The microtiter test plates were set up with each well containing 2X10⁴ cells in 100 µ selection medium, i

178 685 cells were grown overnight. Agonist solutions were prepared in selection medium at X2 target concentration as specified:

μΜ glucagon

200 nM glucagon-like peptide (GLP) nM intestinal vasoactive peptide (VIP)

100 nM calcitonin (CT) μΜ forskolin (CalBiochem, San Diego, Califf.)

Induction was triggered by adding 100 µL of each of the X2 solutions to duplicate wells. Uninduced levels were determined in triplicate wells to which were added 100 µL of DMEM containing 10% fetal calf serum. The plates were incubated for four hours at 37 ° C, 5% CO₂to enable the production of luciferase.

Following induction, the medium was removed and the wells were washed once with 200 µΐ / well PBS. After washing, 25 µL of IX Cell Culture Lysis Reagent (Luciferase Assay System, Promega Corp., Madison, Wis.) Was added to each well and the plates were incubated for 15 minutes at room temperature. The plates were transferred to a Labsystem Luminoscan lunescence plate reader (Labsystems Inc., Morton Grove, lii.) Which added 40 μΐ of Luciferase Assay Substrate (Luciferase Assay System, Promega), mixed the reaction for three seconds, and read the luciferase signal from each well for two seconds. The fold induction of luciferase by each agonist was calculated as follows:

,,, evoked signal - no induced signal induction multiplicity = -------------------------------------- signal not triggered

One of the pLJ4 transfectant clones, KZ6 / rGR-DHFR-2, showed a 6-10 fold luciferase induction by glucagon but was not induced by GLP, VIP or CT.

The cAMP response of the KZ6 / rGR-DHFR-2 clone to glucagon and forskolin was also tested by cAMP radioimmunoassay [^12sI] Scintillation Proximity Assay System (Amersham) according to the manufacturer's instructions. Briefly, 100 μΐ 2X10⁵ KZ6 / rGR-DHFR-2 cells per ml were seeded into the wells of a multi-well culture plate and grown overnight in selection medium. Glucagon and forskolin were prepared in DMEM, 10% fetal calf serum, 10 µM in concentrations from 0.0001-1000 nM and 25 µM.

The medium was changed to 50 µ / well agonist (glucagon or forskolin). Cells were incubated with the agonist for 10 minutes at 37 ° C, 5% CO₂. After incubation, cells were lysed by adding 200 µL of boiling water to each well. After 15 minutes, supernatants were harvested and diluted with L5 or 1:40 in acetate buffer (cAMP [¹²⁵I] Scintillation Proximity Assay System (Amersham)). The samples were acetylated using triethylamine and acetic anhydride according to the protocol provided by the manufacturer.

100 µΐ of acetylated samples were combined with 75 µΐ ^l25I-cAMP, 75 μΐ anti-succinate-cAMP serum and 75 μΐ donkey anti-rabbit IgG conjugated to SPA beads (all solutions were provided in cAMP [¹²⁵I] Scintillation Proximity Assay System (Amersham)) in the LKB T tray well. The trays were closed and incubated overnight with continuous shaking on a shaker set at 200 rpm. The samples were counted in a 1205 BETAPLATE scintillation counter (Pharmacia LKB Instruments Inc., Gaithersburg, Md.). A standard curve was also determined using concentrations from 2 to 128 fmoles of acetylated cAMP. Total bound is also specified^l23I-cAMP and non-specific binding. KZ6 / rGR-DHFR-2 showed a 140-fold induction of cAMP level at saturation with glucagon (10-100 nM) and ED₅₀ equal to 0.25 nM.

E. Determination of Intracellular Calcium Concentration

The intracellular calcium responses of pLJ4 transfectants to glucagon were examined using the method described by Grynkiewicz et al (J Biot Chem 260: 3440-3450, 1985, incorporated herein by reference). PLJ4 transfectants were seeded into 5X10 two-well chambers (NUNC)⁴ cells per chamber. Cells were grown for one to three days under normal conditions

178 685 were cultured in selection medium containing methotrexate. The medium was aspirated and the chambers were washed twice with 1 ml of Imaging Buffer (Table 3). Cells were incubated for 30 minutes in the dark at room temperature with 0.5 ml of Fura-2 AM Solution (Table 3). After incubation, the Fura-2 AM Solution was removed and cells were washed with 1 ml Imaging Buffer three times. After the last wash, 0.5 ml of buffer was left in the chamber. Cells were kept in the dark at room temperature for 30 to 120 minutes.

Imaging was performed on a Nikon Diaphot inverted fluorescent microscope equipped with a mercury lamp and Nikon Fluor dry 10Χ and 40Χ lenses. Experiments were controlled and analyzed using a Sun SPARCII station and Inovision RATIOTOOL software (Research Triangle Park, NC). Variable excitation wavelengths were controlled by this software through an automated filter set containing filters with a spectra of 340 nm and 380 nm. Emission images were directed through a semi-transparent mirror (380 nm) to a Dage-MTI 72 CCD camera equipped with a Genesis II image intensifier and recorded digitally.

The intracellular calcium concentration was monitored by calculating the ratio of the emission intensities at each of the two wavelengths (340/380) for each of the digital field of view image elements (512X480 pixels). Grynkiewicz et al., (Ibid) showed that this ratio is related to the concentration of calcium viewed thanks to the fura-2 dye inside the cells that took up and de-esterified with the acetoxymethyl derivative (fura-2 AM) used for cell loading. The RATIOTOOL software shows this information as a pseudo-color image that can be calibrated to calcium concentration. Images were uploaded and computed every five seconds during each experiment.

Cells were monitored for at least 60 seconds (12 images) to establish a baseline condition prior to stimulation. Stimulation was performed by adding 0.5 ml of Imaging Buffer containing 200 nM glucagon to 0.5 ml of Imaging Buffer in the chamber, resulting in a final concentration of 100 nM. Cells were monitored and images were recorded for at least three minutes after stimulation.

As observed, the intensity ratio images corresponding to the number of cells in each field of view changed dramatically shortly after the addition of glucagon. This was measured by using the RATIOTOOL software to calculate the mean value for specific regions of the intensity ratio image corresponding to each of the corresponding cells. Cells with an average resting ratio of 1.4 rapidly increased to values ​​of 3 to 6. They remained at these high values ​​for 40 to 50 seconds and then gradually decreased to the baseline. Independent kahbration experiments indicated that this reflected changes in intracellular calcium concentration from resting values ​​of 150 nM to peaks of 400 nM.

F. Determination of Inositol Phosphate

BHK570 cells expressing the glucagon receptor from pLJ4 or control transfected were seeded into 24 wells of culture plates at a density of approximately 200,000 cells per well. After 24 hours, cells in each well were labeled by incubation in 0.5 ml of selection medium ΜΤΧ containing 2.0 mlCi of myo- (2-³H) Inositol (specific activity 20 Ci / mmol; Amersham). At the end of the 24 hour incubation, cells were washed with 1 ml of warmed DMEM (Dulbecco's modified Eagle's medium. JHR Biosciences, Lenexa, Kan.,) Buffered with 20 mM Hepes, pH 7.0 (Sigma Chemical Co.) containing 10 mM LiCl. The lavage medium was removed by aspiration and replaced with 900 µl of fresh buffered medium. Cells were incubated for five minutes at 37 ° C. After incubation, each agonist or antagonist was added to the wells in triplicate and incubated at the volumes and conditions specified in Table 8.

178 685

Table 8

<td>Ligand volume</td><td>Incubation period</td>

<td>PLJ4 transfectants 100 μΐ 1000 nM glucagon 100 μΐ 10 nM glucagon 100 μΐ 100 μM forskolm</td><td>10 minutes 10 minutes 10 mmut</td>

<td>Control transfectants 100 μΐ 1000 nM glucagon 100 μΐ 1000 nM isoproterenol</td><td>10 minutes 10 mmut</td>

<td>PLJ4 transectants 100 μΐ 50 nM glucagon 1 9 100 µM dcs-His [Glu] glucagon</td><td>10 minutes 10 minutes</td>

The reaction was terminated by placing the cells on ice. After aspiration of medium, cells were lysed by adding 1 ml of cold DMEM and 1 ml of ice-cold 10% perchloric acid. After ten minutes, cell lysates were transferred to ice tubes containing 500 µL of 10 mM EDTA, pH 7.0. Samples were neutralized by adding 900 µL 1.5 µ KOH in 60 mM Hepes buffer followed by dropwise addition of KOH-Hepes solution until the pFI was between 7 and 7.5. The neutralized samples were frozen at -20 ° C overnight. The frozen samples were melted and the precipitate was allowed to settle to the bottom of the samples. Supernatants were applied to AMPREP minicolumns (Amicon) where they were washed successively with 5 ml of methanol and 1 M KHCO₃and then washed with 15 ml of water. After loading the samples, the effluent was collected. The columns were washed 4 times with 1 ml water and a 1 ml sample was collected after each wash. Inositol phosphate was eluted from the columns by four successive additions of 1 ml 0.25 M KHCO₃ from 1 ml of sample collected after each administration. Ten milliliters of OPTIFLUOR (Packard Instruments Co., Menden, Conn.) Was added to each of the samples and counted. Stimulation of the inositol phosphate pathway was indicated by an increase in the level of labeled inositol phosphate. There was no increase in inositol phosphate production in any of the samples.

G. Human Glucagon Receptor Expression in COS-7 Cells

Plasmid pLJ6 'was transfected into COS-7 cells by the DEAE-dextran method described above. Cells were grown on glass slides (as described in Example 3) for 72 hours after transfection. In situ testing of glucagon binding with¹²⁵I-glucagon followed by autoradiography in the emulsion was performed as described in Example 3. Greater than 50% of the cells transfected with pLJ4 specifically bound glucagon.

H. Expression of the Human Glucagon Receptor in BHK570 Cells

BHK570 cells (deposited with the American Type Culture Collection under number 10314) were transfected with plasmid pLJ6 'by calcium phosphate transfection (Example 8). Transfected cells were selected in the presence of G418 until single colonies were visible. Single colomes were cloned using glass cylinders. The ability of the clones to bind glucagon in an in situ iodinated glucagon binding assay as described above was demonstrated.

Cells transfected with pLJ6 'were tested for cAMP accumulation as described in Example 8.C. The transfectants were tested after stimulation with glucagon, VIP secretin or GLP-I. Tests showed that pLJ6 'transfected cells accumulated increased concentrations of cAMP following stimulation with glucagon compared to control cells. Stimulation of transfectants with secretin, VIP or GLP-I showed no increased accumulation of cAMP. Intracellular calcium response was examined as described in Example 8.E. The glucagon-stimulated cells transfected with pLJ6 'showed an increase in intracellular calcium levels as shown by the sharp increase in fluorescence of the Fura-2 calcium index. These results show that pLJ6 'encodes a functional human glucagon receptor capable of binding to glucagon and signaling

As is apparent from the above, while specific embodiments have been described to illustrate the invention, various modifications are possible without departing from the spirit and scope of the invention. Thus, the invention is only limited by the appended claims.

178 685

1. BASIC INFORMATION (x) APPLICANT: NAME: ZymoGenetics, Inc. STREET: 4224 Roosevelt Way North East CITY: Seattle, Washington COUNTRY: USA POSTAL CODE: 98105 PHONE: (206) 547-80808 (li) (A) ADDRESSEE: SEED AND BERRY (B) STREET: 6300 COLUMBIA CENTER (C) CITY: SEATLLE (D) STATE: WA (E) COUNTRY: USA (F) ZIP CODE: 99104-7092 (v) READABLE FORM BY COMPUTER:

(A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM / PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentLn Release # 1.0, Version # 1.25 (vi) CURRENT DATA ENTRIES: (A) ENTRY NUMBER: 08 / 086,631 (B) ENTRY DATE: June 1, 1993 (C) CLASSIFICATION:

(vii) PRE-DECLARATION DATA: (A) APPLICATION NUMBER: US 07 / 938,331 (B) NOTIFICATION DATE: August 28, 1992

(viii) PROXY / AGENT DETAILS: (A) SURNAME: McMaster, David D.

(B) REGISTRATION NUMBER: 33, 963 (C) CASE NUMBER: 990008.424C1 (ix) TELECOMMUNICATIONS INFORMATION: (A) PHONE: 206-622-4900 (B) TELEFAX: 206-682-6031

178 685 (2) INFORMATION FOR SEQ IDENTIFIER NO. 1:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 17 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC447 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 1: TAACAATTTC ACACAGG (2) INFORMATION FOR SEQ IDENTIFIER NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 18 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC976 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 2: CGTTGTAAAA CGACGGCC (2) INFORMATION FOR SEQ IDENTIFIER NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 71 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: Linear (vii) DIRECT SOURCE:

(B) CLONE: ZC982 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 3:

AATTCCCCTC CCGCGAAGGC GTCGGCGCGG GGCTGGCGTA GGGCCTGCGT CAGCTGCAGC CCGCCGGAGC T (2) INFORMATION FOR SEQ IDENTIFIER NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 63 base pairs (B) TYPE: Nucleic acid

178 685 (C) N1C1NESS: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC983 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 4:

CCGGCGGGCT GCAGCTGACG CAGGCCCTAC GCCAGCCCCG CGCCGACGCC TTCGCGGGAG GGG (2) INFORMATION FOR SEQ ID. NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 38 base pairs (B) TYPE: Nucleic acid (C) STRANDNESS: Single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC3509 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 5:

CAAATTGACG TCATGGTAAA AATTGACGTC ATGGTAAG (2) INFORMATION FOR SEQ IDENTIFIER NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 46 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC3510 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 6:

AATTCTTACC ATGACGTCAA IIII IACCAT GACGTCAATT TGAGCT (2) INFORMATION FOR SEQ ID. NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 42 base pairs (B) TYPE: Nucleic acids (C) THREADNESS: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC3747

178 685 (χί) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 7: GACAGAGCAC AGAATTCACT ACTCGAGTTT TTT1 IIIIII TT (2) INFORMATION FOR SEQ ID. NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 25 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: Linear (vii) DIRECT SOURCE:

(B) CLONE: ZC4701 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 8: ACTCTCCGGT TNARRAAGCA RTANA (2) INFORMATION FOR SEQ ID. NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 26 base pairs (B) TYPE: Nucleic acid (C) THREADNESS: Single (D) TOPOLOGY: Linear (vii) DIRECT SOURCE:

(B) CLONE: ZC 4715 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 9: CATCCACGGT AYACNGTNGG NTAYWS (2) INFORMATION FOR SEQ IDENTIFIER NO: 10:

(i) CHARACTERIZATION OF THE SEQUENCES:

(A) LENGTH: 26 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: Linear (vii) DIRECT SOURCE:

(B) CLONE: ZC4758 (xi) SEQUENCE DESCRIPTION SEQ IDENTIFIER NO: 10: GCGGAATTCK MNAYNGTNGG NYAYWS (2) INFORMATION FOR SEQ ID. NO: 11:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 26 base pairs

178 685 (B) TYPE: nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC4778 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 11: CGCGGATCCY SRTTNMRRAA RCARTA (2) INFORMATION FOR SEQ IDENTIFIER NO: 12:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 21 base pairs (B) TYPE: Nucleic acid (C) STRANDNESS: Single (D) TOPOLOGY: Linear (vii) DIRECT SOURCE:

(B) CLONE: ZC5432 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 12: CAGGACCCGC TACAGCCAGA A (2) INFORMATION FOR SEQ IDENTIFIER NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 22 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC5433 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 13: CAACAGAAGC CCATGTTGTGC A (2) INFORMATION FOR SEQ IDENTIFIER NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1875 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (vi) ORIGINAL SOURCE:

178 685 (F) TISSUE TYPE: Liver (vii) DIRECT SOURCE:

(B) CLONE: pLJ4 (x) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 145..1599 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 14

GAATTCGCGG CCGCCGCCGG GCCCCAGATC CCAGTGCGCG AGGAGCCCAG TCCTAGACCC60

AGCAACCTGA GGAGAGGTGC ACACACCCCC AAGGACCCAG GCACCCAACC TCTGCCAGAT120

GTGGGGGGGT GGCTACCCAG AGGC AT6 CTC CTC ACC CAG CTC CAC TGT CCC171

Met Leu Leu Thr Gin Leu His Cys Pro

TAC CTG CTG CTG CTG CTG GTG GTG CTG TCA TGT CTG CCA AAG GCA CCC 219

Tyr Leu Leu Leu Leu Leu Val Val Leu Ser Cys Leu Pro Lys Ala Pro 10 15 2025

178 685

TCT GCC CAG GTA ATG GAC TTT HG TTT GAG AAG TGG AAG CTC TAT AGT267

Ser Ala Gin Val Met Asp Phe Leu Phe Glu Lys Trp Lys Leu TyrSer

3540

GAC CAG TGC CAC CAC AAC CTA AGC CTG CTG CCC CCA CCT ACT GAG CTG315

Asp Gin Cys His His Asn Leu Ser Leu Leu Pro Pro Thr GluLeu

5055

GTC TGC AAC AGA ACT TTC GAC AAG TAC TCC TGC TGG CCT GAC ACC CCT363

Val Cys Asn Arg Thr Phe Asp Lys Tyr Ser Cys Trp Pro Asp ThrPro

6570

CCC AAC ACC ACT GCC AAC ATT TCC TGC CCC TGG TAC CTA CCT TGG TAC411

Pro Asn Thr Thr Ala Asn Ile Ser Cys Pro Trp Tyr Leu Pro TrpTyr

8085

CAC AAA GTG CAG CAC CGC CTA GTG TTC AAG AGG TGT GGG CCT GAT GGG459

His Lys Val Gin His Arg Leu Val Phe Lys Arg Cys Gly Pro AspGly

95 100105

CAG TGG GTT CGA GGG CCA CGG GGG CAG TCA TGG CGC GAC GCC TCC CAA507

Gin Trp Val Arg Gly Pro Arg Gly Gin Ser Trp Arg Asp Ala SerGin

110 115120

TGT CAG ATG GAT GAT GAC GAG ATC GAG GTC CAG AAG GGG GTA GCC AAG555

Cys Gin Met Asp Asp Asp Glu Ile Glu Val Gin Lys Gly Val AlaLys

125 130135

ATG TAT AGC AGC TAC CAG GTG ATG TAC ACT GTG GGC TAC AGT CTG TCC603

Ket Tyr Ser Ser Tyr Gin Val Met Tyr Thr Val Gly Tyr Ser LeuSer

140 145150

CTG GGG GCC TTG CTC CTG GCG CTG GTC ATC CTG CTG GGC CTC AGG AAG651

Leu Gly Ala Leu Leu Leu Ala Leu Val Ile Leu Leu Gly Leu ArgLys

155 160165

178 685

CTG CAC TGC ACC CGG AAC TAC ATC CAC GGG AAC CTG TTC GCG TCC TTC699

Leu His Cys Thr Arg Asn Tyr Ile His Gly Asn Leu Phe Ala SerPhe

170 175 180185

GTG CTC AAG GCT GGC TCT GTG CTG GTC ATT GAT TGG CTG CTC AAG ACA747

Val Leu Lys Ala Gly Ser Val Leu Val Ile Asp Trp Leu Leu LysThr

190 195200

CGC TAT AGC CAG AAG ATT GGA GAT GAC CTC AGT GTG AGC GTC TGG CTC795

Arg Tyr Ser Gin Lys Ile Gly Asp Asp Leu Ser Val Ser Val TrpLeu

205 210215

AGT GAT GGG GCG GTG GCT GGC TGC AGA GTG GCC ACA GTG ATC ATG CAG843

Ser Asp Gly Ala Val Ala Gly Cys Arg Val Ala Thr Val Ile MetGin

220 225230

TAC GGC ATC ATA GCC AAC TAC TGC TGG TTG CTG GTG GAG GGT GTG TAC891

Tyr Gly Ile Ala Asn Tyr Cys Trp Leu Leu Val Glu Gly ValTyr

235 240245

CTG TAC AGC CTG CTG AGC ATC ACC ACC TTC TCG GAG AAG AGC TTC TTC939

Leu Tyr Ser Leu Leu Ser Ile Thr Phe Ser Glu Lys Cheese PhePhe

250 255 260265

TCC CTC TAT CTG TGC ATC GGC TGG GGA TCT CCC CTG CTG TTT GTC ATC987

Leu Tyr Leu Cys Ile Gly Trp Gly Cheese Pro Leu Leu Phe ValIle

270 275280

CCC TGG GTG GTG GTC AAG TGT CTG TTT GAG AAT GTC CAG TGC TGG ACC1035

Pro Trp Val Val Val Lys Cys Leu Phe Glu Asn Val Gin Cys TrpThr

285 290295

AGC AAT GAC AAT ATG GGA TTC TGG TGG ATC CTG CGT ATC CCT GTA CTC1083

Cheese Asn Asp Asn Met Gly Phe Trp Trp Ile Leu Arg Ile Pro ValLeu

300 305310

178 685

CTG GCC ΑΤΑ CTG ATC AAT TTT TTC ΑΤΟ ΊΤΓ GTC CGC ATC ATT CAT CTT1131

Leu Ala Ile Leu Ile Asn Phe Phe Ile Phe Val Arg Ile Ile HisLeu

315 320325

CTT GTG GCC AAG CTG CGT GCC CAT CAG ATG CAC TAT GCT GAT TAC AAG1179

Leu Val Ala Lys Leu Arg Ala His Gin Met His Tyr Ala Asp TyrLys

330 335 340345

TTC CGG CTA GCC AGG TCC ACG CTG ACC CTC ATT CCT CTG CTG GGA GTC1227

Phe Arg Leu Ala Arg Ser Thr Leu Thr Leu Ile Pro Leu Leu GlyVal

350 355360

CAC GAA GTG GTC TTT GCC TTT GTG ACT GAT GAG CAT GCC CAG 6GC ACC1275

His Glu Val Val Phe Ala Phe Val Thr Asp Glu His Ala Gin GlyThr

365 370375

CTG CGC TCC ACC AAG CTC TTT TTT GAC CTG TTC TTC AGC TCC TTT CAG1323

Leu Arg Cheese Thr Lys Leu Phe Phe Asp Leu Phe Phe Cheese Cheese PheGin

380 385390

GGT CTG CTG GTG GCT GTT CTC TAC TGT TTC CTC AAC AAG GAG GTG CAG1371

Gly Leu Leu Val Ala Val Leu Tyr Cys Phe Leu Asn Lys Glu ValGin

395 400405

GCA GAG CTA CTG CGG CGT TGG AGG CGA TGG CAA GAA GGC AAA GCT CTT1419

Ala Glu Leu Leu Arg Arg Trp Arg Arg Trp Gin Glu Gly Lys AlaLeu

410 415 420425

CAG GAG GAA AGG ATG GCC AGC AGC CAT GGC AGC CAC ATG GCC CCA GCA1467

Gin Glu Glu Arg Met Ala Ser Ser His Gly Ser His Met Ala ProAla

430 435440

GGG ACT TGT CAT GGT GAT CCC TGT GAG AAA CTT CAG CTT ATG AGT GCA1515

Gly Thr Cys His Gly Asp Pro Cys Glu Lys Leu Gin Leu Met SerAla

445 450455

178 685

GGC AGC AGC AGT GGG ACT GGC TGT GAG CCC TCT GCG AAG ACC TCA TTG 1563

Gly Ser Ser Ser Gly Thr Gly Cys Glu Pro Ser Ala Lys Thr Ser Leu

460 465 470

GCC AGT AGT CTC CCA AGG CTG GCT GAC AGC CCC ACC TGAATCTCCA 1609

Ala Ser Ser Leu Pro Arg Leu Ala Asp Ser Pro Thr

<td> 475</td><td> 480</td><td> 485</td>

<td>CTGGACTCCA</td><td>GCCAAGTTGG ATTCAGAAAG</td><td>GGCCTCACAA GACAACCCAG AAACAGATGC 1669</td>

<td>CTGGCCAAGG</td><td>CTGAAGAGGC AAAGCAGCAA</td><td>GACAGCAGCT TGTACTATCC ACACTCCCCT 1729</td>

<td>AACCTGTCCT</td><td>GGCCGGGTAC AGGCCACATT</td><td>GATGGAGTAG GGGCTGGATA TGATGGAGTA 1789</td>

<td>GCCATGCTAT</td><td>GAACTATGGG TGTTCCCATG</td><td>AGTGTTGCCA TGTTCCATGC ACACAGATAT 1849</td>

<td>GACCTTCAGT</td><td>AAAGAGCTCC CGTAGG</td><td> 1875</td>

178 685 (2) INFORMATION FOR SEQ IDENTIFIER NO: 15:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 485 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 15:

Met Leu Leu Thr Gin Leu His Cys Pro Tyr Leu Leu Leu Leu Leu Yal 15 1015

Yal Leu Ser Cys Leu Pro Lys Ala Pro Ser Ala Gin Val Met Asp Phe 20 2530

Leu Phe Glu Lys Trp Lys Leu Tyr Ser Asp Gin Cys His Asn Leu 35 4045

Ser Leu Leu Pro Pro Pro Thr Glu Leu Val Cys Asn Arg Thr Phe Asp 50 5560

Lys Tyr Ser Cys Trp Pro Asp Thr Pro Pro Asn Thr Thr Ala Asn Ile 65 70 7580

Ser Cys Pro Trp Tyr Leu Pro Trp Tyr His Lys Val Gin His Arg Leu 85 9095

Yal Phe Lys Arg Cys Gly Pro Asp Gly Gin Trp Val Arg Gly Pro Arg 100 105 110

Gly Gin Ser Trp Arg Asp Ala Ser Gin Cys Gin Met Asp Asp Asp Glu 115 120 125

Ile Glu Yal Gin Lys Gly Yal Ala Lys Met Tyr Cheese Cheese Tyr Gin Yal 130 135 140

Met Tyr Thr Yal Gly Tyr Ser Leu Ser Leu Gly Ala Leu Leu LeuAla

145 150 155160

Leu Yal Ile Leu Leu Gly Leu Arg Lys Leu His Cys Thr Arg AsnTyr

165 170175

178 685

Ile His Gly Asn Leu Phe Ala Ser Phe 180 185

Leu Val Ile Asp Trp Leu Leu Lys Thr 195 200

Asp Asp Leu Ser Val Ser Val Trp Leu 210 215

Cys Arg Val Ala Thr Val Ile Met Gin 225 230

Cys Trp Leu Leu Val Glu Gly Val Tyr 245

Thr Thr Phe Ser Glu Lys Ser Phe Phe 260 265

Trp Gly Cheese Pro Leu Leu Phe Val Ile 275 280

Leu Phe Glu Asn Val Gin Cys Trp Thr 290 295

Trp Trp Ile Leu Arg Ile Pro Val Leu 305310

Phe Ile Phe Val Arg Ile Ile His Leu 325

His Gin Met His Tyr Ala Asp Tyr Lys 340 345

Leu Thr Leu Ile Pro Leu Leu Gly Val 355360

Leu Lys Ala Gly Ser Val 190

Tyr Ser Gin Lys Ile Gly 205

Asp Gly Ala Val Ala Gly 220

Gly Ile Ile Ala Asn Tyr 235 240

Tyr Ser Leu Leu Ser Ile 255

Leu Tyr Leu Cys Ile Gly 270

Trp Val Val Val Lys Cys 285

Asn Asp Asn Met Gly Phe 300

Ala Ile Leu Ile Asn Phe 315 320

Val Ala Lys Leu Arg Ala 33 R

Arg Leu Ala Arg Ser Thr 350

Glu Val Val Phe Ala Phe 365

Val Thr Asp Glu His Ala Gin Gly Thr Leu Arg Ser Thr Lys Leu Phe

370 375380

178 685

Phe Asp Leu Phe Phe Cheese Phe Gin Gly Leu Leu Val Ala Val Leu

385 390 395400

Tyr Cys Phe Leu Asn Lys Glu Val Gin Ala Glu Leu Leu Arg ArgTrp

405 410415

Arg Arg Trp Gin Glu Gly Lys Ala Leu Gin Glu Glu Arg Met Ala Ser 420 425430

Ser His Gly Ser His Met Ala Pro Ala Gly Thr Cys His Gly Asp Pro 435 440'445

Cys Glu Lys Leu Gin Leu Met Ser Ala Gly Cheese Ser Ser Gly Thr Gly 450 455460

Cys Glu Pro Ser Ala Lys Thr Ser Leu Ala Ser Ser Leu Pro Arg Leu 465 470 475480

Ala Asp Ser Pro Thr

485

178 685 (21 INF0RMATION FOR SEQUENCE ID NO: 16:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 576 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: Linear (vii) DIRECT SOURCE:

(B) CLONE: G30 (ix) FEATURE:

(A) NAME / KEY: intron (B) LOCATION: 225..314 (ix) FEATURE:

(A) NAME / KEY: exon (B) location: 1..225 (ix) FEATURE:

(A) NAME / KEY: exon (B) LOCATION: 315..576 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 16:

GACATGGTAG GTCACAGCCT GTCCCTGGGG GCCCTGCTCC TCGCCTTGGC CATCCTGGGG60

GGCCTCAGCA AGCTGCACTG CACCCGCAAT GCCATCCACG CGAATCTGTT TGCGTCCTTC120

GTGCTGAAAG CCAGCTCCGT GCTGGTCATT GATGGGCTGC TCAGGACCCG CTACAGCCAG180

AAAATTGGCG ACGACCTCAG TGTCAGCACC TGGCTCAGTG ATGGAGTGAG CCCCCCTCGG240

CGGCCCCAGG CAGGTGGGIG GGTGGGCAGC CAGGCAGGTG GCCACGTAGC CGCGTCACAC300

TGCACCTGTA CCAGGCGGTG GCTGGCTGCC GTGTGGCCGC GGTGTTCATG CAATATGGCA360

TCGTGGCCAA CTACTGCTGG CTGCTGGTGG AGGGCCTGTA CCTGCACAAC CTGCTGGGCC420

TGGCCACCTT CCCCGAGAGG AGCTTCTTCA GCCTCTACCT GGGCATCGGC TGGGGTGCCC480

CCATGCTGTT CGTCGTCCCC TGGGCAGTGG TCAAGTGTCT GTTCGAGAAC GTCCAGTGCT540

GGACCAGCAA TGACAACATG GGCTTCTGGT GGATCC

576

178 685 (2) INFORMATION FOR SEQ IDENTIFIER NO: 17:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 487 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: 40-2-2 (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 1..486 (xi) SEQUENCE IDENTIFIER DESCRIPTION SEQ. NO: 17:

GAC ATG GTA GGT CAC AGC CTG TCC CTG GGG GCC CTG CTC CTC GCC TTG 48

Asp Met Val Gly His Ser Leu Ser Leu Gly Ala Leu Leu Leu Ala Leu 15 1015

GCC ATC CTG GGG GGC CTC AGC AAG CTG CAC TGC ACC CGC AAT GCC ATC96

Ala Ile Leu Gly Gly Leu Ser Lys Leu His Cys Thr Arg Asn AlaIle

2530

CAC GCG AAT CTG TTT GCG TCC TTC GTG CTG AAA GCC AGC TCC GTG CTG144

His Ala Asn Leu Phe Ala Ser Phe Val Leu Lys Ala Ser ValLeu Ser

4045

GTC ATT GAT GGG CTG CTC AGG ACC CGC TAC AGC CAG AAA ATT GGC GAC192

Val Ile Asp Gly Leu Leu Arg Thr Arg Tyr Ser Gin Lys Ile GlyAsp

5560

GAC CTC AGT GTC AGC ACC TGG CTC AGT GAT GGA GCG GTG GCT GGC TGC240

Asp Leu Ser Val Ser Thr Trp Leu Ser Asp Gly Ala Val Ala GlyCys

70 7580

CGT GTG GCC GCG GTG TTC ATG CAA TAT GGC ATC GTG GCC AAC TAC TGC288

Arg Val Ala Ala Val Phe Met Gin Tyr Gly Ile Val Ala Asn TyrCys

9095

TGG CTG CTG GTG GAG GGC CTG TAC CTG CAC AAC CTG CTG GGC CTG GCC336

Trp Leu Leu Val Glu Gly Leu Tyr Leu His Asn Leu Leu Gly LeuAla

100 105110

178 685

ACC TTC CCC GAG AGG AGC TTC TTC AGC CTC TAC CTG GGC ATC GGC TGG

Thr Phe Pro Glu Arg Cheese Phe Phe Cheese Leu Tyr Leu Gly Ile Gly Trp

115 120 125

384

<td rowspan="3">GGT Gly</td><td rowspan="3">GCC CCC ATG Ala Pro Met 130</td><td colspan="3">CTG TTC GTC</td><td colspan="6">GTC CCC TGG GCA GTG GTC AAG TGT</td><td rowspan="3">CTG Leu</td>

<td rowspan="2">Leu</td><td rowspan="2">Phe</td><td rowspan="2">Val 135</td><td>Val Pro Trp</td><td rowspan="2">Ala</td><td rowspan="2">Val 140</td><td rowspan="2">Val</td><td rowspan="2">Lys</td><td rowspan="2">Cys</td>

<td></td>

<td>TTC</td><td>GAG AAC GTC</td><td>CAG</td><td>TGC</td><td>TGG</td><td>ACC AGC AAT</td><td>GAC</td><td>AAC</td><td>ATG</td><td>GGC</td><td>TTC</td><td>TGG</td>

<td>Phe 145</td><td>Glu Asn Val</td><td>Gin</td><td>Cys 150</td><td>Trp</td><td>Thr Ser Asn</td><td>Asp 155</td><td>Asn</td><td>Underworld</td><td>Gly</td><td>Phe</td><td>Trp 160</td>

432

480

TGG ATC C

Trp Ile

487

178 685 (2) INFORMATION FOR SEQ IDENTIFIER NO: 18:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 162 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 18:

Asp Met Val Gly His Ser Leu Ser Leu Gly Ala Leu Leu Leu Ala Leu 15 1015

Ala Ile Leu Gly Gly Leu Ser Lys Leu His Cys Thr Arg Asn Ala Ile 20 2530

His Ala Asn Leu Phe Ala Ser Phe Val Leu Lys Ala Ser Ser Val Leu 35 4045

Val Ile Asp Gly Leu Leu Arg Thr Arg Tyr Ser Gin Lys Ile Gly Asp 50 5560

Asp Leu Ser Val Ser Thr Trp Leu Ser Asp Gly Ala Val Ala Gly Cys 65 70 7580

Arg Val Ala Ala Val Phe Met Gin Tyr Gly Ile Val Ala Asn Tyr Cys 85 9095

Trp Leu Leu Val Glu Gly Leu Tyr Leu His Asn Leu Leu Gly Leu Ala 100 105 110

Thr Phe Pro Glu Arg Cheese Phe Phe Cheese Leu Tyr Leu Gly Ile Gly Trp 115 120 125

Gly Ala Pro Met Leu Phe Val Val Pro Trp Ala Val Val Lys Cys Leu 130 135140

Phe Glu Asn Val Gin Cys Trp Thr Ser Asn Asp Asn Met Gly Phe Trp

145 150 155160

Trp Ile

178 685 (2) INFORMATION FOR SEQ IDENTIFIER NO: 19:

(i) CHARACTERISTICS OF THE SEQUENCES.

(A) LENGTH: 21 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC4812 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 19: GTGAGTTCAC GAATTCCATG G (2) INFORMATION FOR SEQ IDENTIFIER NO: 20:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) THREADNESS: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE (B) CLONE: ZC4814 (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 20: AGTTCACGAA TTCCATGGCC CCCCCCCCCC CCCC (2) SEQ IDENTIFIER DLEA INFORMATION NO: 21:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC5624 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 21: CGCATCTCTT GAACACGAAG (2) INFORMATION FOR SEQ IDENTIFIER NO: 22:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 41 base pairs (B) TYPE: Nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear

178 685 (vii) DIRECT SOURCE:

(B) CLONE: ZC5763 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 22: GAGAGAGAGA GAGAATTCGG AGGAGCGTAC ACACACACCA G (2) INFORMATION FOR SEQ IDENTIFIER NO: 23:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (vii) DIRECT SOURCE:

(B) CLONE: ZC5849 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 23: AGAGAGAGAGAGAGCTCGAG TTTATTGTTG GAGGACATTT CCAT (2) INFORMATION FOR SEQ IDENTIFIER NO: 24:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1809 bp (B) TYPE: Nucleic acid (C) STRETCHITY: Double (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (vii) DIRECT SOURCE:

(B) CLONE: pLJ6 '(ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 53..1486 (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 24:

<td colspan="2">GAATTCTGTG CAGCCCCTGC CAGATGTGGG AGGCAGCTAG CTGCCCAGAG GC ATG Met</td>

<td></td><td> 1</td>

<td>CCC CCC TGC CAG CCA CAG CGA</td><td>CCC CTG CTG CTG TTG CTG CTG CTG CTG</td>

<td>Pro Pro Cys Gin Pro Gin Arg</td><td>Pro Leu Leu Leu Leu Leu Leu Leu Leu</td>

<td> 5</td><td> 10 15</td>

<td>GCC TGC CAG CCA CAG GTC CCC</td><td>TCC GCT CAG GTG ATG GAC TTC CTG TTT</td>

<td>Ala Cys Gin Pro Gin Val Pro</td><td>Cheese Ala Gin Val Met Asp Phe Leu Phe</td>

<td> 20</td><td> 25 30</td>

103

151

178 685

GAG AAG TGG AAG CTC TAC GGT GAC CAG Glu Lys Trp Lys Leu Tyr Gly Asp Gin 3540

CTG CCC CCT CCC ACG GAG CTG GTGTGC

Leu Pro Pro Pro Thr Glu Leu ValCys

5055

TCC TGC TGG CCG GAC ACC CCC GCC AAT

Ser Cys Trp Pro Asp Thr Pro Ala Asn

CCC TGG TAC CTG CCT TGG CAC CAC AAA

Pro Trp Tyr Leu Pro Trp His His Lys

8590

AAG AGA TGC GGG CCC GAC GGT CAGTGG

Lys Arg Cys Gly Pro Asp Gly GinTrp

100105

CCT TGG CGT GAT GCC TCC CAG TGCCAG

Pro Trp Arg Asp Ala Ser Gin CysGin

115120

GTC CAG AAG GAG GTG GCC AAG ATGTAC

Val Gin Lys Glu Val Ala Lys MetTyr

130135

ACA GTG 6GC TAC AGC CTG TCC CTGGGG

Thr Val Gly Tyr Cheese Leu Cheese LeuGly

150

ATC CTG GGG GGC CTC AGC AAG CTGCAC

How many Leu Gly Gly Leu Ser Lys LeuHis

165170

TGT CAC CAC AAC CTG AGC CTG 199

Cys His His Asn Leu Ser Leu 45

AAC AGA ACC TTC GAC AAG TAT 247

Asn Arg Thr Phe Asp Lys Tyr 60 65

ACC ACG GCC AAC ATC TCC TGC 295

Thr Thr Ala Asn Ile Ser Cys 75 80

GTG CAA CAC CGC TTC GTG TTC 343

Val Gin His Arg Phe Val Phe 95

GTG CGT GGA CCC CGG GGG CAG 391

Val Arg Gly Pro Arg Gly Gin 110

ATG GAT GGC GAG GAG ATT GAG 439

Met Asp Gly Glu Glu Ile Glu 125

AGC AGC TTC CAG GTG ATG TAC 487

Cheese Phe Gin Val Met Tyr cheese 140 145

GCC CTG CTC CTC GCC TTG GCC 535

Ala Leu Leu Leu Ala Leu Ala

155 160

TGC ACC CGC AAT GCC ATC CAC 583

Cys Thr Arg Asn Ala Ile His 175

178 685

GCG AAT CTG TTT GCG TCC TTC GTG CTG AAA GCC AGC TCC GTG CTG GTC631

Ala Asn Leu Phe Ala Ser Phe Val Leu Lys Ala Ser Val LeuVal

180 185190

ATT GAT GGG CTG CTC AGG ACC CGC TAC AGC CAG AAA ATT GGC GAC GAC679

Ile Asp Gly Leu Leu Arg Thr Arg Tyr Ser Gin Lys Ile Gly AspAsp

195 200205

CTC AGT GTC AGC ACC TGG CTC AGT GAT GGA GCG GTG GCT GGC TGC CGT727

Leu Ser Val Ser Thr Trp Leu Ser Asp Gly Ala Val Ala Gly CysArg

210 215 220225

GTG GCC GCG GTG TTC ATG CAA TAT GGC ATC GTG GCC AAC TAC TGC TGG775

Val Ala Ala Val Phe Met Gin Tyr Gly Ile Val Ala Asn Tyr CysTrp

230 235240

CTG CTG GTG GAG GGC CTG TAC CTG CAC AAC CTG CTG GGC CTG GCC ACC823

Leu Leu Val Glu Gly Leu Tyr Leu His Asn Leu Leu Gly Leu AlaThr

245 250255

CTC CCC GAG AGG AGC TTC TTC AGC CTC TAC CTG GGC ATC GGC TGG GGT871

Leu Pro Glu Arg Cheese Phe Phe Cheese Leu Tyr Leu Gly Ile Gly TrpGly

260 265270

GCC CCC ATG CTG TTC GTC GTC CCC TGG GCA GTG GTC AAG TGT CTG TTC919

Ala Pro Met Leu Phe Val Val Pro Trp Ala Val Val Lys Cys LeuPhe

275 280285

GAG AAC GTC CAG TGC TGG ACC AGC AAT GAC AAC ATG GGC TTC TGG TGG967

Glu Asn Val Gin Cys Trp Thr Ser Asn Asp Asn Met Gly Phe TrpTrp

290 295 300305

ATC CTG CGG TTC CCC GTC TTC CTG GCC ATC CTG ATC AAC TTC TTC ATC1015

Ile Leu Arg Phe Pro Val Phe Leu Ala Ile Leu Ile Asn Phe PheIle

310 315320

178 685

TTC GTC CGC ATC Phe Yal Arg Ile 325

ATG CAC CAC ACA Met His His Thr 340

CTC ATC CCT CTG

Leu Ile Pro Leu 355

GAC GAG CAC GCC Asp Glu His Ala 370

CTC TTC CTC AGC Leu Phe Leu Ser

TTC CTC AAC AAG Phe Leu Asn Lys 405

GTT CAG CTG CTC GTG GCC AAG CTG CGG GCA CGG CAG

Yal Gin Leu Leu Yal Ala Lys Leu Arg Ala Arg Gin

330335

GAC TAC AAG TTC CGG CTG GCC AAG TCC ACG CTGACC

Asp Tyr Lys Phe Arg Leu Ala Lys Ser Thr LeuThr

345350

CTG 6GC GTC CAC GAA GTG GTC TTT GCC TTC GTGACG

Leu Gly Yal His Glu Val Yal Phe Ala Phe ValThr

360365

CAG GGC ACC CTG CGC TCC GCC AAG CTC TTC TTCGAC

Gin Gly Thr Leu Arg Ser Ala Lys Leu Phe PheAsp

375 380385

TCC TTC CAG GGC CTG CTG GTG GCT GTC CTC TACTGC

Cheese Phe Gin Gly Leu Leu Val Ala Val Leu TyrCys

390 395400

GAG GTG CAG TCG GAG CTG CGG CGG CGT TGG CACCGC

Glu Yal Gin Ser Glu Leu Arg Arg Arg Arg Trp HisArg

410415

1063 them

1159

1207

1255

1303

TGG CGC CTG GGC AAA GTG CTA TGG

Trp Arg Leu Gly Lys Yal Leu Trp

420425

AGG GCC TCA TCT TCG CCC GGCCAC

Arg Ala Ser Ser Ser Pro GlyHis

435440

TTT GGG AGG GGT GGT GGC AGCCAG

Phe Gly Arg Gly Gly Gly SerGin

450455

GAG GAG CGG AAC ACC AGC AAC CAC Glu Glu Arg Asn Thr Ser Asn His 430

GGC CCT CCC AGC AAG GAG CTG CAG Gly Pro Pro Ser Lys Glu Leu Gin 445

6AT TCA TCT 6CG GAG ACC CCC TTG Asp Ser Ser Ala Glu Thr Pro Leu 460 465

1351

1399

1447

178 685

GCT GGT GGC CTC CCT AGA TTG GCT GAG AGC CCC TTC TGAACCCTGC1493

Ala Gly Gly Leu Pro Arg Leu Ala Glu Ser Pro Phe 470 475

TGGGACCCCA GCTAGGGCTG GACTCTGGCA CCCAGAGGCG TCGCTGGACA ACCCAGAACT1553

GGACGCCCAG CTGAGGCTGG GGGCGGGGGA GCCAACAGCA GCCCCCACCT ACCCCCCACC1613

CCCAGTGTGG CTGTCTGCGA GATTGGGCCT CCTCTCCCTG CACCTGCCTT GTCCCTGGTG1673

CAGAGGTGAG CAGAGGAGTC CAGGGCGGGA GTGGGGGCTG TGCCGTGAAC TGCGTGCCAG1733

TGTCCCCACG TATGTCGGCA CGTCCCATGT GCATGGAAAT GTCCTCCAAC AATAAAGAGC 1793

TCAAGTGGTC ACCGAG1809

178 685 (2) INFORMATION FOR SEQ IDENTIFIER NO: 25:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 477 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 25

Met Pro Pro Cys Gin Pro Gin Arg Pro Leu Leu Leu Leu Leu Leu Leu ¹ 5 1015

Leu Ala Cys Gin Pro Gin Val Pro Ser Ala Gin Val Met Asp PheLeu

2530

Phe Glu Lys Trp Lys Leu Tyr Gly Asp Gin Cys His His Asn Leu Ser 35 4045

Leu Leu Pro Pro Pro Thr Glu Leu Val Cys Asn Arg Thr Phe Asp Lys 50 5560

Tyr Ser Cys Trp Pro Asp Thr Pro Ala Asn Thr Thr Ala Asn Ile Ser 65 70 7580

Cys Pro Trp Tyr Leu Pro Trp His His Lys Val Gin His Arg Phe Va1 85 9095

Phe Lys Arg Cys Gly Pro Asp Gly Gin Trp Val Arg Gly Pro Arg Gly

100 105110

Gin Pro Trp Arg Asp Ala Ser Gin Cys Gin Met Asp Gly Glu Glu Ile 115 120 125

Glu Val Gin Lys Glu Val Ala Lys Met Tyr Cheese Cheese Phe Gin Val Met 130 135 140

Tyr Thr Val Gly Tyr Ser Leu Ser Leu Gly Ala Leu Leu Leu AlaLeu

145 150 155160

Ala Ile Leu Gly Gly Leu Ser Lys Leu His Cys Thr Arg Asn AlaIle

165 170175

178 685

His Ala Asn Leu Phe Ala Ser Phe 180

Val Ile Asp Gly Leu Leu Arg Thr 195 200

Asp Leu Ser Val Ser Thr Trp Leu 210 215

Arg Val Ala Ala Val Phe Het Gin 225 230

Trp Leu Leu Val Glu Gly Leu Tyr 245

Thr Leu Pro Glu Arg Ser Phe Phe 260

Gly Ala Pro Met Leu Phe Val Val 275280

Phe Glu Asn Val Gin Cys Trp Thr 290 295

Trp Ile Leu Arg Phe Pro Val Phe 305 310

Ile Phe Val Arg Ile Val Gin Leu 325

Gin Met His His Thr Asp Tyr Lys 340

Thr Leu Ile Pro Leu Leu Gly Val 355360

Thr Asp Glu His Ala Gin Gly Thr 370 375

Leu Lys Ala Cheese Cheese Val Leu 190

Tyr Ser Gin Lys Ile Gly Asp 205

Asp Gly Ala Val Ala Gly Cys 220

Gly Ile Val Ala Asn Tyr Cys 235 240

His Asn Leu Leu Gly Leu Ala 250 255

Leu Tyr Leu Gly Ile Gly Trp 270

Trp Ala Val Val Lys Cys Leu 285

Asn Asp Asn Met Gly Phe Trp 300

Ala Ile Leu Ile Asn Phe Phe 315 320

Val Ala Lys Leu Arg Ala Arg 330 335

Arg Leu Ala Lys Ser Thr Leu 350

Glu Val Val Phe Ala Phe Val 365

Arg Cheese Ala Lys Leu Phe Phe 380

Asp Leu Phe Leu Cheese Cheese Phe Gin Gly Leu Leu Val Ala Val Leu Tyr

385 390 395400

178 685

<td rowspan="2">Cys</td><td rowspan="2">Phe</td><td colspan="13">Leu Asn Lys Glu Val Gin Ser Glu Leu Arg Arg Arg Trp His</td>

<td colspan="4"> 405</td><td colspan="7"> 410</td><td colspan="2"> 415</td>

<td>Arg</td><td>Trp</td><td>Arg</td><td>Leu 420</td><td>Gly</td><td>Lys</td><td>Val</td><td>Leu</td><td>Trp 425</td><td>Glu</td><td>Glu</td><td>Arg</td><td>Asn Thr 430</td><td>Cheese</td><td>Asn</td>

<td>His</td><td>Arg</td><td>Ala 435</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>Gly 440</td><td>His</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Lys cheese 445</td><td>Glu</td><td>Leu</td>

<td>Gin</td><td>Phe 450</td><td colspan="5">Gly Arg Gly Gly Gly 455</td><td>Cheese</td><td>Gin</td><td>Asp</td><td>Cheese</td><td>Cheese 460</td><td>Ala Glu</td><td>Thr</td><td>Pro</td>

<td>Leu</td><td>Ala</td><td colspan="5">Gly Gly Leu Pro Arg</td><td>Leu</td><td>Ala</td><td>Glu</td><td>Cheese</td><td>Pro</td><td>Phe</td><td></td><td></td>

'65 470 475

178 685

<img file="PL178685B1_D0001.tif" />

<img file="PL178685B1_D0002.tif" />

HYDROPHILICITY

HYDROPHILICITY

178 685

SPECIFIC BINDING, SPECIFIC BOUND

FIG. 3

<img file="PL178685B1_D0003.tif" />

GLUCAGON, M

GLUKAGON, M

178 685 connected / free BOUND / FREE

<img file="PL178685B1_D0004.tif" />

BOUND bound

178 685

Figure 5

Figure 5

Met Leu Leu Thr Gin Leu His Cys Pro Tyr Leu Leu Leu Leu Leu Val 15 1015

Val Leu Ser Cys Leu Pro Lys Ala Pro Ser Ala Gin Val Met Asp Phe 20 2530

Leu Phe Glu Lys Trp Lys Leu Tyr Ser Asp Gin Cys His Asn Leu 35 4045

Ser Leu Leu Pro Pro Pro Thr Glu Leu Val Cys Asn Arg Thr Phe Asp 50 5560

Lys Tyr Ser Cys Trp Pro Asp Thr Pro Pro Asn Thr Tnr Ala Asn Ile 65 70 7530

Ser Cys Pro Trp Tyr Leu Pro Trp Tyr His Lys Val Gin His Arg Leu 85 9095

Val Phe Lys Arg Cys Gly Pro Asp Gly Gin Trp Val Arg Gly Pro Arg 100 105 110

Gly Gin Ser Trp Arg Asp Ala Ser Gin Cys Gin Met Aso Aso Asp Glu 115 120 125

Ile 'Glu Val Gin Lys Gly Val Ala Lys Met Tyr Cheese Ser Tyr Gin Val

130 135140

Met Tyr Thr Val Gly Tyr Ser Leu Ser Leu Gly Ala Leu Leu Leu Ala

145 150 155160

Leu Val Ile Leu Leu Gly Leu Arg Lys Leu His Cys Thr Arg Asn Tyr

165 170175

Ile His Gly Asn Leu Phe Ala Ser Phe Val Leu Lys Ala Gly Ser Val

180 185190

178 685

<td>Leu Val</td><td>How much 195</td><td colspan="2">Asp Trp Leu</td><td>Leu</td><td>Lys 200</td><td>Thr</td><td>Arg Tyr Ser Gin 205</td><td>Lys</td><td>How much</td><td>Gly</td>

<td>Asp Asp 210</td><td>Leu</td><td>Val cheese</td><td>Cheese</td><td>Val 215</td><td>Trp</td><td>Leu</td><td>Asp Gly Ala cheese 220</td><td>Val</td><td colspan="2">Ala Gly</td>

<td>Cys Arg 225</td><td>Val</td><td>Ala Thr</td><td>Val 230</td><td>How much</td><td>Underworld</td><td>Gin</td><td>Tyr Gly Ile Ile 235</td><td colspan="3">Ala Asn Tyr 240</td>

<td colspan="2">Cys Trp Leu</td><td>Leu Val 245</td><td>Glu</td><td colspan="2">Gly Val</td><td>Tyr</td><td>Leu Tyr Ser Leu 250</td><td>Leu</td><td>Cheese 255</td><td>He</td>

Thr Thr Phe Ser Glu Lys Cheese Phe Phe Ser Leu Tyr Leu Cys Ile Gly

260 265270

Trp Gly Ser Pro Leu Leu Phe Val Ile Pro Trp Val Val Val Lys Cys 275 230 235

Leu Phe Glu Asn Val Gin Cys Trp Thr Ser Asn Asp Asn Met Gly Phe 230 295300

<td rowspan="2">Trp Trp Ile Leu 305</td><td colspan="2">Arg He Pro Val Leu Leu Ala Ile</td><td rowspan="2">Leu</td><td rowspan="2">How Much Asn Phe 320</td>

<td> 310</td><td> 315</td>

<td>Phe Ile Phe Val</td><td>Arg Ile Ile His</td><td>Leu Leu Val Ala</td><td>Lys</td><td>Leu Arg Al</td>

<td></td><td> 325</td><td> 330</td><td></td><td> 335</td>

<td>His Gin Met His</td><td colspan="2">Tyr Ala Asp Tyr Lys Phe Arg Leu</td><td>Ala</td><td>Arg Ser Thr</td>

<td> 340</td><td></td><td> 345</td><td></td><td> 350</td>

Leu Thr Leu Ile Pro Leu Leu Gly Val His Glu Val Val Phe Ala Phe

355 360365

Val Thr Asp Glu His Ala Gin Gly Thr Leu Arg Ser Thr Lys Leu Phe

370 375380

Phe Asp Leu Phe Phe Cheese Phe Gin Gly Leu Leu Val Ala Val Leu

385 390 395400

178 685

Tyr Cys Phe Leu Asn Lys Glu Val Gin Ala Glu Leu Leu Arg Arg Trp 405 410 415

Arg Arg Trp Gin Glu Gly Lys Ala Leu Gin Glu Glu Arg Met Ala Ser 420 425430

Ser His Gly Ser His Met Ala Pm Ala Gly Thr Cys His Gly Asp Pm 435 440445

Cys Glu Lys Leu Gin Leu Met Ser Ala Gly Cheese Ser Ser Gly Thr Gly 450 455460

Cys Glu Pro Ser Ala Lys Thr Ser Leu Ala Ser Ser Leu Pm Arg Leu 465 470 475480

Ala Asp Ser Pro Thr

485

178 685

FIGURE

<img file="PL178685B1_D0005.tif" />

CYTOPLASM

The Publishing Department of the Polish Patent Office of the Republic of Poland Circulation 70 copies Price PLN 6.00.