Glucagon receptors.

Description: TECHNICAL FIELD [Detailed description of the invention] Technical field The present invention relates generally to cell surface receptors, and more particularly to glucagon receptors. Background of the invention Glucagon is a 29-amino acid hormone produced by alpha cells in the islets of the pancreas. Glucagon is responsible for maintaining normal levels of glucose in many animals, including humans, by acting as an insulin reaction hormone. In particular, insulin is known to rapidly reduce blood glucose levels, but glucagon reversely balances these effects by contributing to its elevated blood glucose levels. The interaction of glucagon and insulin is very important for maintaining glucose levels in the body. Glucagon or insulin imbalances are believed to play a role in several diseases, such as diabetic and diabetic ketoacidosis. According to one theory, hyperglycemic conditions in diabetes mellitus can be brought about not only by underutilization of glucose (due to decreased insulin), but also by overproduction of glucose due to elevated levels of glucagon ("Diabetes and". The alpha cell, "Diabetes 25: 136-151,1976; Under 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, as well as in the role of glucagon in diseases, such as true diabetes, is the conversion of signals into its cells during binding to glucagon, thereby glucogenolysis (glycogenolysis). And a glucagon receptor that triggers gluconeogenesis. It is conventionally believed that the effects of glucagon are partially mediated by elevated intracellular levels of cyclic adenosine monophosphate (cAMP). In particular, glucagon binding to its cellular receptor activates adenylate cyclase to produce cAMP, thus increasing intracellular cAMP levels. This increase in intracellular levels of cAMP is believed to result in glycogenolysis and gluconeogenesis due to the increase obtained in glucose production by the liver (Unson et al., "Biological Activities of des-His").¹[Glu⁹] Glucagon Amide, a Glucagon Antagonist, "Peptides 10: 1171-1177, 1989.). However, additional pathways have also been suggested for stimulation of glycogenolysis and gluconeogenesis. In particular, glucagon has been reported to bind to receptors within the hepatocellular membrane bound to phospholipase C via G-protein. During stimulation, this protein causes the degradation of phosphatidylinositol 4,5biphosphate to produce the second messenger inositol and 1,2 diacylglycerol (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; and Pittner and See Fain, Biochem.J. 277: 371378,1991. ). Stimulation of inositol and phospholipid metabolism with glucagon can thereby be an additional pathway by which glucagon can stimulate glycogenolysis and gluconeogenesis. The present invention discloses glucagon receptors (s) and further provides other related benefits. Abstract of the invention In aspects of the invention, the isolated DNA molecule encoding the glucagon receptor provided. The term "isolated DNA molecule" as used herein refers to a DNA molecule or sequence that is separate, separated and placed alone, or separated from other components. For example, a DNA molecule is isolated when it contains other chromosomal sequences to which it naturally associates within its genome, and is particularly isolated from other DNA molecules that do not contain other structural genes. .. The isolated DNA molecule can contain 5'and 3'untranslated sequences to which it naturally associates. In one aspect of the invention, the glucagon receptor is selected from the group consisting of rat and human glucagon receptors. In another embodiment, the DNA molecule comprises the sequence of the molecule of SEQ ID NO: 14 from nucleotide 145 to nucleotide 1599. In another embodiment, the DNA molecule encodes a glucagon receptor comprising a sequence of amino acids of SEQ ID NO: 15 from amino acid number 1 to threonine, amino acid number 485. In another embodiment, the DNA molecule comprises the nucleotide sequence of the molecule of SEQ ID NO: 24 from nucleotide 53 to nucleotide 1486. In yet another embodiment, the DNA molecule encodes a glucagon receptor comprising a sequence of amino acids of SEQ ID NO: 25 from amino acid number 1 to phenylalanine, amino acid number 477. A DNA construct comprising a first DNA segment encoding a Glucagon receptacle operably linked to an additional DNA segment required for expression of the first DNA segment, a host cell containing such a DNA construct. Also provided is a method for producing a Glucagon Receptor, comprising culturing a host cell under conditions that promote expression of the DNA segment encoding the Glucagon Receptor. In another aspect of the invention, an isolated glucagon receptor is provided. In some embodiments, an isolated glucagon receptor comprising a sequence of glutamine, nucleotides of SEQ ID NO: 15 from amino acid number 28 to tyrosine, amino acid number 142 is provided. In another aspect of the invention, an isolated antibody that specifically binds to the glucagon receptor is provided. In some embodiments, the antibody is a monoclonal antibody. In an additional aspect, a monoclonal antibody capable of blocking the binding of glucagon to the glucagon receptor is provided. Hybridomas that produce the monoclonal antibodies described above are also provided. In a further aspect of the invention is a method of detecting the presence of a glucagon antagonist, which is sufficient to (a) allow the compound to bind to its receptor in the presence of the glucagon agonist. Exposure to recombinant glucagon receptors bound to the response pathway and related responses through that pathway over time and conditions, and (b) to the glucagon receptor for stimulation of the response pathway by glucagon agonist alone. Provided is a step-by-step method of detecting a decrease in the stimulation of the response pathway resulting from the binding of the glucagon antagonist and then measuring the presence of the glucagon antagonist. In various aspects of the invention, the response pathway is a membrane-bound adenylate cyclase response pathway, and its detection step comprises measuring the reduction in cyclic AMP production by the membrane-bound adenylate cyclase response pathway. In another aspect of the invention, the response pathway comprises a luciferase reporter system. In yet another aspect of the invention, a probe having at least 12 nucleotides is provided that can hybridize to the nucleic acid encoding the glucagon receptor. These and other aspects of the invention will be clarified with reference to the following detailed description and accompanying drawings. In addition, specific procedures or compositions (eg, plasmids, etc.) are described in more detail, and therefore various references incorporated by citation as a whole are described below. A brief description of the drawing FIG. 1 illustrates the structure of a typical glucagon receptor. The symbols used are EATD (extracellular amino-terminal domain) surrounded by a dotted line; CM (cell membrane); ED (effector domain) surrounded by a dash line; 1ID, first intracellular loop domain; 2ID, Second intracellular loop domain; 3ID, third intracellular loop domain; C-ID, carboxy-terminal intracellular domain; 1ELD, first cell loop domain; 2ELD, second extracellular loop domain; 3ELD, first Three extracellular loop domains; TMD1, first transmembran domain; TMD2, second transmembran domain; TMD3, third transmembran domain; TMD4, fourth transmembran domain; TMD5, fifth transmembran domain. Domains; TMD6, 6th transmembran domain; and TMD7, 7th transmembran domain. FIG. 2 illustrates the hydrophobicity of the rat glucagon receptor. Figure 3 shows the glucagon receptor¹²⁵The binding of I-glucagon is illustrated. FIG. 4 is a Scatchard analysis of the apparent Kd for the glucagon receptor. FIG. 5 shows the amino acid sequence of the rat glucagon receptor, the transmembran domain of which is underlined. Detailed description of the invention As mentioned earlier, the present invention provides an isolated DNA molecule encoding a glucagon receptor. In their natural configuration, the glucagon receptor is believed to exist as a membrane-bound protein consisting of an extracellular amino-terminal domain and several smaller external and internal domains (see Figure 1). In the context of the present invention, "glucagon receptor" refers to such proteins and substantially similar derivatives. Derivatives include allelic and genetically engineered variants that contain conservative amino acid substitutions and / or slight additions, substitutions or deletions of amino acids. The glucagon receptor according to the present invention can bind to glucagon and convert the signal provided by glucagon into cells. Preferably, the glucagon receptor according to the invention can bind to a glucagon having a Kd of 100 nm or less, more preferably 50 nM or less, and most preferably 33 nM or less. Representative tests that can be used to measure glucagon binding by the glucagon receptor are described in more detail below in Examples 3 and 6. Signal conversion typically occurs when the response pathway is activated by an external stimulus that is generally, but not always, bound directly to the membrane-bound receptor. Response pathways generally cause cellular responses, such as extracellular matrix secretion from responsive cell lines, hormone secretion, chemotactic, differentiation, or initiation or inhibition of cell division of responsive cells. As used herein, binding of a receptor to a response pathway refers to direct activation of the response pathway or activation of the cellular response pathway by translating a signal via a second messenger, eg, G-protein. It means the conversion. Various cellular response pathways can be utilized by the glucagon receptor to convert glucagon binding signals into the cell, including, for example, the adenylate cyclase response pathway and the intracellular calcium response pathway. Tests for measuring adenylate cyclase activity are well known in the art and, for example, Lin et al. (Biochemistry). 14: 1559-1563, 1975). The present invention measures the biological activity of the glucagon receptor based on intracellular calcium concentration (see Grynkiewics et al., J. Biol. Chem. 260: 3440-3450, 1985), and more details below. Measurements are also provided through the use of the luciferase reporter system described in. In addition, the biological response that occurs via the inositol trisphosphate pathway is described by Subers and Nathanson (J.Mol.Cell.Cardiol.20: 131-140,1988) or Pittner and It can be evaluated by measuring inositol-phosphate metabolism as commonly described in Fain (Biochem. J. 277: 371-378, 1991). It should be noted that in the context of the present invention, not all response pathways need to be present for the glucagon receptor to convert signals into cells. For example, an increase in a cellular response, eg, intracellular calcium levels, can also be triggered by glucagon binding to its receptor or an inositol phosphate signal in the absence of cAMP. Isolation of glucagon receptor cDNA clones As mentioned earlier, the present invention provides an isolated DNA molecule encoding a glucagon receptor. Simply put, the genome or cDNA molecule encoding the glucagon receptor can be obtained from a library prepared from cells and tissues according to procedures as described below and in the examples. The cells and tissues that can be used in the present invention can be obtained from various mammals, including, for example, humans, macaques (monkeys), cows, pigs, horses, dogs, rats, and mice. .. Preferred cells and tissues that can be used include fat, kidney, pancreas, heart, and liver. In one aspect of the invention, the rat glucagon receptor is isolated and cloned using the procedures described herein. Simply put, poly (A)⁺RNA is isolated from Sprague Dawley rats and used as a template for cDNA synthesis as essentially described by Houamed et al (Science 252: 1318-1321, 1991) to generate full-length cDNAs. did. Then about 1x10⁶A library containing clones was constructed within a mammalian cell expression plasmid by directional cloning of cDNAs larger than 800 base pairs. Plasmid DNAs prepared from pools, each containing 5,000 clones, were then transfected into COS-7 cells, selected and grown on microscopic slides. 72 hours after this transfected cell¹²⁵Binding to I-glucagon, followed by emulsion radiography (McMahan et al., EMBO J. 10: 2821-2832, 1991). Positive pools were sorted in sequence until a single clone was isolated. The plasmid derived from this clone, named pLJ4, contains an approximately 2.0 kilobase insert encoding a 485 amino acid protein with an expected molecular weight of 54,962 daltons (see SEQ ID NO: 15). In another aspect of the invention, a method of isolating and cloning a human glucagon receptor is provided. The various techniques provided herein can be used, which will also be used, for example, in identifying a library containing the sequence encoding the human glucagon receptor. Includes the use of a polymerase chain reaction ("PCR") to amplify the glucagon receptor encoding sequence (Example 4), followed by cloning of the receptor (Example 5). Particularly preferred strategies for cloning the human glucagon receptor are described in Examples 4 and 5 below. Alternatively, an expression library containing human cDNAs is prepared from a suitable RNA source as essentially described in Example 1, and clones expressing a functional human glucagon receptor are described in Example 3. Screen as it is. Production of recombinant glucagon receptor The present invention cultivates a host cell containing a DNA construct comprising a first DNA segment encoding a Glucagon receptor operably linked to an additional DNA segment required for expression of the first DNA segment. Providing the production of recombinant Glucagon Receptor. As mentioned earlier, in the context of the present invention, "glucagon receptors" are understood to include substantially similar derivatives thereof. Moreover, the glucagon receptor can be encoded by a DNA sequence that is substantially similar to the DNA sequence disclosed herein. As used herein, a DNA sequence is obtained from (a) the coding region of the native Glucagon-Receptor gene (eg, including an allelic variant of the sequence disclosed below); b) The DNA sequence can be hybridized to the DNA sequence of the present invention under high or low stringency (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory). Press, NY, 1989); or if the DNA sequence is reduced to the DNA sequence defined in (a) or (b) as a result of its genetic code, it is considered to be "substantially similar". .. Mutations in the nucleotide sequence constructed for expression of the mutant glucagon receptor will preserve the reading frame for that coding sequence. In addition, this mutation is preferably complementary to which it can hybridize to produce a secondary mRNA structure, eg, a loop or hairpin that would adversely affect the translation of its receptor mRNA. Will not create a new area. Although the mutation site can be predicted, it is not necessary to predict the nature of the mutation itself. For example, a random mutation can be made at its target codon to select the optimal characteristics of the mutation at a given site, and its expressed glucagon receptor mutant is screened for biological activity. Mutations can be introduced at a particular locus by synthesizing an oligonucleotide containing a mutant sequence flanking a restriction site that allows ligation to a fragment of its native sequence. After ligation, the resulting reconstituted sequence encodes a derivative with the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-designated site-specific mutagenesis procedure can be used to provide a modified gene with a particular codon modified according to the required substitution, deletion, or insertion. An exemplary method for making the aforementioned changes is Walder et al., (Gene 42: 133,1986): Bauer et al. (Gene 37: 73, 1985); Craik (Bio Techniques, January 1985,). 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al. (Supra). Modify the primary amino acid structure of the glucagon receptacle by forming covalent or collective conjugates with other chemical moieties such as glycosyl groups, lipids, phosphates, acetyl groups, or other proteins or polypeptides. You can also do it. In a further embodiment, the glucagon receptor can be fused with other peptides that facilitate purification or identification of the glucagon receptor. For example, the glucagon receptor can be prepared as a fusion protein with a FLAG polypeptide sequence (see US Pat. No. 4,851,341; also Hopp et al., Bio / Thchnology 6: 1204,1988). This FLAG polypeptide sequence is highly antigenic and provides an epitope for binding by a specific monoclonal antibody that allows rapid purification of the expressed recombinant protein. In addition, this sequence is specifically cleaved by bovine mucosal enteropeptidase at the residue immediately after the Asp-Lys pair. A large number of DNA constructs containing all or part of the nucleotide sequence of the native or mutant glucagon receptor as discussed above can be conveniently prepared. In the context of the present invention, DNA constructs are DNA molecules modified through human intervention to contain segments of DNA that are bound and juxtaposed in a manner that would not otherwise exist in nature, or this. A clone of such a molecule (either single-or double-stranded). The DNA structure of the present invention comprises a primary DNA segment encoding a glucagon receptor operably linked to an additional DNA segment required for expression of the primary DNA segment. In the context of the present invention, additional DNA segments will generally include promoters and transcription terminators, and can further include enhancers and other elements. DNA constructs, also known as expression vectors, can also contain the DNA segments needed to direct the secretion of the polypeptide in question. Such a DNA segment can contain at least one secretory signal sequence. Preferred secretory signals are the Glucagon secretory signal (pre-pro sequence), alpha factor signal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30: 933-943,1982; Kurjan et al., US Pat. No. 4,546,082; Brake. , European Patent No. 116,201), PH05 signal sequence (Beck et al., WO86 / 00637), BAR1 secretory signal sequence (MacKay et al., US Pat. No. 4,613,572; MacKay, 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), α-2 plasmin inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 102: 1033-1042,1987), tissue plasminogen activator signal sequence (Yuan et al., J. Biol. Chem. 265: 13528-13552, 1990) or, for example, the bacterial signal sequence reviewed by Oliver (Ann. Rev. Microbiol. 39: 615-649, 1985). including. Alternatively, the secretory signal sequence can be synthesized, for example, according to the rules established by von Heinje (Eur.J.Biochem.133: 17-21,1983: J.Mol.Biol.184: 99-105, 1985; Nuc. Acids Res. 14: 4683-4690,1986). The secretory signal sequence can be used alone or in combination. For example, a combination of the first secretory sequence with the sequence encoding the third domain of Barrier (described herein in US Pat. No. 5,037,243, which is incorporated herein by reference in its entirety). Can be used in. The sequence encoding the third domain of Barrier is placed in a suitable reading frame at 3'or 5'for that DNA segment of the DNA sequence in question, and in a suitable reading frame with its secretory signal sequence and the problem. It can be arranged to have both of the DNA segments of. For expression, the DNA molecule encoding the glucagon receptor is inserted into a suitable DNA construct, which is then used to transform or transfect the appropriate host cell for expression. Host cells for use in the practice of the present invention include mammalian, avian plant, insect, bacterial and fungal cells. Preferred eukaryotic cells are cultured mammalian cell lines (eg, rodent or human cell lines) and fungal cells of yeast species (eg, Saccharomyces spp.), Especially Saccharomyces cerevisiae (S). Includes .cerevisiae), Schizosacchromyces spp., Or Kluyveromyces spp.) Or filamentous fungi (eg, Aspergillus spp., Neurospora spp.) Including things. Methods for producing recombinant proteins in various prokaryotic and eukaryotic host cells are generally known in the art ("Gene Expression Technology," Methods in. Enzymology, Vol.185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990; Also, "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds.) Academic Press, San See Diego, Calif., 1991. ). In general, host cells will be selected based on their ability to produce the protein in question at high levels or to perform at least some of the processing steps required for the biological activity of the protein. Let's go. In this method, the number of cloned DNA sequences that must be transfected into the host cell can be minimized, and the overall yield of biologically active protein can be maximized. it can. Vectors suitable for use in the present invention are YRp7 (Struhl et al., Proc.Natl.Acad.Sci.USA.76:1035-1039,1978), YEp13 (Broach). et al., Gene 8: 121-133,1979), POT Vector (Kawasaki et al., US Pat. No. 4,931,373 (incorporated herein by reference)), pJDB249 and pJDB219 (Beggs, Nature). 275: 104-108,1978) and their derivatives. Such a vector can generally be one of any number of genes exhibiting a predominant phenotype in which a phenotypic test exists to allow selection of transformants. Will include selectable markers. Preferred selectable markers are those that supplement the autotrophic nature of the host cell, provide antibiotic resistance, or allow the cell to utilize a particular carbon source, and LEU2 (Broach et al., Ibid). ), URA3 (Botstein et al., Gene 8: 17,1979), HIS3 (Struhl et al., Ibid) or POT1 (Kawasaki et al., Ibid.). Another suitable selectable marker is the CAT gene, which confer chloramphenicol resistance on yeast cells. The preferred promoter for use in yeast is the yeast glycogen (Hitzeman et al., J.Biol.Chem.255:1207312080,1980; Alber and Kawasaki, J.Mol.Appl.Genet.1: 419 434,1982; Kawasaki, US Pat. No. 4,599,311) or the alcohol dehydrogenase gene (Young et al., In Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (eds.), P.355, Plenum, New York, 1982. Includes Ammerer, Meth. Enzymol. 101: 192-201, 1983). Particularly preferred promoters in this regard are the TPI1 promoter (Kawasaki, US Pat. No. 4,599,311, 1986) and ADH2-4.^CPromoter (Russell et al., Nature 304: 652-654, 1983; Irani and Kilgore, US Patent Application Sequential No. 07 / 784,653 (incorporated herein by reference)). The expression unit can 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 can be expressed in strains of filamentous fungi, such as Aspergillus (McKnight et al., US Pat. No. 4,935,349, which is incorporated herein by reference). ). Examples of useful promoters include those derived from the Aspergillus nidulans glycogene, such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985) 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 that can be inserted into the Aspergillus chromosomal DNA. Techniques for transforming fungi are well known in the literature and, for example, Beggs (ibid.), Hinnnen 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 cell will generally include a genetic defect that is supplemented by a selectable marker present on its expression vector. The selection of specific hosts and selectable markers is well within the level of one of ordinary skill in the art. To optimize the production of heterologous proteins in yeast, for example, the host strain is mutated, eg, yeast pep4 mutation (Jones, Genetics 85: 23-33, 1977) to reduce proteolytic activity. It is preferable to carry what it brings. In addition to fungal cells, cultured mammalian cells can be used as host cells in the present invention. Preferred cultured mammalian cells for use in the present invention are COS-1 (ATCC No.CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCC No.CRL 1632), and 293 (ATCC No.CRL 1573). Graham et al., J.Gen.Virol. 36: 59-72, 1977) Includes cell lines. The preferred BHK cell line is the BHK570 cell line (deposited by the American Type Culture Collection under deposit number CRL 10314). In addition, Rat Hep I (ATCC No.CRL 1600), Rat Hep II (ATCC No.CRL 1548), TCMK (ATCC No.CCL 139), Human Lung (ATCC No.CCL 75.1), Human Liver Cancer (ATCC No.HTB) 52), Hep G2 (ATCC No.HB 8065), Mouse Liver (ATCC No.CCL 29.1), NCTC 1469 (ATCC No.CCL) 9.1), SP2 / 0-Ag14 (ATCC No.1581), HIT-T15 (ATCC No.CRL 1777), and RINm 5AHT₂A number of other mammalian cell lines can be used in the present invention, including B (Orskov and Nielson, FEBS 299 (1): 175-178, 1988). A mammalian expression vector for use in the practice of the present invention will include a promoter capable of designating transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. The viral promoters are the immediate early cytomegalovirus promoter (Boshart et al., Cell 41: 521-530,1985) and the SV40 promoter (Subramani et al., Mol.Cell.Biol. 1: 854-864, 1981). including. Cell promoters are the mouse metallothionein-1 promoter (Palmiter et al., US Pat. No. 4,579,821), the mouse Vκ promoter (Bergman et al., Proc. Natl. Acad. Sci. USA. 81: 7041-7045, 1983; Grant et al., Nuc. Acids Res.15: 5496,1987) and Mouse V_HIncludes promoter (Loh et al., Cell 33: 85-93, 1983). A particularly preferred promoter is the major late promoter from adenovirus 2 (Kaufman and Sharp, Mol.Cell. Biol. 2: 1304-13199, 1982). Such an expression vector can also include a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the peptide or protein in question. This preferred RNA splice site can be obtained from the adenovirus and / or immunoglobulin gene. Also included in its expression vector is a polyadenylation signal located downstream of the coding sequence in question. Suitable polyadenylation signals are early and late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), Polyadenylation signals from the adenovirus 5 E1B region and human growth hormone gene terminators (DeNoto et et. al., Nuc. Acids Res. 9: 3719-3730, 1981) is included. The expression vector can include a non-coding virus leader sequence, eg, an adenovirus 2 tripartite reader located between its promoter and its RNA splice site. Preferred vectors can also include enhancer sequences, such as SV40 enhancers and mouse μ enhancers (Gillies, Cell 33: 717-728,1983). The expression vector can also include sequences encoding adenovirus VA RNAs. Suitable vectors can be obtained from commercial sources (eg Stratagene, La Jolla, CA). Clone DNA sequences in cultured mammalian cells, eg, calcium phosphate mediated transfection (Wigler et al., Cell 14: 725,1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981; Graham and van der Eb, Virology 52: 456,1973), Electroporation (Neumann et. al., EMBO J.1: 841-845,1982), or DEAE-dextran-mediated transfection (Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987) (This is incorporated herein by reference.). To identify cells that have stably integrated the cloned DNA, selectable markers are generally introduced into the cells along with the gene or the cDNA in question. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs such as neomycin, hygromycin, and methotrexate. This selectable marker can be an amplifyable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable marker is Thilly (Mammalian Cell Technology, Butterworth) Review by Publishers, Stoneham, MA (incorporated herein by reference). The selection of selectable markers is well within the level of those skilled in the art. Selectable markers can be introduced intracellularly on separate vectors at the same time as the glucagon receptor sequence, or they can be introduced into the same vector. When on the same vector, the selectable marker and glucagon receptor sequence can be under the control of different promoters or the same promoter, the latter organization producing a two-cistron message. This type of construct is known in the art (eg, Levinson and Simonsen, US Pat. No. 4,713,339). It is also advantageous to add additional DNA, known as "carrier DNA", to the mixture introduced into the cell. Transfected mammalian cells are subjected to growth for a period of time, eg 1-2 days, to initiate expression of the DNA sequence in question (s). Next, drug selection. It is applied to select for the growth of cells expressing a selectable marker in stable fusion. For cells transfected with an amplifyable selectable marker, the drug concentration is increased in a stepwise manner for selection for increased copy number of the cloned sequence, thereby increasing expression levels. Cells expressing the introduced sequence are selected and screened for the production of the protein in question in the desired form or at the desired level. Cells that meet these criteria can then be cloned and scaled up for production. A preferred prokaryotic host cell for use in the practice of the present invention is a strain of bacterial Escherichia coli. However, Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing cloned foreign DNA sequences therein are well known in the art (eg, Maniatis et. al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982 (incorporated herein by reference); or Sambrook et al., See above. ). Vectors used to express cloned DNA sequences in a bacterial host will generally include selectable markers, such as genes for antibiotic resistance, and promoters that function in the host cell. Suitable promoters are trp (Nichols and Yanofsky, Meth.Enzymol.101: 155-164,1983), lac (Casadaban et al., J. Bacteriol.143: 971-980,1980), and λ phage (Queen.J). .Mol.Appl.Genet.2: 1-10,1983) Includes promoter system. A useful plasmid for transforming bacteria is pBR322 (Bolivar et al., Gene). 2: 95-113,1977), pUC plasmid (Messing, Meth.Enzymol.101: 20-78,1983; Vieria and Messuing, Gene 19: 259-268,1982), pCQV2 (Queen, ibid.) And theirs. Contains derivatives. The plasmid can contain both viral and bacterial elements. Given the teachings provided herein, methods of introducing expression vectors encoding promoters, terminators and glucagon receptors of the invention into plant, bird and insect cells will be apparent to those of skill in the art. .. For example, the use of baculovirus as a vector for the expression of heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28: 215-224, 1990). In addition, the use of Agrobacterium rhizogenes as a vector for gene expression in plant cells is described by Sinker et. It has been reviewed by al (J. Biosci. (Bangalore) 11: 47-58, 1987). Host cells containing the DNA constructs of the invention are then cultured to express the DNA segment encoding the glucagon receptor. The cells are cultured according to standard methods in culture medium containing the nutrients required for the growth of the selected host cells. A variety of suitable media are known in the art and are generally carbon sources, nitrogen sources, essential amino acids, vitamins and minerals, as well as other components such as growth factors or formations of a particular host cell. Including those that can be requested by. Culture medium generally contains cells containing the DNA construct (s), for example, due to defects in essential nutrients supplemented by drug selection or selection markers co-transfected with or co-transfected with the DNA construct. Will be selected for. Suitable culture conditions for yeast cells are those that include, for example, a chemically defined medium that includes a non-amino acid nitrogen source or a nitrogen source that can be a yeast extract, inorganic salts, vitamins and essential amino acid supplements. It involves culturing in a temperature between 4 ° C. and 37 ° C., particularly preferably 30 ° C. The pH of the medium is preferably above 2 and maintained at a pH below 8, more preferably pH 5-6. Methods of maintaining a stable pH include buffering and constant pH control. A preferred agent for pH control is sodium hydroxide. Preferred buffering agents are succinic acid and Bis-Tris (Sigma Chemical). Co., St. Louis, MO) is included. Since yeast host cells tend to add heterologous proteins to hypersaccharides, it is preferable to express the glucagon receptor of the present invention in yeast cells having a defect in the gene required for asparagine-binding sugar addition. Such cells are preferably cultured in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol supplemented in the medium at a concentration between 0.1M and 1.5M, preferably at a concentration of 0.5M or 1.0M. Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free medium. The choice of medium and culture conditions suitable for the particular cell line used is within the level of one of ordinary skill in the art. The glucagon receptor can also be expressed in non-human-transgenic animals, especially in transgenic warm-blooded animals. Methods for producing transgenic animals, including mice, rabbits, sheep and pigs, are known in the art and are described, for example, in Hammer et al (Nature 315: 680-683,1985), Palmiter et al. (Science). 222: 809-814,1983), Brinster et al. (Proc.Natl.Acad.Sci.USA 82: 4438-4442,1985), Palmiter and Brinster (Cell 41: 343-345,1985) and US Pat. No. 4,736,866. Disclosed by issue (incorporated herein by reference). Simply put, an expression unit containing a DNA sequence to be expressed along with an appropriately arranged expression regulatory sequence is introduced into the pronucleus of a fertilized egg. DNA introduction is generally done by microinjection. Incorporation of injected DNA is detected by blot analysis of DNA from tissue samples, typically tail tissue samples. It is generally preferred that the introduced DNA be incorporated into the animal's reproductive system so that it can be passed on to the animal's offspring. In a preferred embodiment of the invention, transgenic animals such as mice are developed by targeting mutations to disrupt the glucagon receptor sequence (Mansour et al., "Disruption of the". protooncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes, "nature 336: 348-352,1988). Can be easily used as a model for. Glucagon Receptor Peptide As mentioned earlier, the invention also provides a glucagon receptor peptide. In the context of the present invention, the glucagon receptor peptide is understood to include the previously discussed portion of the glucagon receptor or a derivative thereof that does not contain the transmembran domain and is at least 10 amino acids in length. .. Simply put, the structure of the glucagon receptor as well as the putative transmembran domain can be constructed, for example, using the hydrophobic plot function of the P / C Gene or Intelligenetics Suite (Interigenetics, Mt.View, CA) or Kyte and. It can be predicted from its primary translation product according to the method described by Doolittle (J. Mol. Biol. 157: 105-132, 1982). A hydrophobic plot of the rat glucagon receptor is shown in FIG. Based on this hydrophobic analysis, the glucagon receptor is believed to have the general structure shown in FIG. 1, although it does not want to be bound by the chart. In particular, these receptors are believed to contain one extracellular amino-terminal domain, three extracellular loop domains and four intracellular loop domains, each separated by a transmembran domain. There is. In one aspect of the invention, an isolated glucagon receptor peptide comprising an extracellular amino-terminal domain of a glucagon receptor is provided. In a preferred embodiment, an isolated glucagon receptor peptide comprising the amino acid sequence of SEQ ID NO: 15, glutamine, amino acid number 28 to tyrosine, amino acid number 142 is provided. Also provided are other isolated glucagon receptor peptides that can be selected from the extracellular and intracellular loop domains of the glucagon receptor (see FIGS. 1 and 5). In one embodiment, the glucagon receptor peptide is 1ID (SEQ ID NO: 15, lysine, amino acid number 169 to histidine, amino acid number 178), 1ELD (SEQ ID NO: 15, tyrosine, amino acid number 203, isoleucine, amino acid. Number 231), 2ID (SEQ ID NO: 15, phenylalanine, amino acid number 259 to serine, amino acid number 266), 2ELD (SEQ ID NO: 15, valine, amino acid number 293 to isoleucine, amino acid number 307), 3ID (sequence It is selected from the group consisting of number 15, leucine, amino acid number 334 to lysine, amino acid number 345), and 3ELD (SEQ ID NO: 15, aspartic acid, amino acid number 371 to serine, amino acid number 380). The glucagon receptor peptide of the present invention can be produced using the recombinant techniques discussed above or by synthetic methods and can be further purified as described below. Purification of glucagon receptor peptide The isolated glucagon receptor peptide can be produced, among other things, by culturing a suitable host / vector system to produce the recombinant translation product of the invention. The supernatant from such cell lines can then be treated by various purification procedures for isolating the glucagon receptor peptide. For example, the supernatant can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. After concentration, the concentrate can be applied to a suitable purification matrix, eg, glucagon or anti-glucagon antibody bound to a suitable support. Alternatively, an anion or cation exchange resin can be used to purify its receptor or peptide. Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps can be used to further purify the glucagon receptor peptide. Glucagon receptor peptides are considered "isolated" or purified in the context of the present invention if only one band is detected after SDS-polyacrylamide gel analysis followed by staining with Coomassie Brilliant Blue. Antibodies to glucagon receptors In one embodiment of the invention, an antibody that specifically binds a derivative thereof and a portion or fragment of a protein thereof, eg, a glucagon receptor comprising the glucagon receptor peptide previously discussed, to the glucagon receptor. Can be used to prepare. In the context of the present invention, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F (ab')._２And Fab fragments, as well as recombinantly made binding partners. These binding partners incorporate variable regions from genes encoding specifically binding monoclonal antibodies. Antibodies, they are 10 to the glucagon receptor⁷M^-1Above K_aIt is defined as specifically binding when binding with. The affinity of a monoclonal antibody or binding partner can be readily measured by one of ordinary skill in the art (see Scatchard, Ann. Acad. Sci. 51: 660-672, 1949). Polyclonal antibodies can be readily produced by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats. Simply put, glucagon receptors are used to immunize animals through intraperitoneal, intramuscular, intraocular, or subcutaneous injections. The immunogenicity of the glucagon receptor or glucagon receptor peptide can be increased through an adjuvant, such as Freund's complete or incomplete adjuvant. After some booster immunosensitization, a small sample of serum is taken and tested for its responsiveness to the glucagon receptor. Various assays can be used to detect antibodies that specifically bind to the glucagon receptor. An exemplary test is Antibodies: A Laboratory Manual, Harlow and It is described in detail in Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Typical examples of such assays are: countercurrent immunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), dot blot assay, inhibition or competition assay, and sandwich. Testing (US Pat. Nos. 4,376,110 and 4,486,530; also Antibodies: A Laboratory Manual, see above). A particularly preferred polyclonal antiserum will give a signal at least 3 times greater than the background. Once the animal's titer reaches a plateau with respect to its responsiveness to the glucagon receptor, large amounts of polyclonal antisera can be readily obtained by weekly bleeding or total blood sampling of the animal. Monoclonal antibodies can also be easily produced using well-known techniques (US Pat. Nos. RE32,011, 4,902,614, 4,543,439, and 4,411,993; and Monoclonal. Antibodies, Hybridomas: A New Dimension in Biological Analyses, Pleneum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988) See. ). Briefly, in one embodiment, a test animal, eg, a rat or mouse, is injected with a form of glucagon receptor suitable for producing an immune response against the glucagon receptor. Representative examples of suitable forms include, among other things, cells expressing the glucagon receptor, or a peptide based on the glucagon receptor. In addition, the resulting immune response, eg, its receptor or receptor peptide, can be another protein, egg white albumin or Keyhole. Many techniques are known for increasing by binding with limpet hemocyanin (LLH) or through the use of an adjuvant, such as Freund's complete or incomplete adjuvant. The first immune sensitization can be through the peritoneal, intramuscular, intraocular, or subcutaneous pathways. Animals can be re-immunized by other booster immunizations after 1-3 weeks of the first immunization. Animals are then tested for blood draw and their sera tested for binding to the glucagon receptor using a test as described above. Additional immunosensitization can be performed until the animal becomes a plateau in its responsiveness to the glucagon receptor. The animal can then be given a final boost of glucagon receptor or glucagon receptor peptide and killed after 3-4 days. At this time, the spleen and lymph nodes are harvested and the organs are passed through a mesh screen, or the spleen and lymph node membranes that enclose the cells are disrupted to destroy a single cell suspension. In one embodiment, the erythrocyte cells are then lysed by the addition of a hypotonic solution followed by a return to the hypertonic solution. In another embodiment, cells suitable for the preparation of monoclonal antibodies are acquired through the use of immunosensitization techniques in vitro. Simply put, the animal is killed and its spleen and lymph node cells are removed as described above. A single cell suspension is prepared and the cells are placed in a culture containing the form of a glucagon receptor suitable for producing an immune response as described above. The lymphocytes are then harvested and fused as described below. Cells obtained through the use of immunosensitization in vitro as described above or from immunosensitized animals can be immortalized by transfection with a virus, such as the Epstein-Barr virus (EBV) (Epstein-Barr virus (EBV)). Glasky and Reading, Hybridoma 8 (4): 377-389,1989). Alternatively, in a preferred embodiment, the harvested spleen and / or lymph node cells are fused with suitable myeloma cells to produce a "hybridoma" that secretes a monoclonal antibody. Suitable myeloma strains are preferably defective in the construction or expression of antibodies and are further syngeneic with cells from their immunosensitized animals. Numerous such myeloma cell lines are well known in the art and sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (Catalogue of Cell Lines &amp; Hybridomas, 6th ed., ATCC, 1988). Can be obtained from). Typical myelomas are: for humans, 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-Ag14 (ATCC No.CRL 1581) and P3X64Ag8 (ATCC) No.TIB 18); and for rats, including Y3-Ag1.2.3 (ATCC No.CRL 1631) and YB2 / 0 (ATCC No.CRL 1662). Particularly preferred fusion systems are NS-1 (ATCC No. TIB 18) and P3X63-Ag8.653 (ATCC No. CRL 1580), which are used for fusion with either mouse, rat, or human cell lines. Including those that can. Fusion between cells from the Immieroma cell line and immunosensitizers can be described by polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988) or electro. It can be done by a variety of methods, including fusion (see Zimmerman and Vienken, J. Membrane Biol. 67: 165-182, 1982). After the fusion, the cells are placed in a suitable medium, eg RPMI 1640 or DMEM (Dulbecco's Modified Eagles). Place in a culture plate containing Medium) (JRH Biosciences, Lenexa, Kan.). This medium comes from additional components, such as Fetal Bovine Serum (FBS, ie from Hyclone, Logan, Utah, or JRH Biosciences), baby animals of the same species used for immunosensitization. It can also contain harvested thymocytes, or agar to solidify its medium. In addition, the medium must contain reagents that selectively allow the growth of fused spleen and myeloma cells. Particularly preferred is the use of HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Missouri). After about 7 days, the resulting fusion cells or hybridomas can be screened to determine the presence of antibodies that recognize the glucagon receptor. After dilution and retesting of some clones, hybridomas that produce antibodies that bind to the glucagon receptor can be isolated. Other techniques can also be used to construct monoclonal antibodies (William D. Huse et al., "Generation of a Large Combinational Library of the Immunogloblin Repertoire in Phage Lambda," Science 246: 1275-1281, December. See also 1989; L. Sastry et al. "Cloning the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad. Sci. USA 86: 5728 See 5732, August 1989; and Michelle Alting Mees et al., Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas, "Strategies in Molecular Biology 3: 1-9, january 1990; these references are available from Stratacyte, La Jolla, Clifornia, which enable antibody production through recombinant technology. Describes a commercial system.). Briefly, mRNA is isolated from a B cell population and a heavy and light chain immunoglobulin cDNA expression library is created within the λIMMUNOZAP (H) and λIMMUNOZAP (L) vectors. These vectors can be screened individually or co-expressed to make Fab fragments or antibodies (Huse et al., Supra; and Sastry et al., Supra). ). Positive plaques can then be converted to non-lytic plasmids that allow high levels of expression of monoclonal antibody fragments from E. coli. Similarly, binding partners can be constructed using recombinant DNA technology to incorporate the variable region of the gene encoding the specific binding antibody. Construction of these proteins can be facilitated by those skilled in the art (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; Richemann et al., "Reshaping Human Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Enginerring," Nature 328: 731-734,1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity, "Science 239: 15341-536, 1988; Chaudhary et al.," A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin, "Nature 339: 394-397, 1989; US Pat. No. 5,132,405, entitled "Biosynthetic Antibody Binding Sites"). These disclosures are provided herein. Simply put, in one embodiment, the Glucagon-Receptor-specific binding domain is encoded. DNA segments are amplified from hybridomas that produce specifically binding monoclonal antibodies and inserted directly into the genome of cells that produce human antibodies (Verhoeyen et al., See supra; also Reichmann et al.). al., see above). This technique allows the antigen binding site of a specifically binding mouse or rat monoclonal antibody to be transferred into a human antibody. Such antibodies are preferably for therapeutic use in humans. This is because they do not have the same antigenicity as rat or mouse antibodies. Alternatively, the antigen binding site (variable region) can either be bound to or inserted into another completely different protein (Chaudhary et al., See supra) and is an antibody. It results in new proteins with antigen-binding sites and completely different protein functional activities. As will be appreciated by those skilled in the art, the antigen binding site or glucagon receptor binding domain of an antibody can be within the variable region of the antibody. In addition, DNA sequences encoding smaller moieties or variable regions of the antibody that specifically bind to the mammalian glucagon receptor can also be used in the context of the present invention. These moieties can be readily tested for specific binding to the glucagon receptor using the tests described below. In a preferred embodiment, the gene encoding the variable region from the hybridoma producing the monoclonal antibody in question can be amplified using oligonucleotide primers for that variable region. These primers can be synthesized by one of ordinary skill in the art or purchased from commercially available sources. Stratacyte (La Jolla, Calif.), Above all, V_Ha, V_Hb, V_Hc, V_Hd, C_H1, V_LAnd C_LWe sell primers for mouse and human variable regions that contain primers for the region. These primers can be used to amplify heavy or light chain variable regions, which are then inserted into vectors, such as IMMUNOZAP * (H) or IMMUNOZAP * (L) (Stratacyte), respectively. be able to. These vectors can then be introduced into E. coli for expression. Using these technologies, V_HAnd V_LLarge numbers of single-stranded proteins, including domain fusions, can be produced (see Bird et al., Science 242: 423-426, 1988). In other embodiments, the binding partner is fused within its expression vector to another protein, such as a toxin. Cells bound by this binding partner can therefore be killed by uptake of its toxin (Chaudhary et al., See supra). Once suitable antibodies or binding partners have been obtained, they can be isolated or purified by many techniques well known to those of skill in the art (Antibodies: A Laboratory Manual, see supra). Suitable techniques include purification on peptide or protein affinity columns, HPLC or RP-HPLC, protein A or protein G columns, or a combination of any of these techniques. In the context of the present invention, when used to define an antibody or binding partner, the term "isolated" means "substantially free of other blood components". The antibody and binding partner according to the present invention have many uses. For example, the antibody can be used in flow cytometry to separate glucagon receptor-bearing cells or to histologically stain glucagon receptor-bearing tissue. Simply put, to detect a glucagon receptor on a cell, the cell can be incubated with a labeled monoclonal antibody that specifically binds to the glucagon receptor, followed by detection of the presence of the bound antibody. it can. These steps can also be performed by additional steps, eg, washing to remove unbound antibody. Labels suitable for use in the present invention are well known in the art, among others, fluorescein isothiocyanate (FITC), phycoerythrin (PE), phos radish peroxidase (HRP), and collidal gold. )including. Particularly preferred for use in flow cytometry is FITC, which is the "Conjugation of Fluorescein Isothiocyanate to". Antibodies.I.Experiments on the Conditions of Conjugation, "Immunology 18: 865-873, 1970 can be bound to purified antibody according to the method of Keltkamp. (Also, Keltkamp," Conjugation of Fluorescein Isothicyanate to Antibodies, II. .A Reproducible Methods, "Immunology 18: 875-881, 1970; and Goding," Conjugation of Antibodies with Fluorochromes: Modification to the Strandard Methods, "J.Immunol.Methods 13: 215-226, 1970.) The preferred histological staining, HRP, can be combined with the purified antibody according to the method of Nakane and Kawaoi ("Peroxidase-Labeled Antibody: A New method of". Conjugation, "J.Histochem.Cytochem.22: 1084-1091,1974; and Tijssen and Kurstak," Highly Efficient and Simple Methods for Preparation of Peroxidase and Active Peroxidase Antibody Conjugates for Enzyme Immunoassays, "Anal.Biochem.136: 451- See 457,1984). In addition, purified antibodies or binding partners can be used therapeutically to block the binding of glucagon to the glucagon receptor in vitro or in vivo. Simply put, a blocking antibody is an antibody that binds to a glucagon receptor epitope in such a way as to prevent glucagon from binding to its receptor, or to prevent its glucagon from affecting signal conversion. .. As mentioned earlier, various assays can be used to detect antibodies that block or inhibit the binding of glucagon to the glucagon receptor, and these are, among other things, inhibition as described above. And competing tests. In one embodiment, the monoclonal antibody (prepared as described above) is tested for binding to the glucagon receptor in the absence of glucagon and in the presence of variable concentrations of glucagon. A blocking antibody or binding partner is identified, for example, as one that binds to a glucagon receptor and, in the presence of glucagon, blocks or inhibits the binding of glucagon to that glucagon receptor. An antibody or binding partner to be used therapeutically is preferably provided in a therapeutic composition comprising the antibody or binding partner and a physiologically acceptable carrier or diluent. Suitable carriers or diluents include, among other things, neutral buffered saline or saline, and additional excipients or stabilizers such as buffers, sugars such as glucose, sucrose, or dextranase. Includes chelating agents such as EDTA, as well as various preservatives. Glucagon antagonist As mentioned earlier, the present invention provides a method for detecting glucagon antagonists. In the context of the present invention, an antagonist is understood to mean a molecule that is capable of binding to a receptor but does not stimulate or diminishes the intracellular response pathway. In particular, glucagon antagonists are generally identified by their ability to bind to glucagon receptors and thereby reduce stimulation of intracellular response pathways. In one aspect of the invention, (a) to a recombinant glucagon receptacle bound to a response pathway and associated response through that pathway for conditions and time sufficient to allow the compound to bind to the receptacle. The compound is exposed in the presence of the glucagon agonist, and (b) a decrease in the stimulation of the response pathway resulting from the binding of the compound to the glucagon receptor as compared to the stimulation of the response pathway by the glucagon agonist. A stepwise method of detecting and then detecting the presence of a glucagon antagonist is provided for detecting the presence of a glucagon antagonist. In the context of the present invention, glucagon antagonists include molecules that can bind to glucagon receptors (including glucagon itself) and stimulate intracellular response pathways. Various compounds can be screened using such a method. Representative examples include blocking antibodies, glucagon receptor peptides, and glucagon analogs (including both peptide and non-peptide ligands) as discussed above. US Sequential Number 07 / 741,931 provides, for example, a way to create a large number of glucagon analogs using a pool of DNA sequences encoding such analogs. Such pools of Glucagon-encoding DNA sequences are saturated mutagenesis of Glucagon-encoding DNA sequences (eg, Little, Gene 88: 113-115, 1990; Hambers et al., Gene). 88: 143-151,1989) by segment-designated mutagenesis (eg, Shortle et al., Proc.Natl.Acad.Sci.USA 77: 5375-5379,1980) by forced nucleotide erroneous uptake (eg, eg). Liao and Wise Gene 88: 107-111,1990) or by the use of random mutagenesis oligonucleotides (Hutchison et al., Proc. Natl. Acad.Sci.USA.USA 83: 710-714,1986). it can. Individual transformants expressing the glucagon analog can be cloned or pooled as previously discussed. The compound is exposed to the recombinant glucagon receptor bound to the response pathway and the relevant response through that pathway in the presence of the glucagon agonist for conditions and time that allow binding of the compound to the receptor. When used in the present invention, the conditions and time sufficient for the glucagon antagonist to bind to its receptor will vary depending on the source of the receptor, but suitable conditions for its binding are generally defined. Is between 0 and 2M NaCl, preferably between 0 and 0.9M NaCl, particularly preferably 0.1M. It occurs in a buffer solution in NaCl between 4 ° C. and 55 ° C. and within the pH range between 5 and 9, preferably between 6.8 and 8. Sufficient time for binding and response will generally be between 5 and 15 minutes after exposure. Once a compound is exposed to a recombinant glucagon receptor in the presence of a glucagon agonist under conditions and for a period of time sufficient for the compound to bind to its receptor, the reduction in stimulation of its response pathway, the compound. Can be detected if the recombinant glucagon receptor competes with the glucagon agonist. In one embodiment of the invention, the response pathway is a membrane-bound adenylate cyclase response pathway, and this detection step is that membrane binding compared to cyclic AMP production in the presence of glucagon agonist alone. Includes measurement of reduction in cAMP production by the adenylate cyclase response pathway. For the purposes of the present invention, the reduction in stimulation of its response pathway is death-His.¹-Preferably a compound equal to or greater than the decrease associated with glucagon. Adenylate cyclase activity assay can be performed, for example, by Lin et al. (Biochem. 14: 1559-1563, 1975) and using the methods described in this example. These methods measure the level of stimulation of cAMP against native glucagon and, in general, radiolabel a preparation of cells expressing a biologically active recombinant glucagon receptor. In the presence of ATP, it involves exposure to a mixture of glucagon and the test compound. Alternatively, cAMP production is a well-known method in the art, eg, Salomon et al. (Anal. Biochem. 58: 541-548,1976) or Krishna et al. (J. Pharmacol. Exp. Ther). .163: 379,1968), or preferably a commercially available kit, eg, Scintillation Proximity Assay from Amersham Corporation. It can also be easily measured using the Kit. This Scintillation Proximity Assay Kit measures the production of cAMP by competing for iodination-cAMP with anti-cAMP antibodies. A particularly preferred glucagon receptor is an ED of less than 1 nM.₅₀(Effective dose for 50% response), more preferably less than 0.7 nM ED₅₀By, and most preferably less than 0.25 nM ED₅₀Has biological activity in the above assay. In a further aspect of the invention, the response pathway comprises a luciferase reporter system. Simply put, luciferase is an enzyme that catalyzes the release of photons by luciferin, and therefore its expression in the presence of luciferin can be easily detected (Alam and). Cook, Anal. Biochem. 188: 245-254, 1990). As described in more detail below, in a particularly preferred embodiment of the invention, a cyclic AMP response element, such as a proenkephalin cyclic AMP response element, is bound in a state capable of acting on the luciferase cDNA. A DNA construct comprising one is provided. A DNA construct containing luciferase cDNA is stably transfected into a host cell. The host cell is then transfected with a second DNA construct containing the first DNA segment encoding the glucagon receptor operably linked to the additional DNA segment required for expression of that receptor. During binding of the glucagon receptor agonist, elevated cAMP levels induce luciferase expression. The luciferase is exposed to luciferin, and the photons released during the oxidation of luciferin by the luciferase are measured. In another aspect of the invention, activation of the response pathway results in an increase in the intracellular concentration of free calcium. Various tests can be performed to measure the concentration of intracellular calcium, which are described, for example, by Charest et al. (J. Biol. Chem. 259: 8769-8773, 1983). Includes the QuinZ method or the aequorin photoprotein method described by Nakajima-Shimada (Proc.Natl.Acad.Sci.USA 88: 6878-6882,1991). A particularly preferred method is the intracellular calcium photoimaging method, which will be described in more detail in Example 6. Briefly, in one embodiment, cells are transformed with a glucagon receptor expression plasmid and grown under normal culture conditions for 3 days. This growth medium is then removed and replaced with a solution containing 10 μM fura-2AM (Grynkiewicz et. See al., J. Biol. Chem. 260: 3440-3450, 1985). The cells are then incubated in the dark for 30 minutes, then rinsed and further incubated for 30-120 minutes. Optical image formation can be performed by a Nikon Diaphot inverted fluorescence microscope equipped with a mercury discharge lamp. Cells can first be monitored for at least 60 seconds to establish a baseline and then stimulated with a glucagon-containing buffer. Images are typically recorded for at least 3 minutes after stimulation. Software, such as Inovision (Research Triangle Park, NC), can be used to process and quantify the image. The glucagon antagonists detected as discussed above are described by, for example, Coy et al. (Peptides Structure and Function, Pierce Chemical Company, Rockford IL., Pp. 369-372, 1983) for ion exchange and partition chromatography. By graphics, reverse phase chromatography (Andreu and It can be purified by Merrifield.Eur.J.Biochem.164: 585-590,1987) or by HPLC (eg, Kofod et al., Int.J.Peptide Protein Res.32: 436-440,1988). .. Additional purification can be achieved by conventional chemical purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis. Methods of protein purification are known in the art (generally see Scopes, R., Protein Purification, Springer-Verlag, NY, 1982) and the recombination described herein. It can be applied to the purification of glucagon antagonists. Alternatively, glucagon antagonists (in The Peptides, vol.2A, Gross and Meienhofer, eds., Academic Press, NY, pp.1-284, 1979) by the solid phase method of Barany and Merrifield, or by automated peptide synthesizers. Can be synthesized by using. Substantially pure recombinant glucagon antagonists with at least about 50% homogeneity are preferred, at least about 70% -80% homogeneity is more preferred, and at least 95% -99%, especially for pharmaceutical applications. The above homogeneity is most preferable. Once purified, or to the desired homogeneity, the glucagon antagonist can be used therapeutically. In general, the antagonist can be administered parenterally or by infusion. Typically, this antagonist is present as a free warm group or acid salt. Suitable salts must be pharmaceutically acceptable. Representative examples include metal salts, alkaline and alkaline earth metal salts, such as potassium or sodium salts. Other pharmaceutically acceptable salts include citric acid, succinic acid, lactic acid, hydrochloride and hydrobromide. The pharmaceutical composition can be formulated in an aqueous isotonic solution with a pH between 5.6 and 7.4. Suitable isotonic solutions can include sodium chloride, dextrose, sodium tetratartrate borate, and polyethylene glycol solutions. A therapeutic dose of the antagonist can be administered simultaneously with insulin, either in the same composition or in separate compositions. Diagnostic use of glucagon receptor probe In another aspect of the invention, probes and primers are provided to detect the glucagon receptor. In one aspect of the invention, a probe capable of hybridizing to glucagon receptor DNA or RNA is provided. For the purposes of the present invention, probes can "hybridize" to glucagon receptors when they hybridize under high or low stringency conditions (Sambrook et al., See supra). .). Preferably, the probe is used to hybridize to a suitable nucleotide sequence in the presence of 6x SCC, 1x Denhardt's (Sambrook et al., Supra), 0.1% SDS at 65 ° C., and 2x SCC at 65 ° C. Wash excess probe at least once in the presence of 1x Denhardt's, 0.1% SDS. The probe sequence is preferably designed to allow hybridization to the glucagon / receptor sequence, but not to hybridize to the secretin, calcitonin or parathyroid hormone / receptor sequence. The probes of the invention can consist of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and are as short as about 12 nucleotides in length, typically about 14-18 nucleotides in length, and if possible. For example, it can be as large as the complete array of glucagon receptacles. The choice of probe size depends somewhat on the application of the probe. For example, a probe consisting that actually contains the full length of the glucagon receptor coding sequence is preferred to measure the presence of various polymorphic forms of the glucagon receptor within an individual. Glucagon receptor probes can be used to identify polymorphisms bound to the glucagon receptor gene (eg, Weber, Genomics 7: 524-530, 1990; and Weber and May, Amer.J. See Hum.Gen.44: 388-396,1989.). Such polymorphisms can be associated with genetic disorders, such as diabetes. Probes can be constructed and labeled using techniques well known in the art. For example, shorter probes of 12 bases can be synthetically produced. Longer probes of about 75 bases less than 1.5 kb, preferably labeled precursors, eg³²Produced by PCR amplification in the presence of P-dCTP, digoxigenin-dUTP, or biotin-dATP. Probes larger than 1.5 kb generally transfect cells with a plasmid containing the associated probe, grow the transfected cells to a large extent, and purify the associated sequence from the transfected cells. This is easily amplified (Sambrook et al., See above). The probe can be labeled with a variety of markers, including, for example, radiation markers, fluorescence markers, enzyme markers, and color development markers.³²The use of P is particularly preferred for marking or labeling a particular probe. The probes of the invention can also be used to detect the presence of glucagon receptor mRNA or DNA in a sample. However, if there are only a limited number of glucagon receptors, or if it is desirable to detect selective mutant sequences that are present in only a limited number, or clone glucagon receptors from selected warm-blooded animals. If desired, it can be beneficial to amplify the relevant sequence such that it can be more easily detected or acquired. For example, RNA amplification (Lizardi et al., Bio / Technology 6: 1197-1202, 1988; Kramer et al., Nature 339: 401-402, 1989; Lomeli et al., Clinical See Chem.35 (9): 1826-1831, 1989; U.S. Pat. No. 4,786,600. ) And DNA amplification using the polymerase chain reaction ("PCR") (see, eg, US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159) (also US Pat. Nos. 4,876,187 and 5,011,769, which are fragile bindings. Various methods can be used to amplify the selected sequence, including those described for other detection / amplification systems comprising.). In a particularly preferred embodiment, PCR amplification is used to detect or obtain glucagon receptor DNA. Briefly, DNA samples are denatured at 95 ° C. to produce single-stranded DNA, as described in more detail below. Specific primers as described below are then annealed at 37 ° C. to 70 ° C., depending on the AT / GC ratio within the primers. This primer is extended at 72 ° C. with Taq polymerase to produce strands opposite the template. These steps consist of one cycle, which can be repeated to amplify the selected sequence. Primers for amplification of selected sequences must be selected from sequences that are highly specific and form a stable double strand with their target sequence. This primer is also non-complementary, especially at its 3'end, must not form a dimer with itself or with other primers, and forms a secondary structure or double strand with other regions of DNA. should not be done. In general, primers of about 18-20 nucleotides are preferred and can be readily synthesized using techniques well known in the art. Particularly preferred primers are shown in Table 1 below and include their degenerate oligonucleotides ZC4715 and ZC4701 (SEQ ID NOs: 9 and 8 respectively) and oligonucleotides ZC4758 and ZC4778 (SEQ ID NOs: 10 and 11 respectively). Additional Uses of Glucagon Receptor Nucleotide Sequence In yet another aspect of the invention, a virus that can be used to treat a disease in which the glucagon receptor (or mutant glucagon receptor) is overexpressed or no glucagon receptor is expressed at all. Provide a vector. Simply put, in one embodiment of the invention, a viral vector directed to the production of antisense glucagon receptor RNA to prohibit overexpression of the glucagon receptor or expression of the mutant glucagon receptor. I will provide a. In another aspect, a viral vector directed to the expression of glucagon receptor cDNA is provided. Suitable viral vectors for use in the present invention are, among other things, recombinant vaccinia vectors (US Pat. Nos. 4,603,112 and 4,769,330), recombinant varicella virus vectors (PCT application number WO89 / 01973), and preferably , Recombinant Retroviruses with Amphotropic and Ecoptropic Host Ranges, "PCT Publication No. WO 90/02806;" Retroviral Packaging Cell Lines and Processes of Using Same, "PCT Publication No. WO 89/07150; and" Antisense RNA for Treatment of Retroviral Disease States, "PCT Publication No. WO / 03451 )including. As mentioned earlier, the viral vectors of the invention treat a disease condition in which either the glucagon receptor is overexpressed, the mutant glucagon receptor is expressed, or the glucagon receptor is not expressed at all. Can be used in. The following examples are provided by description and without limitation. Example Example 1 Synthesis of cDNA and preparation of cDNA library A. Rat liver cDNA synthesis Livers from 30 grams of female Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) were removed and immediately placed in liquid nitrogen. Total RNA was prepared from its liver tissue using guanidine isothiocyanate (Chirgwin et al., Biochemistry 18: 52-94, 1979) and CsCl centrifugation. Poly (A)⁺RNA was isolated using oligo d (T) cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-1412, 1972). Double the first strand cDNA poly d (T) -selected liver poly (A)⁺Synthesized from RNA. 10 μg liver poly (A)⁺A solution of 10 microtiters containing RNA was mixed with 20 μl of 20 pmol / μl first strand primer ZC3747 (SEQ ID NO: 7) and 4 μl of diethyl pyrocarbonate-treated water. The mixture was heated at 65 ° C. for 4 minutes and cooled by cold hardening on ice. First Stond cDNA synthesis was performed with 8 μl of 5X SUPERSCRIPT buffer (GIBCO BRL, Gaitthersburg, Md.), 4 μl of 100 mM dithiotratel and 2.0 μl of deoxynucleotide triphosphate solution of 10 mM dATP, dGTP, dTTP and 5 respectively. -Methyl-dCTP (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) was initiated by addition to the RNA-primer mixture. The reaction mixture was incubated for 3 minutes at 42 ° C. After incubation, 6.0 μl of 200 U / μl of SUPER SCRIPT reverse transcriptase (GIBCO BRL) was added. The efficiency of first strand synthesis is 10 μCi to label the reaction product.³²Analysis was performed in a parallel reaction by adding P-αdCTP to a 10 μl aliquot of the reaction solution. The first strand synthetic reaction mixture was incubated for 45 minutes at 45 ° C. and then at 50 ° C. for 15 minutes. The reaction was terminated by the addition of water to a final volume of 100 μl, followed by two phenol / chloroform (1: 1) extractions and one chloroform / isoamyl alcohol (24: 1) extraction. Non-incorporated nucleotides were removed from each reaction by precipitating the cDNA twice in the presence of 6 μg glycogen carrier, 2.5 M ammonium acetate and 2.5 volumes of ethanol. This unlabeled cDNA was resuspended in 50 μl of water and used for second strand synthesis. The length of the first strand cDNA was evaluated by resuspending the labeled cDNA in 20 μl of water and measuring its cDNA size by agarose gel electrophoresis. Second-strand synthesis was performed on the RNA-DNA hybrid from the first-strand synthesis reaction under conditions that promoted first-strand priming of the second-strand synthesis resulting in a DNA hairpin format. 20.0 μl of the reaction mixture in 5X polymerase I buffer (100 mM Tris, pH 7.4,500 mM KCl, 25 mM MgCl)₂, 50mM (NH₄）₂SO₄, 4.0 μl 100 mM dithiotateol, 1.0 μl solution containing 10 mM deoxynucleotide triphosphate, 3.0 μl β-NAD, 15.0 μl 3 U / μl E. coli DNA ligase ( NBL Enzymes Ltd., Cramlington, Northumbria, England), 5.0 μl of 10 U / μl E. coli DNA polymerase I (GIBCO BRL)) and 50.0 μl of unlabeled first strand DNA. 10 μl alicot of second strand synthesis is 10 μCi³²A parallel reaction labeled with the addition of P-αdCTP was used to monitor the efficiency of second strand synthesis. The reaction mixture was incubated at room temperature for 4 minutes, after which 1.5 μl of 2 U / μl RNase H (GBCO BRL) was added to each reaction mixture. The reaction was incubated at 15 ° C. for 2 hours and then at room temperature for 15 minutes. Each of these reactions was terminated with 4 μl of 500 mM EDTA, followed by phenol / chloroform and chloroform / isoamyl alcohol extraction in sequence as described above. DNA from each reaction was precipitated in the presence of ethanol and 2.5 ammonium acetate. DNA from the unlabeled reactant was resuspended in 50 μl of water. The labeled DNA was resuspended as described above and electrophoresed. Single-stranded DNA in the hairpin structure was cleaved using mung bean nuclease. This reaction mixture was prepared in 10 μl of 10X Mung Bean Nuclease Buffer (Stratagene Cloning Systems, La). Jolla, Calif.), 4 μl of 200 mM dithiothreitol, 34 μl of water, 50 μl of second strand cDNA and 2 μl of Mung Bean nuclease (Promega Corp., Madison. Wis.) In Stratagene MB dilution buffer (Stratagene Cloning Systems). ) 1:10 Containing dilution. The reaction was incubated at 37 ° C. for 15 minutes, and the reaction was terminated by the addition of 20 μl of Tris-HCl, pH 8.0, followed by sequential phenol / chloroform and chloroform / isoamyl alcohol extraction as described above. went. After extraction, the DNA was precipitated in ethanol and resuspended in water. The resuspended cDNA was resuspended in 192 μl of water and 50 μl of 5X T4 DNA polymerase buffer (250 mM Tris-HCl, pH 8.0, 250 mM KCl, 25 mM MgCl).₂), 3 μl of 100 mM dithiothreitol, 3 μl of solution containing 10 mM of each deoxynucleotide triphosphate, and 2 μl of 6.7 U / μl of T4 DNA polymerase (Pharmacia LKB Biotechnology Inc.). After incubation at 15 ° C. for 30 minutes, the reaction was terminated by the addition of 2 μl of 500 mM EDTA, followed by phenol / chloroform and chloroform / isoamyl alcohol extraction in sequence as described above. The DNA was ethanol precipitated and resuspended in 30 μl of water.³²Based on the uptake of P-dCTP, the cDNA yield was estimated to be 4 μg from a 10 μg starting mRNA template. B. Preparation of rat liver cDNA library An Eco RI adapter (Invitrogen, San Diego, Calif.) Was added to the previously prepared cDNA to facilitate cloning of the cDNA into a mammalian expression vector. A 10 μl alicot of this cDNA and an 800 pmol adapter (12 μl) to 4.0 μl of 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; Stratagene). Mixed with Cloning Systems). The reaction was incubated for 16 hours in a temperature gradient of 4 ° C to 15 ° C. This reaction was performed with 185 μl of water and 25 μl of REACT 2 buffer (GIBCO). It was terminated by the addition of BRL) and then incubated at 65 ° C. for 30-60 minutes. After incubation, the reaction was subjected to phenol / chloroform extraction, then chloroform / isoamyl alcohol extraction, and ethanol precipitation as described above. After centrifugation, the DNA pellet was washed with 70% ethanol and air dried. The pellet was resuspended in 180 μl of water. To facilitate directional insertion of the cDNA into the mammalian expression vector, the cDNA was digested with Xho I to yield a cDNA with a 5'Eco RI attachment and a 3'Xho I attachment. The Xho I restriction site at the 3'end of this cDNA was introduced through the ZC3747 primer (SEQ ID NO: 7). Restriction digestion was terminated by sequential phenol / chloroform and chloroform / isoamyl alcohol extraction. The cDNA was ethanol precipitated and the resulting pellet was resuspended in 1X loading buffer (10 mM phosphate buffer, pH 8.8, 5% glycerol, 0.125% bromphenol blue). Resuspended cDNA is heated to 65 ° C. for 10 minutes, cooled on ice, and 0.9% low melting point using BRL 1 kb ladder (GIBCO BRL) and Pharmacia 100 base pair ladder (Pharmacia LKB Biotechnology Inc.) as size markers. It was electrophoresed on an agarose gel (Seaplaque GTG Low Melt Agarose, FMC Corp., Rockland, Me.). Contamination adapters and by-product fragments less than 800 base pairs in size were excised from the gel. The electrodes were reversed and the cDNA was electrophoresed until concentrated near its lane origin. The area of the gel containing the concentrated DNA was excised, placed in a microfuge tube, and the approximate volume of the gel slice was measured. An aliquot of TE equal to half the volume of the gel slice was added to the tube and the agarose was melted by heating to 65 ° C. for 15 minutes. After equilibrating the sample to 42 ° C, approximately 5 units of β-Agarase I (New England) Biolabs, Beverly, Mass) was added. The sample was incubated for 90 minutes to digest the agarose. After incubation, 0.1x volume of 3M sodium acetate was added to the sample and the mixture was incubated on ice for 15 minutes. After incubation, the sample was centrifuged at 14,000 xg for 15 minutes at 4 ° C. to remove undigested agarose. The cDNA in the supernatant was precipitated with ethanol. The cDNA pellet was washed with 70% ethanol, air dried and resuspended in 10 μl of water. The resulting cDNA was cloned into a derivative of pCDNA1 (Invitorogen) in which the E. coli vector pZCEP, its M13 origin of replication and its SupF selectable marker were replaced by a pUC18-derived beta-lactamase cassette. The plasmid pZCEP linearized by digestion with Eco RI and Xho I was ligated with Eco RI-Xho I cDNA. The obtained plasmid was used as E. coli strain DH10B ELECTROMAX cells (GIBCO). Electropolated into BRL). C. Synthesis of human islet cell cDNA Island cells were isolated from human pancreas obtained from organ transplant donors for which no matching receptor was available. After in-situ perfusion with cold UW solution (Du Pont, Boston, Mass), each pancreas was carefully excised, cannulated into the pancreatic duct, and 4 mg / ml collagenase solution (type V, Sigma, St). .Louis, MO.) Was injected at a constant rate, first at 4 ° C and then at 39 ° C. The glands were torn, and the released fragments were washed by centrifugation, ground through a reduced inner diameter needle, and purified by intermittent Ficoll density centrifugation (Warnock, Diabetes). 35 (Suppl.1): 136-139,1989). Materials harvested from the upper interface were pooled and counted after measurement of island purity by dithiazone staining. The islands used in the library construction exceeded 65% purity, while the islands used in Northern blots exceeded 40% purity. The average island diameter was 175 μm. In addition, the isolated islands exhibited both first and second insulin secretory function after perfusion with either high glucose or isobutylmethylxanthin (IBMX). Poly (A)⁺RNA was isolated using the FASTTRACK mRNA isolation kit (Invitrogen) according to the manufacturer's instructions. Simply put, 30,000 purified islands are rapidly dissolved in a lysis buffer, homogenized using a reduced inner diameter needle, and digested in the presence of proteinase K and RNasin, then poly (A).⁺RNA was selected by oligo-d (T) cellulose chromatography. Set the concentration and purity of the eluted fraction to 0D_260/280Measured in. Approximately 2.5 μg of poly (A) from human islands⁺RNA was used for cDNA library construction using the LIBRARIAN II cDNA Library Construction System (Invitrogen) and DH10B ELECTROMAX E. coli (E. coli) cells (GIBCO BRL) according to the manufacturer's instructions. In summary, about 2.5 μg of poly (A) isolated from human islands.⁺RNA was converted to double-stranded cDNA, followed by the addition of Bst XI non-palindrome linker (Invitrogen). The cDNA was size fractionated and the unreacted linker was removed by agarose gel electrophoresis and electroelution. Complementary DNA strands larger than 600 base pairs were selected. Example 2 Isolation of rat glucagon receptor cDNA by polymerase chain reaction amplification Rat hepatic cDNA was used as a template for amplification of glucagon receptor sequences using degenerate oligonucleotides (ZC4715 and ZC4701; SEQ ID NOs: 8 and 9, respectively) corresponding to highly conserved regions within the members of the SEECLETIN gene family. did. 5 ng template cDNA (Example 1A); 100 pmol of each oligonucleotide ZC4715 (SEQ ID NO: 9) and ZC4701 (SEQ ID NO: 8); 0.25 mM of each deoxynucleotide triphosphate (Cetus, Emeryville, CA); 1x Promega 10x buffer (Promega Corp.); and 50 μl of reaction containing 1.25 units of Taq polymerase (Promega). The PCR sequences were run for 40 cycles (1 minute at 95 ° C, 1 minute at 42 ° C, and 2 minutes at 72 ° C) and then incubated at 72 ° C for 7 minutes. A 650 base pair PCR product was isolated by gel electrophoresis and ligated by pCR1000 (Stratagene Cloning Systems). The resulting plasmid E. coli XL-1 cells were used to transform. Plasmid DNA was prepared from selected transformed cells, named G13 / pCR1000, and sequenced (SEQ ID NO: 14). Sequence analysis of this clone showed that the insert encoded the polypeptide associated with the secretin receptor. Example 3 Cloning of full-length rat glucagon receptor cDNA Full-length rat glucagon receptor cDNA was obtained by screening the library described in Example 1B in the glucagon binding assay. This labyrinth was plated to obtain one million independent clones. 10 ml of LB-Amp (Sambrook et. Al, above) scraped during. The cells were precipitated by centrifugation and the medium was discarded. Cell pellets were resuspended in 4 ml LB-Amp, 15% glycerol, and 4 1 ml aliquots were stored at 80 ° C. The first glycerol stock was titrated and 100 pools of 5000 colonies were plated per plate. After growing the colonies, each plate was scraped into 10 ml LB-amp. Aliquots of cells from each pool were removed for use in the preparation of plasmid DNA. The remaining cell mixture was brought to a final concentration of 15% glycerol, divided equally and frozen at -80 ° C. Plasmid DNA was prepared from each pool of cells and the DNA was digested with RNAse (Boehringer Mannheim, Indianapolis, Ind.) According to the manufacturer's instructions. This RNAse reaction was terminated by extraction with phenol / chloroform / isoamyl alcohol (24: 24: 1) and the DNA was ethanol precipitated. The plasmid DNA from each pool is transfected into COS-7 cells (ATCC CRL 1651), and the transfected form is transfected.¹²⁵The presence of the glucagon receptor was screened by the I-glucagon binding test. In summary, about 2x10 one day before transfection⁵COS-7 cells were plated on sterile single chamber slides (Nunc AS, Roskilde, Denmark) coated with 10 μg / ml human fibronectin (Table 1) for 30 minutes at room temperature and phosphate buffered saline. Washed with water (PBS, Sigma Chemical Co., St. Louis, Mo.). 2 μg of plasmid DNA from each pool is incorporated herein by reference essentially by McMahan et al. (EMBO J. 10: 2821-2832, 1991; (incorporated herein by reference in its entirety). )) Was used to transfect cells cultured on individual chamber slides. After transfection, cells are 5% CO₂The cells were cultured at medium 37 ° C. for 72 hours.<img file="000002.tif" id="000002" he="050" wi="151" img-format="tif" img-content="drawing" /> The lyophilized powder is dissolved in the buffer solution. Ammonium sulphate is then added to a concentration of 25% and the solution is allowed to settle at 4 ° C. for 2 hours. Fibronectin is pelleted by centrifugation in a Bench Top centrifuge (Beckman Instruments, Inc., Irvine, Calif.) At 1,000 rpm for 15 minutes. Discard the supernatant and pour the pellet into 10 ml NaPO₄Dissolve in buffer solution (above). 1 liter of fibronectin in a final volume of 16.9 ml (described above) NaPO₄Dialyze against the buffer solution overnight. 1 mM NaPO on dialyzed material₄Dilute 3-fold with pH 7.4, 1 mM NaPO₄Make a solution of pH 7.4,100 mM NaCl. Fibronectin is then diluted 2-fold with distilled water. Remove the thread-like insoluble precipitate with a glass rod. Fibronectin is subjected to FPLC across a 50 ml DEAE FF Sepharose column (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) equilibrated with 3 volumes of 10 mM Tris, pH 8.1, 50 mM NaCl. The column is washed with 10 mM Tris, pH 8.1, 50 mM NaCl until a baseline sample is generated, after which fibronectin is eluted with a salt gradient up to 10 mM Tris, pH 8.1, 300 mM NaCl. Fractions are collected, aliquots of the fractions are electrophoresed on an acrylamide gel, and the gel is analyzed by Coomasie Blue staining and Western analysis. Pool peak fractions and 10 mM CAPS (3- (cyclohexylamine) -1 propane-sulfonic acid, Sigma), pH 11.0, 10 mM CaCl₂, Dialysis against 150 mM NaCl. Store this solution at -80 ° C. ¹²⁵To prepare transfectants for the I-glucagon binding assay, consumption medium was aspirated from the cells and the cells were washed 3 times with cold (4 ° C.) PBS. After the final wash, cells were covered with binding medium (Table 2) and incubated for 10 minutes at room temperature. Medium, 0.5 ml, 0.5 nM¹²⁵It was replaced with a binding medium containing I-glucagon (Amersham receptor grade, specific activity 2000 Ci / mM; Amersham). The cells were then shaken at 30 ° C. for 1 hour. Medium was aspirated from the cells, glucagon-free cold (4 ° C.) bound medium was added, and the cells were incubated at room temperature for 5 minutes. The medium was aspirated from the cells and the cells were washed 3 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 rinsed 3 times with PBS. The slides were air dried at room temperature for 1 hour, soaked in a liquid photographic emulsion (eastman Kodak Co., Rochester, NY) according to the manufacturer's instructions, and dried at room temperature in the dark for at least 30 minutes. Cells capable of binding glucagon were detected at 2.5X magnification under brightfield illumination. One pool, # 57, was identified as containing cells capable of binding glucagon.<img file="000003.tif" id="000003" he="080" wi="100" img-format="tif" img-content="drawing" />1M sodium bicarbonate 8.4 grams of solid NaCO₃ Sodium bicarbonate was poured into a 100 ml stoppered prefiller and 80 ml of distilled water was added. Mix the solution until the solid dissolves. Add up to 100 ml of distilled water. The solution is mixed again and stored in a stoppered bottle at 4 ° C. Culture medium Add 1 milliliter of 1M sodium bicarbonate per liter of distilled water. 4 liters are either prepared with the above solution, which has been pre-cooled overnight, or with chilled distilled water. 69% sucrose solution 69 grams of sucrose Dissolve sucrose in 31 ml of distilled water while heating. The concentration of this solution is measured with a refractometer. Solid sucrose is added as appropriate to adjust the concentration to 69 +/- 0.5%. 42.3% sucrose 42 grams of sucrose Dissolve sucrose in 57 ml of distilled water while heating. The concentration of this solution is measured with a refractometer. Add 69% sucrose solution or water as appropriate to adjust the concentration to 42.3 +/- 1%. 2x bond buffer 100mM HEPES, pH 7.3 300 mM NaCl 2 mM EDTA 2% bovine serum albumin 1.6mg / ml bacitracin Image formation buffer 140 mM NaCl 10mM HEPES 5.6 mM glucose 5mM KCl 1mM DDL₄1mM CaCl₂Fura-2 AM solution 50mg fura-2 AM (Molecular Probes) 50ml DMSO 5 ml image formation buffer Dissolve 50 mg of fura-2 AM in 50 ml of DMSO. After the solid has dissolved, the solution is mixed with 5 ml of imaging buffer. Aliquots of plasmid DNA from the # 57 pool were subjected to PCR amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID NOS: 8 and 9, respectively). # 57 plasmid DNA between 200 ng and 400 ng from pool; 100 p mol of each oligonucleotide ZC4701 and ZC4715 (SEQ ID NOS: 8 and 9 respectively); 50 mM KCl; 10 mM Tris-HCl, pH 9.0 (20 ° C); 1.5 mM MgCl₂A 50 μl reaction mixture was prepared containing 0.01% gelatin; 0.1% Triton X-100; 0.2 mM of each deoxynucleotide triphosphate (Pharmacia LKB Biotechnology Inc) and 1 unit of Taq polymerase (Promega). The PCR reaction was run for 30 cycles (2 minutes at 95 ° C, 2 minutes at 45 ° C, and 2 minutes at 72 ° C) and then incubated at 72 ° C for 7 minutes. The reaction mixture was stored at 4 ° C. Analysis of the PCR product by gel electrophoresis showed the presence of a 700 base pair band, which was about the same size as the product described in Example 2. Glycerol stock from pool # 57 was titrated and 20 plates of 500 colonies were plated. Colonies were pooled and glycerol stock and plasmid DNA were prepared as described above. The plasmid DNA was transfected into COS-7 cells and the transfectants were screened using the glucagon binding assay as described above. One pool, # 57-18, was identified as containing cells capable of binding glucagon. Alicots of plasmid DNA from pool # 57-18 were subjected to PCR amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID NOs: 8 and 9, respectively) as described above. Analysis of the PCR product by gel electrophoresis showed the presence of a 700 base pair band, confirming the presence of the glucagon receptor DNA sequence. Glycerol stock from pool # 57-18 was titrated and 6 plates of 500 colonies and 47 plates of 20 colonies were plated. Colonies were pooled and glycerol stock and plasmid DNA were prepared as described above. Alicots from each pool of plasmid DNA were transfected into COS-7 cells and the transfectants were screened using the glucagon binding test as described above. In addition, aliquots from each pool of plasmid DNA were subjected to PCR amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID NOs: 8 and 9, respectively) as described above. Four positive pools (# 57-18-16, # 57-18-18, # 57-18-36 and # 57-18-48) were identified as containing cells capable of binding glucagon, and PCR amplification showed that it contained a definitive 700 base pair band. To isolate this cDNA, two 150 mm plates were plated with 2,500 colonies in the # 57-18 pool, respectively. Hanahan and filter lift Prepared using the methods essentially described by Meselson (Gene 10: 63, 1980) and Sambrook et al. (Ibid.), Which are incorporated herein by reference in their entirety. Hybridization probes were obtained by PCR amplification of plasmid DNA from the # 57 pool using oligonucleotides and methods as described above. The PCR product was gel-purified from a low melting point agarose gel and randomly-primed using the MEGAPRIME kit (Amersham, Arlington Heights, Ill.) According to the manufacturer's instructions. Filter 6x SCC, 5x Denhardt's, 5% SDS, 200 μg / ml sonicated salmon sperm DNA and 2x10⁵cpm / ml³²Hybridized in solution containing P-labeled PCR fragments. The filters were hybridized overnight at 65 ° C. Excess labeling was removed by 3 washes at 65 ° C. with 2x SCC, 1% SDS. The filter was exposed to film by two screens at 80 ° C. for 4 hours. Positive clones containing the plasmid pLJ4 were identified and screened. The plasmid pLJ4 was deposited by American Type Culture Coollection (12301 Parklawn Dr., Rockville, MD 20852) on August 21, 1992 as a transformant for E. coli under deposit number 69056. Restricted endonuclease analysis and sequence analysis showed that pLJ4 contained an insert of approximately 2 kb encoding a 485 amino acid protein with an expected molecular weight of 54,962 daltons. This nucleic acid sequence and deductive amino acid sequence are shown in SEQ ID NOs: 14 and 15. Kyte and Hydrotherapy analysis using Doolittle's method (J. Mol. Biol. 157: 105-132,1982; (which is incorporated herein by reference) corresponds to the amino-terminal signal sequence. Eight clusters of hydrophobic amino acids and seven transmembran domains were revealed (Fig. 2). In addition, analysis of this degenerative amino acid sequence revealed the addition of four potential N-linked sugars located within the elongated hydrophilic sequence. The presence of the site and the presence of 6 cysteines within the same region were shown. Example 4 Isolation of human glucagon receptor cDNA by polymerase chain reaction amplification Human islet cell cDNA (Example 1C) was used as a template for amplification of human glucagon receptor sequences using degenerate oligonucleotides ZC4715 and ZC4701 (SEQ ID NOs: 8 and 9, respectively). 5 ng template cDNA (Example 1C); 100 p mol of each oligonucleotide ZC4715 (SEQ ID NO: 9) and ZC4701 (SEQ ID NO: 8); 0.25 mM of each deoxynucleotide triphosphate (Cetus, Emeryville, Calif.); 1x Promega 50 μl of reaction was set up containing 10x buffer (Promega Corp.); and 1.25 units of Taq polymerase (Promega). The PCR reaction was run for 40 cycles (1 minute at 95 ° C, 1 minute at 42 ° C, and 2 minutes at 72 ° C) and then incubated at 72 ° C for 7 minutes. About 750 base pairs of PCR product was isolated by gel electrophoresis. One tenth of the isolated PCR product was used as a template for other PCR reactions using oligonucleotides ZC4758 and ZC4778 (SEQ ID NOs: 10 and 11). It was designed to insert a BamHI restriction site at the 3'end and an Eco RI restriction site at the 5'end of the PCR product for subcloning. 50 μl of the reaction mixture was set as described above. The PCR reaction was run for 40 cycles (1 minute at 95 ° C, 1 minute at 50 ° C, and 1.5 minutes at 72 ° C) and then incubated at 72 ° C for 7 minutes. To screen the transformants for the human glucagon receptor sequence, the inserted DNA present in each transformant was used as a generic pUC sequencing primer for oligonucleotides ZC447 and ZC976 (with SEQ ID NO: 1, respectively). Amplified using 2) and primed to the pUC sequence flanking the PCR-generated insert. Forty-eight of transformants, 20 pmoles of each oligonucleotide; 0.125 mM of each deoxynucleotide triphosphate (Cetus, Emeryville, Calif.); 1x Promega 10x buffer (Promega); and 1.25 units of Taq polymerase (Promega). ), Each of which was picked up in 25 μl of the reaction mixture. The PCR reaction was run for 30 cycles (1 minute at 95 ° C, 1 minute at 45 ° C, and 1.5 minutes at 72 ° C) and then incubated at 72 ° C for 7 minutes. This PCR product was then labeled with random priming using the Amersham MEGAPRIME kit (Amersham) as a probe with a 1.9 kb Eco R of pLJ4. Southern Hallation using I-Xho I fragments (Southern, J. Mol. Biol. 98: 503, 1975; and Sambrook et al, ibid .; (these are incorporated herein by reference as a whole). )). One clone, G30, was shown to hybridize to a full-length rat cDNA probe. The nucleotide sequence of G30 is shown in SEQ ID NO: 16. Example 5 Cloning of full-length human glucagon cDNA Oligonucleotide primers ZC5433 and ZC5432 (SEQ ID NOS: respectively) designed to contain sequences from clone G30 as discussed above to identify libraries containing sequences encoding human glucagon receptors. A series of primers was screened by PCR using 13 and 12). Purchased and prepared human liver, islet cells, HepG2 cells, human genome from brain and placenta and cDNA libraries were screened (Table 3). Each 50 μl PCR reaction was set up using DNA from each library in the volumes listed in Table 4. 20 pmoles of ZC5433 and ZC5432 (SEQ ID NOs: 13 and 12 respectively), 0.25 mM of each deoxynucleotide triphosphate, 5 μl of 10x Taq I buffer (Promega), 15 mM MgCl.₂, 19.5 μl distilled water and 0.5 μl 5 U / μl Taq I polymerase (Promega). In addition, the reactants were set to include pLJ4 as a positive control and no DNA as a negative control.<img file="000004.tif" id="000004" he="145" wi="153" img-format="tif" img-content="drawing" /><img file="000005.tif" id="000005" he="130" wi="155" img-format="tif" img-content="drawing" /> The PCR reaction was run for 30 cycles (1 minute at 94 ° C, 1 minute at 50 ° C, and 1.5 minutes at 72 ° C) and then incubated at 72 ° C for 10 minutes. The PCR product was then analyzed by agarose gel electrophoresis. Only the NIH liver library produced a band of 320-410 base pairs of size equal to that seen with the pLJ4 positive control. A human liver library cloned into plasmid pcD2 (Chen and Okayama, Mol.Cell.Biol. 7: 2745-2752, 1987) from Dr. Roger Bertolloti (National Institute of Health, Bethesda, Md.) , Used to obtain full-length cDNA clones encoding the human glucagon plasmid. This library was plated to obtain 1 million independent clones. 10 ml of transformant colonies from each plate LB-Amp (Sambrook et Al., ibid.) Scraped during. The cells were precipitated by centrifugation and the medium was discarded. Cell pellets were resuspended in 4 ml LB-Amp, 15% glycerol, and 4 1 ml alicots were stored at 80 ° C. The first glycerol stock was titrated and 100 pools of 5000 colonies were plated per plate. After growing the colonies, each plate was scraped into 10 ml LB-amp. Aliquots of cells from each pool were removed for use in the preparation of plasmid DNA. The remaining cell mixture was brought to a final concentration of 15% glycerol, divided equally and frozen at -80 ° C. Plasmid DNA was prepared from each pool of cells and the DNA was digested with RNAse (Boehringer Mannheim, Indianapolis, In.) According to the manufacturer's instructions. This RNAse reaction was terminated by extraction with phenol / chloroform / isoamyl alcohol (24: 24: 1) and the DNA was precipitated with ethanol. Allicots of resuspended plasmid DNA from each pool were combined into groups of 10 (ie, 1-10, 11-20, 21-30, 31-40, etc.). The plasmid DNA was diluted 1:20 and 1 μl of DNA from each pool was used in the same PCR reaction mixture as the reaction mixture described above. The reaction mixture was subjected to amplification under the conditions described above. Analysis of the PCR product by agarose gel electrophoresis showed that pool 31-40 contained a band of 320-410 base pairs of the same size as the positive control. The plasmid DNA prepared from the original pools 31-40 was diluted 1:20 and 1 μl of each pool was used in the same reaction mixture as described above. PCR amplification of the reaction mixture was performed using the conditions described above. Analysis of the PCR product by agarose gel electrophoresis showed that pool # 40 gave a band size of approximately 310-420 base pairs. The concentration of plasmid DNA was approximately 70 ng / μl by 1 μl agarose gel electrophoresis of pool # 40 diluted at 1:10. 70 ng of pool # 40 plasmid DNA was electroporated into E. coli strain DH10B at 2.3 kV, 400 Ω, 25 μF and resuspended in 1 ml SOC (Sambrook et al., Ibid.). 10 dilutes of 3 cells^-2、10^-3And 10^-4Prepared in. Each 100 μl dilution was plated on 4 plates. Number of colonies, 10^-2About 10,000 colonies per plate for 4 plates containing cells from the dilution, and 10^-3It was estimated that there were about 1000 colonies per plate for the four plates containing cells from the dilution. Two filter lifts, four 10^-3From each of the plates, and 10^-2Prepared from one of the plates and named pools # 1 to pool # 5. One filter from each double set was laid on solid medium and grown until colonies were formed. The colonies were then scraped and used to prepare plasmid DNA for PCR amplification. In addition, 10^-2The three remaining plates of the plate were scraped and plasmid DNA was prepared from the cells. The remaining filter was prewashed with 3x SSC + 0.5% SDS at 65 ° C. with stirring for 12 hours. The filter was then prehybridized in Ulllrich's Buffer (Ulllrich, EMBO J.3: 361-364,1984) + 50% formaldehyde, 1% SDS overnight at 37 ° C. Boil the labeled clone G30 DNA using the Amersham MEGAPRIME kit (Amersham) according to the conditions suggested by the manufacturer, and 6x10⁵It was added to the hybridization solution (Ullrich's Buffer + 50% formamide) to the final concentration of cpm / ml. After overnight incubation, the probe solution was removed and the filter was washed in 2x SSC + 0.1% SDS at 65 ° C. for 5 minutes. After the first wash, the filters were washed in the same solution for 15 minutes at 65 ° C. and then at room temperature for 5 minutes with shaking. The final wash protocol was repeated two more times. The filter was exposed to the filter overnight at room temperature. Single colonies on plate # 2 corresponding to pool # 2 were positive by hybridization to G30. This colony was picked up and smeared to obtain an independent colony. Plasmid DNA prepared from several independent colonies was subjected to DNA sequence analysis. One clone 40-2-2 was subjected to further sequence analysis, and on August 21, 1992, as an E. coli transformant under deposit number 69055, the American Type Culture Collection (12301 Parklawn Dr., Rockville,). MD 20852) deposited. The partial DNA sequence of clone 40-2-2 and its deductive amino acid sequence are shown as SEQ ID NOs: 17 and 18. To confirm the presence of the glucagon receptor sequence in filter hybridization, the PCR amplification sequence was subjected to each plasmid DNA preparation (plasmid DNA prepared from each double filter and three 10s.^-2It was performed on plasmid DNA) prepared from each of the plates. Each pooled plasmid DNA was diluted 1:20 and 1 μl of each DNA was used as a template in PCR settings and runs as described above. Agarose gel electrophoresis of the resulting PCR product showed that 3 of the 4 pools of pool # 2 and 10,000 contained PCR generation bands for between 310-420 base pairs. The presence of the PCR-generated band in pool # 2 corresponding to plate # 2 confirmed the presence of the glucagon receptor DNA sequence. F. Strategy for cloning 5'sequence of human glucagon receptor Analysis of the partial cDNA sequence of clone 40-2-2 and the full-length rat glucagon receptacle cDNA sequence showed that the 40-2-2 clone lacked the amino-terminal sequence corresponding to about 25 amino acids. .. This 5'human glucagon receptor cDNA sequence was obtained using the application of the method described by Frohman et al. (Proc. Natl. Acad. Sci. USA 85: 8988-9002, 1988). Simply put, oligonucleotide primers were designed so that their sequences hybridize to sequences near the 5'end of the 40-2-2 coding sequence. Primers were hybridized to the G-tail human liver first strand cDNA template and the primers were extended towards its 5'end using Taq I DNA polymerase. A second poly d (C) primer was annealed to the G- tail cDNA template, polymerase chain sequence amplification, the were subjected to splicing to the coding sequence present cloning after, in the sequencing and clone 40-2-2 .. Three cDNA templates were prepared. The first template was commercially available human liver first strand cDNA. The second and third cDNA templates are commercially available human liver using either oligonucleotides containing sequences specific for the human glucagon receptor or traditional oligo d (T) priming. Each was prepared by synthesizing the first strand cDNA from mRNA (Clontech). A second cDNA template was prepared by synthesizing from human liver mRNA using oligonucleotide ZC5433 (SEQ ID NO: 13) specific for the human glucagon receptor coding sequence. A reaction mixture containing 2 μl of 1 μg / μl of human liver mRNA, 8 μg / ml of 20 pmol / μl of ZC5433 (SEQ ID NO: 13) and 0.5 μl of 10 mM Tris, pH 7.4, 0.1 mM EDTA was then added at 68 ° C. for 7 minutes. Incubated on ice for 2 minutes. After incubation, the reaction mixture is 4 μl 5X SUPERSCRIPT buffer (GIBCO-BRL), 1 μl of 200 mM dithiothreitol, 1 μl of solution containing 10 mM of each dNTP, 1 μl of 0.25 μCi / μl of α³²Received P-dCTP and 5 μl SUPERSCRIPT reverse transcriptase. After incubation, the reaction was incubated at 45 ° C. for 1 hour. The reaction was terminated by the addition of 80 μl of TE. RNA was hydrolyzed by the addition of 1 μl of 0.5 M EDTA and 1 μl of KOH. The hydrolyzate was incubated at 65 ° C. for 5 minutes. After incubation, the sample was diluted to 1 ml with 50 mM KOH, 0.1 mM EDTA and the sample was passed over a CENTRICON 100 concentrator (Amicon, Danvers, Mass.). The column was washed with 1 ml of 50 mM KOH, 0.1 mM EDTA. Concentrated cDNA was collected and neutralized with half the volume of 100 mM HCl. The neutralized cDNA sample was precipitated with ethanol and the precipitate was resuspended in 26 μl of distilled water. It was prepared by synthesizing human liver mRNA using oligo d (T) primers for which a third cDNA template was purchased. 2 μl of 1 μg / μl human liver mRNA, 1 μl of 1 μg / μl oligo d (T) 18 (New) England Biolabs, Beverly, Mass.) And 5 μl of 10 mM Tris, pH 7.4, 0.1 mM EDTA. This cDNA synthesis was performed using the conditions described for the synthesis described above. The first strand cDNA was then G-tailed. 4 μl of human liver first strand cDNA (QUICK CLONE; Clontech, Palo Alto, Calif.), 4 μl of human liver first strand cDNA from ZC5433 (SEQ ID NO: 13) or oligo d (T) primer in the reaction tube. Human liver first strand cDNA was set to contain. Each reaction mixture receives 22 μl of water, 8 μl of 5X buffer (Promega), 4 μl of 10 mM dGTP and 2 μl of 15 U / μl of terminal transferase, and the reaction is incubated at 37 ° C. for 30 minutes, followed by Incubated at 65 ° C. for 10 minutes. The reaction was then diluted to 90 μl with 10 mM Tris, pH 7.4, 1 mM EDTA and ethanol precipitated. Second strand synthesis of all G-tail cDNAs was performed identically. Each cDNA was first suspended in 50 μl of distilled water. Each cDNA then received 100 pmoles of ZC4814 (SEQ ID NO: 20) in 5 μl. The cDNA was annealed by heating each mixture to 68 ° C. for 5 minutes and then incubating on ice for 2 minutes. After annealing the primers to the cDNA, each mixture is 20 μl of 5x Polymerase I buffer (Example 1), 1 μl of 100 mM dithiothreitol, 2 μl of solution containing 10 mM of each dNTP, 2 μl of 0.5 μCi / μl α.³²We received 1 μl of 3U / μl E. coli DNA ligase (New England Biolabs) and 5 μl of 7 U / μl E. coli DNA polymerase I (Amersham). The reaction was incubated at 22 ° C. for 5 minutes, then 1.5 μl of 2 U / μl of RNase H (GIBCO-BRL) was added, after which the incubation was restarted at 16 ° C. for 2 hours. Reaction with 200 μl of 10 mM Tris (pH 8.0), 1 mM EDTA was then terminated by the addition of 150 μl of saturated water phenol and 150 μl of chloroform. The mixture was spun and then centrifuged at 22 ° C. for 3 minutes for phase separation. The aqueous phase was removed and re-extracted with phenol and chloroform as described above. After the second phenol-chloroform extraction, the aqueous phase was extracted with chloroform. The cDNA in the aqueous phase is precipitated by the addition of 5 μg of mussel glycogen, 100 μl of 8M ammonium acetate and 300 μl of isopropanol, and the larger nucleic acid is selectively precipitated while leaving unincorporated oligonucleotides in the supernatant. It was. The cDNA was pelleted by centrifugation and the pellet was washed with 70% ethanol and then air dried. The cDNA pellet was resuspended in 15 μl of double-distilled water. Oligonucleotide primers containing a restricted endonucleosis site homologous to the 5'end of ZC4814 (SEQ ID NO: 20) and facilitating subcloning in a 5-parallel reaction of double-stranded cDNA, as well as a 40-2-2 clone. Amplified using oligonucleotide primers specific for the very 5'end of the coding sequence present within. Each reaction mixture is 5 μl of 10X Taq I buffer, 3 μl of 25 mM MgCl.₂, 5 μl solution containing 2.5 mM each dNTP, 1 μl double-stranded cDNA, and 0.5 μl Taq I polymerase (Promega). The reaction was denatured at 98 ° C. for 5 minutes prior to the addition of 1 μl each of 10 pmol / μl ZC5624 (SEQ ID NO: 21) and 20 pmol / μl ZC4812 (SEQ ID NO: 19). Each sample was layered with 70 μl of mineral oil held at 90 ° C. The reaction was amplified for 30 cycles (95 ° C. for 60 seconds, 57 ° C. for 40 seconds, 72 ° C. for 60 seconds) and then extended at 72 ° C. for 7 minutes. Each PCR product was subjected to agarose gel electrophoresis, and each amplified fragment was excised and subcloned into pCR1000 using a TA cloning kit (Invitrogen). Oligonucleotide primers ZC5624 (SEQ ID NO: 21) and ZC4812 (SEQ ID NO: 19) in an independent PCR reaction mixture containing three clones from each ligation, each containing an inoculum from the clone as a source of template DNA. Was selected and analyzed for the presence of inserts by using. This PCR reaction was performed as described above. However, only 30 cyclos were amplified. Single inserts from each original PCR reaction-positive clones were subjected to DNA sequence analysis. Of the two clones shown to contain the error-free 5'human glucagon receptor coding sequence, one clone was selected to provide the 5'human glucagon receptor coding sequence. Clone 9A Eco RI and Kpn Digestion with I gave a 551 base pair fragment containing the 5'coding sequence of the glucagon receptor. A 3'glucagon receptor coding sequence was obtained as a 561 base pair Kpn I-Bam HI fragment from 40-2-2. The Eco RI and Kpn I fragments and the 561 base pair Kpn I-Bam HI fragment were ligated in the presence of Eco RI and Bam HI to prevent concatenation. The product of ligation, the 1112 base pair fragment, was gel purified and named 9A1. Conveniently, fragment 9A1 was ligated into Eco RI and Bam HI digestion pUC18. The ligation mixture was transformed into E. coli strain DH10b cells and selected clones were analyzed for the presence of inserts. One clone 9A11 contained the insert. The glucagon receptor coding sequence was constructed in a mammalian expression vector using plasmid p9A11 and clone 40-2-2 to give the complete glucagon receptor coding sequence. Plasmid p9A11 to Pvu II and Bam H Digested with I, 967 base pair fragments were isolated. Clone 40-2-2 was digested with Bam HI and Sac I to isolate an 828 base pair fragment containing the 3'human glucagon receptor coding sequence. The mammalian expression vector pHZ1 was linearized by digestion with Eco RI, and its ends were then blunted using T4 DNA polymerase. This blunt-terminated linear vector was then digested with Sac I. A 967 base pair of Pvu II-Bam HI fragment and an 828 base pair of Bam HI-Sac I fragment were ligated with a blunt-Sac I digested pHZ1 vector. The plasmid pHZ1 is an expression vector that can be used to express proteins from mRNA transcribed in vitro in mammalian cells or in the frog oocyte translation system. This pHZ1 expression system is a mouse metallothionein-1 promoter, a bacteriophage T7 promoter adjacent to a multicloning bank containing a unique restriction site for insertion of coding sequences, a human growth hormone terminator and a bacteriophage T7. Includes a terminator. In addition, pHZ1 contains an E. coli replication origin; a bacterial beta-lactamase gene; an SV40 promoter and origin, a neomycin resistance gene and a mammalian selection marker expression unit comprising an SV40 transcription terminator. The pHZ1 plasmid containing the glucagon receptor cDNA sequence in the correct direction for the promoter was named pLJ6'. The insert-vector junction was sequenced to confirm the presence of the correct sequence. The plasmid pLJ6'was placed under Deposit No. 69183 on January 15, 1993, American Type. Deposited by the Culture Collection (12301 ParkLawn Drive, Rockville, MD 20852). The DNA sequence and deductive amino acid sequence of the glucagon receptor cDNA present in pLJ6'are shown in SEQ ID NO: 24 and SEQ ID NO: 25. Example 6 Cloning of glucagon receptor cDNA from human islet cells In addition to cloning the glucagon receptor from human hepatocytes, glucagon receptor cDNA was obtained from the human islet cell library. Alicot of the human island cell cDNA library (Example 1C) is an oligonucleotide ZC5763 (SEQ ID NO: 1C), which is a sense oligonucleotide containing an Eco RI site flanking a sequence from the 5'untranslated sequence of human glucagon receptacle cDNA. 22) and Xho flanking the sequence from the 3'untranslated sequence of human glucagon receptacle cDNA It was subjected to PCR amplification using oligonucleotide ZC5849 (SEQ ID NO: 23), which is an antisense oligonucleotide containing the I site. Including a restriction site within this oligonucleotide primer facilitates directional cloning of the resulting PCR product into a suitable plasmid vector. PCR reaction mixture of 4 μl island cell cDNA library (Example 1C), 8 μl 10X Promega PCR Buffer (Promega), 20 pmol ZC5763 (SEQ ID NO: 22), 20 p mol ZC5849 (SEQ ID NO: 23), 20 mM. It was set to contain 1 μl of solution containing each deoxyribonucleotide and 46.5 μl of water. The mixture was heated to 95 ° C. for 3 minutes and then the heat was reduced to 80 ° C. for 3 minutes. The mixture was kept at 80 ° C until ready for use. 2 μl of 10X Promega PCR Buffer (Promega), 2 μl of 5 U / μl Taq to initiate the reaction A 20 μl enzyme mixture consisting of I polymerase (Cetus) and 16 μl water was added to the reaction mixture. The reaction was layered with 50 μl of mineral oil (Sigma) and the reaction was carried out for 30 cycles (95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 2 minutes and 15 seconds, where 72 ° C incubation was performed 3 times per cycle. It was subjected to a second increase), followed by a 10-minute incubation at 72 ° C. After the final 72 ° C. incubation, the reaction was kept at 4 ° C. 10 μl agarose gel electrophoresis of this PCR product demonstrated the presence of an approximately 1.8-1.9 kb fragment. Based on the human glucagon receptor cDNA present in pLJ6', a fragment of approximately 1846 base pairs was predicted. The PCR reaction product was extracted with chloroform, followed by phenol: chloroform extraction and the final chloroform extraction. After the final extraction, 5 μl of 4 μg / μl glycogen carrier (Boerhinger Mannheim) Corporation) was added and the mixture was precipitated in the presence of ammonium acetate and ethanol. The DNA was pelleted by centrifugation and the pellet was washed with 70% ethanol. The DNA pellet was reconstituted in water and digested with Xho I and Eco RI. The plasmid pBLUESCRIPT SK, which was gel-purified DNA and linearized with Xho I and Eco RI⁺Subcloned into (Stratagene Cloning Systems). The ligation mixture was transformed into E. coli strain DH10B cells (GIBCO-BRL). Plasmid DNA was prepared from selected transformants and the DNA was subjected to restriction enzyme and Southern blot analysis. These clones were compared to human glucagon receptor cDNA inserts present within pLJ6'. Selected clones were subjected to sequence analysis based on diagnostic restriction enzyme digestion. Sequence analysis showed that one clone, pSLIGR-1, contained the glucagon receptor coding sequence. The coding region of pSLIGR-1 contained a 3 nucleotide change within the glucagon receptor coding region when compared to the liver cDNA present within pLJ6'. One of these nucleotide changes was a silent mutation and the other two resulted in conservative amino acid changes as shown in Table 5. Analysis of these changes suggests that they could be the result of polymorphic differences that present allelic variation.<img file="000006.tif" id="000006" he="055" wi="153" img-format="tif" img-content="drawing" />Example 7 Cloning of human glucagon receptor gene To obtain human glucagon receptor genomic clones, two libraries of genomic DNA were screened using human glucagon receptor cDNA as a probe. Amplified human λFIX II Caucasian male placental genome library and amplified human lung λFIX genome library (both libraries were obtained from Stratagene Cloning Systems; Catalog numbers 946203 and 944201, respectively) were obtained from humans. Screened on the Glucagon receptor gene. Titrate the amplified human lung genomic library, and about 4x10⁴Plaque-forming units (pfu) were plated with E. coli strain LE392 cells (Stratagene Cloning Systems) on each of the 150 mm diameter plates. An additional 10 plates with E. coli strain LE392 cells and approximately 6x10 per plate⁴Plated with pfu. These plates were subjected to overnight incubation at 37 ° C. and 30 plates were selected for screening. Dual filters were prepared for each of the 30 plates. Each filter was prepared by layering plates with HYBOND nylon membrane 8 Amersham) according to the procedure recommended by the manufacturer. The filter was lifted from the plate and cells were lysed in 1.5M NaCl, 0.5M NaOH for 5 minutes at room temperature. The filter was neutralized in 1M Tris-HCl (pH 7.5), 1.5M NaCl for 5 minutes and the filter was fixed with 1200 μjoules of UV energy in STRATALINKER (Stratagene Cloning Systems). After pre-washing, the filter is filtered through a 0.45 μm filter, and just before use, a pre-hybridization solution (5x SSC, 5x Denhardt's solution, 0.2% SDS, 1 mM) supplemented with heat-denatured salmon sperm DNA at a final concentration of 100 μg / ml. Divided into 6 batches out of 10 filters prehybridized in EDTA). These filters were hybridized overnight at 65 ° C. Human glucagon receptor cDNA from p40-2-2 was randomly-primed using the Amersham MEGAPRIME kit (Amersham) using the method recommended by the manufacturer. 28.5x10 prehybridization solution from each batch of filters⁶It was replaced with a new prehybridization solution containing a cpm probe. These filters were hybridized at 65 ° C. for 24 hours. After hybridization, the hybridization solutions were removed and these filters were rinsed 4 or 5 times each at room temperature in a wash solution containing 0.25 x SSC, 0.2% SDS, 1 mM EDTA. After rinsing, these filters were washed in 8 consecutive washes at 65 ° C. in the wash solution, followed by a final wash at 70 ° C. After this 70 ° C. wash, these filters were exposed to autoradiograph film (XAR-5; Eastman Kodak Co .; Rochester, NY) at 70 ° C. for 4 days with a sensitizing screen. Examination of the autoradiograph revealed the present of four regions of hybridization with radiolabeled probes. Agar plugs from each of these four regions were picked up for purification. Each agar plug is 1 ml SM (Maniatis et al., Eds., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982; (incorporated herein by reference)), immersed in 1% chloroform overnight. After overnight incubation, phage from each plug was diluted to 1: 1,000 SM. 5, 25 and 50 μl aliquots were plated with E. coli strain LE392 cells. These plates were incubated and a single lift was prepared from the plates from 5 and 25 μl plating. These filters were prepared as described above, prehybridized and washed. These filters were exposed on an autoradiograph film. Autoradiograph examination revealed positively labeled regions from each of the above four clones. A total of 10 agar plugs were picked up from positive regions that presented at least two positive regions for each of their original clones. Each agar plug was treated as described above. Phage from each plug was diluted 1: 10,000 in SM. 2.5 and 10 μl aliquots were plated with E. coli strain LE392 cells. These plates were incubated and a single lift was prepared from plates from plates with preferably separated plaques. The autoradiographs of these filters showed areas of exposure corresponding to separate plaques. Twelve positive plaques presenting at least one clone from each of the above four original positives were picked up. One plaque from each plate was selected for further analysis. Agar plugs from phage clones 2-2-1, 3-1-1, 14-2-1 and 11-2-1 were treated as described above. Phage were diluted 1: 1000 in SM and inoculated into the culture of E. coli strain LE392 cells. Double-stranded DNA was prepared as essentially described by Grossberger (Nuc. Acids Res. 15: 6737,1987; (which is incorporated herein by reference in its entirety)). This double-stranded DNA was digested with Xba I to release its genomic insert. Agarose gel electrophoresis included clones 2-2-1 and 11-2-1 containing 9 kb Xba I inserts, clones 14-2-1 containing 15 kb inserts, and 3-3-1. It proved to contain a 13 kb insert. Southern blot analysis of Xba I digestion and Xba I-Bam HI digestion clones showed that the human glucagon receptor cDNA hybridized to the fragments shown in Table 6.<img file="000007.tif" id="000007" he="070" wi="153" img-format="tif" img-content="drawing" /> Clones 11-2-1 and 14-2-1 were selected for further analysis. Clone 2-2-1 appeared to be identical to Kron 11-2-1 and was not subjected to further analysis. For convenience, the clone name has been changed to be reflected in Table 6. Double-stranded DNA was prepared from each phage clone for subcloning into a plasmid vector using the method essentially described by Maniatis. This DNA was digested with Xba I, gel purified, and linearized by Xba I digestion and treated with bovine alkaline phosphatase to prevent recyclization pBLUESCRIPT SK⁺Subcloned into (Stratagene Cloning Systems). The ligation mixture was electroporated into ELECTROMAX DH10B cells (GIBCO-BRL) in BioRad GENEPULSER (Bio-Rad Laboratories; Richmond, CA) at 400 ohms, 25 μfarads and 2.3 kvolts. Plasmid DNA was prepared from selected transformants. The clones containing the genomic inserts from clones 6 and 2 were named pSLHGR6 and pSLHGR2, respectively. The clones pSLHGR6 and pSLHGR2 were sequenced. Sequence analysis and comparison with the coding region of the human glucagon receptor cDNA revealed the presence of 12 exons containing this coding region. Chromosome position analysis by Dunam et As essentially described by al. (Mol.Cell.Biol.8: 1863-1867,1988 (collectively incorporated herein by reference)), biotinylated human glucagon. It was performed on a metaphase chromosome spread prepared using a receptacle gene probe and a chromosome 17-specific centromere probe. Chromosome degeneration, hybridization and monochromatic detection by Pinkel et al. (Proc. Natl. Acad. Sci. USA 83: 2934-2938,1986 (collectively incorporated herein by reference)). Performed as essentially described and modified by Kievits et al. (Cytogenet. Cell Genet. 53: 134-136, 1990, which is incorporated herein by reference in its entirety). did. However, hybridization is performed in 65% (vol / vol) formamide / 10% dextran sulfate / 2x SCC, and post-hybridization washing is performed by Palmiter et al. (Proc. Natl. Acad. Sci. USA). To 65% formamide / 2x SSC at 42 ° C and subsequently to 0.1x SSC at 55 ° C as described by 89: 6333-6337,1992 (collectively incorporated herein by reference). It was good. q²⁵The location was hybridized several times as described by Testa et al. (Cytogenet. Cell Genet. 60: 247-249,1992, which is incorporated herein by reference in its entirety). The medium-term spread was confirmed by DAPI staining. This DAPI staining produced a Q-band-like pattern. The amplified human placental genomic library (Stratagene) was essentially screened for the glucagon receptor gene using the method described above. Simply put, the library is titrated, and E. coli strain LE392 cells and about 5x10⁴pfu was plated on each of the 30 150 mm plates. In addition, 11 150 mm plates were plated with approximately 105 pfu and E. coli strain LE392 cells, respectively. These plates were incubated overnight at 37 ° C. and 38 plates were selected for screening. Nylon filter lifts were prepared, washed and prehybridized as described above. The human island glucagon receptor cDNA fragment, G30 (Example 4), was randomly-primed using the Amersham MEGAPRIME kit (Amersham) using the method recommended by the manufacturer. 1.1x10 prehybridization solution from each batch of filters⁶It was replaced with a new prehybridization solution containing a cpm probe. The filters were hybridized overnight at 65 ° C. After hybridization, the hybridization solution was removed and the filter was washed in a wash solution containing 0.25 x SSC, 0.25% SDS in 8 consecutive washes at 65 ° C. After washing at 70 ° C., the filter was exposed to an autoradiograph film (XAR-5; Eastman Kodak co.) At 70 ° C. for 4 hours with an intensifying screen. This autoradiograph test did not show any convincing positive signals; however, seven regions corresponding to the weakly labeled regions were picked up for further analysis. The clones were subjected to a second screen as described above, but the autoradiograph on this second screen did not show any labeled areas. We did not track these clones. Example 8 Expression of glucagon receptor cDNA in mammalian cells A. Expression of rat glucagon receptor in BHK570 cells The plasmid pLJ4 was placed in BHK570 cells by plasmid pLJ1 (deposited in the American Type Culture Collection under Deposit No. 10314) by Graham and Van der Eb (Virology 52: 456,1973 (as a whole, by reference herein). Incorporated.)) Simultaneously transfected using the calcium phosphate method as described essentially. The plasmid pLJ1 was derived from the plasmid p416. It is adenovirus 5 ori, SV40 enhancer, adenovirus 2 major late promoter, adenovirus 2 tripartite leader, 5'and 3'splice sites, DHFR^r It contains cDNA, SV40 polyadenelation signal and pML-1 vector sequence (Lusky and Botchan, Nature 293: 79-81, 1981). The Eco RI-Xba I DHFR expression unit from p416 was ligated to pUC18 linearized by digestion with Eco RI and Xba I to construct the plasmid pLJ1. Transfected cells were grown in growth medium (10% fetal bovine serum, 1x PSN antibiotic mixture (GIBCO BRL 600-5640), and Dulbecco's modified Eagle's medium (DMEM) containing 2.0 mM L-glutamine). After a few days in non-selective growth medium, the growth medium was replaced with selective medium (growth medium containing 250 mM methotrexate (MTX)). Cells were separated and diluted 1:20 and 1:50 in selective medium in 10 cm plates. 250mM After selection in MTX for 7-10 days, colonies were picked up into wells of a 24-well plate using a cloning cylinder. The resulting colonies were tested for their ability to bind glucagon as described above (Example 3). Also, glucagon bonds were placed in the wells of the 24-well plate with 2x10 of each clone.⁵This was done for all cells by plating the cells. 5% CO on the plate₂Incubated at 37 ° C for 72 hours. Glucagon binding was performed as described in Example 3. However, after the final PBS wash, cells were removed from the well into the tube by trypsinization. Tubes were counted in a gamma counter. Cells with the highest counts and hence the ability to bind glucagon best were selected for further characterization. The selected transformants were also assayed for a glucagon-mediated cAMP response as described in Example 8D and a glucagon-mediated intracellular calcium response as described in Example 8E. In addition, a glucagon binding test was performed on membrane preparations from transformants as described below. B. Glucagon binding using membrane preparation Membrane from pLJ4 BHK transformant¹²⁵The ability to bind I-glucagon was compared with rat liver membrane preparations. Rodbell et Membrane Prepared as essentially described by al. (J. Biol. Chem. 246: 1861-1871, 1971 (collectively incorporated herein by reference)). Two batches of liver membrane were prepared from 12-16 decapitated juvenile female rats weighing approximately 80 grams each. The liver was quickly removed and placed in a beaker containing ice. The liver was divided into 10 gram portions. These pieces were chopped with scissors and connective tissue was removed during this chopping procedure. These parts were homogenized separately within the Dounce homogenizer. For each portion, 25 ml of medium (Table 2) was added to the chopped tissue in its homogenizer, and the tissue was homogenized in an ice bucket with eight vigorous strokes of a loose pestle. After homogenization, the homogenized product was pooled in 450 ml cold medium (Table 2). The pooled homogen was stirred for 3 minutes and filtered through two layers of cheesecloth and then through four layers of cheesecloth. The homogenized product was then centrifuged at 1500 xg for 30 minutes at 4 ° C. The supernatant was discarded and the pellet was pooled in a clean Dounce homogenizer. The pellet was resuspended with three gentle strokes of the loose pestle. The resuspended pellet was decanted into a 250 ml gradient containing 62 ml of 69% sucrose solution (Table 2). Distilled water was added to a final volume of 110 ml and the mixture was mixed well and kept cool. The concentration of the solution was adjusted to 44.0% +/- 0.1% using 69% sucrose or water as measured by a refractometer (Bausch &amp; Lomb, Rochester, NY). The sucrose suspension was evenly distributed in a 25 x 89 mm ultracentrifugation tube. Each suspension was carefully layered with 20 ml of a 42.3% sucrose solution (Table 2), and the suspension was placed in a SW28 rotor (Sorvall, DuPont Company, Wilmington, Del.) At 4 ° C. 24, After centrifugation, suspended matter from each tube was removed by suction into a 10 ml syringe through an 18-gauge needle. The material from each tube was pooled and resuspended in about 10 ml of medium (Table 2) by suction and removal of the mixture through the above needle into a centrifuge tube. The tube was filled with medium (Table 2) and centrifuged in a SS-34 rotor (Sorvall) at 15,000 RPM for 15 minutes. The supernatant was carefully decanted and discarded. The pellet was resuspended in medium (Table 2) and diluted 1: 1000 with distilled water. Absorbance was read in a 1 cm cuvette to measure protein concentration. The following formula for protein:<img file="000008.tif" id="000008" he="025" wi="145" img-format="tif" img-content="drawing" />Was measured using. Membrane preparations were divided into equal parts, snap frozen in a dry ice / ethanol bath and stored at 80 ° C. Membranes were prepared from BHK transfects grown to confluence within 150 mm plates in selective medium. Rinse the two plates of the densely transfected product twice with cold phosphate buffer saline (PBS: Sigma Chemical Co., St. Louis, Mo.), And add 10 ml PBS containing 1 mM PMSF to each plate. Added. Cells from each plate were scraped into PBS solution and cells from each plate were transferred into new tubes. Each plate was rinsed with 5 ml PBS containing 1 mM PMSF and the rinse was pooled with their respective cells. The cells were centrifuged at 2,000 rpm in a tabletop centrifuge at 4 ° C. Discard the supernatant and discard the cells in 30 ml 5 mM Hepes, 1 mM Resuspended in PMSF, pH 7.5. Cells were incubated on ice for 15 minutes and then centrifuged at 47,800 xg at 4 ° C. Each pellet was resuspended in solution in PBS containing 1 M PMSF, divided equally and frozen at -80 ° C. Competitive analysis using the glucagon binding assay was performed on rat liver and BHK transformant membrane preparations. Simply put, 10 diluted in 10 mM HOAc^-11M ~ 10^-6A reaction tube containing 20 μl glucagon of M or 20 μl BSA was set. In each tube, 100 μl of 2x binding buffer; 20 μl¹²⁵I-Glucagon (Amersham); 20 μl 1 mM LVDS₃20 μl of 10 mM HOAc, 0.5% BSA (Novo Nordisk N / A, Bagsvaerd, Denmark); and 40 μl of distilled water were added. The binding reaction was initiated by adding 20 μl of the membrane preparation to the reaction tube. The reaction was incubated at 30 ° C. for 30 minutes. Membranes were centrifuged in a microfuge at high speed for 10 minutes at 4 ° C. The supernatant from each sample was aspirated and pellets were counted. Competition with unlabeled glucagon produced almost identical sigmoid curves for glucagon binding (Fig. 3). Scatchard analysis (Scatchard, Ann.NYAcad.Sci.51: 660-672,1949 (collectively incorporated herein by reference)) shows that the apparent Kd is at 50 nM for the cloned receptacle. Yes, and 49 nM for rat liver membrane (Fig. 4). The peculiarity of the receptor encoded by pLJ4,¹²⁵The ability of related peptide hormones to compete for I-glucagon binding was tested. Micromolar amounts of glucagon and related peptides (Table 7), respectively¹²⁵Together with I-glucagon, it was added to the membrane preparation of the pLJ4 lancefect form in the binding assay described above. Natural glucagon and antagonist des-His¹[Glu⁹] Only glucagon amide binds to its membrane¹²⁵I was able to compete with I-Glucagon.<img file="000009.tif" id="000009" he="090" wi="155" img-format="tif" img-content="drawing" />Expression of rat glucagon receptor in C. COS-7 cells The ability of pLJ4-transfected COS-7 cells to be stimulated by glucagon to increase cAMP levels was assessed using the Amersham SPA kit (Amersham) as described below. This assay showed that the glucagon-stimulated pLJ4 transfectant accumulated about 5-fold more cAMP than COS-7 cells transfected with the control vector alone. The relevant peptides secretin, VIP, PTH, GLP and calcitonin were added to the transformants at concentrations of 100 nM to 1000 nM, respectively, and their ability to induce cAMP levels was tested. The results of the test showed that none of the related peptides could induce a significant increase in cAMP levels. D. Luciferase and adenylate cyclase activity test for whole cells Rat glucagon receptor cDNA is expressed in a stable transfected BHK570 cell line with ZK6, an expression unit comprising a promoter containing at least one cyclic AMP response element, a luciferase cDNA and an hGH terminator. It was. This cell line allows measurement of luciferase activity, adenylate cyclase activity and intracellular calcium concentration in response to glucagon's binding to its receptor. The proenkephalin cyclic AMP response element (CRE) within plasmid ZK6 was obtained from Zem233. Zem233 was obtained from Zem67 and Zem106. The plasmid Zem106 was constructed from the precursor Zem93. To construct Zem93, a Kpn I-Bam HI fragment containing the MT-1 promoter was isolated from MThGH111 (Palmiter et al., Science 222: 809-814, 1983) and inserted into pUC18. Plasmid Zem93 then Sst Digested with I and religated to produce plasmid Zem106. Here, the sequence of 600 base pairs on the 5'side of the MT-1 promoter was removed. Proenkephalin CRE was inserted into the 5'end of the SV40 promoter within Zem106 by initial digestion of Zem106 with Eco RI and Sst I to isolate vector-containing fragments. When the oligonucleotides ZC982 and ZC983 (SEQ ID NOs: 3 and 4 respectively) are annealed, the nucleotides -71 to -133 adjacent to the 5'Eco RI and 3'Sst I sites (Comb et al., Nature 323: 353). Designed to encode pro-enkephalin CRE from 356,1986). Oligonucleotides ZC982 and ZC983 (SEQ ID NOs: 3 and 4, respectively) were ligated, annealed and ligated with linearized Zem106 to give plasmid Zem224. The plasmid Zem67 was transferred to pIC19R (Marsh et al., Gene) by Sma I and Hind III. It was obtained by digesting 32: 481-486,1984). The ori region of SV40 from map position 270 (Pvu II) to position 5171 (Hind III) was then ligated to linear pIC19R to create plasmid Zem67. A HindIII-Bam HI neomycin resistance gene-SV40 terminator fragment from the plasmid pSV2-neo (American Type Culture Collection Accession NO.37149) was ligated to HindIII-Bgl II digested Zem67 to give Zem220. The SV40 promoter-neomycin resistance gene SV40 terminator expression unit from plasmid Zem220 was isolated as an Eco RI fragment. The plasmid Zem224 was digested with Eco RI and treated with bovine alkaline phosphatase to prevent recyclization. This neomycin expression unit and linear Zem224 were reigated. The plasmid containing the SV40 plotter near the CRE was named Zem233. Exactly 3'side the plasmid Zem233 with respect to the additional CRE sequence, TATA box, and its proenkephalin CRE sequence so that the resulting expression unit is in the opposite direction to the neomycin resistance unit present within Zem233. Lac Z coding and modified to insert part of the poly (A) sequence. The plasmid Zem233 was linearized by digestion with Sst I and Bam HI. When the oligonucleotides ZC3509 and ZC3510 (SEQ ID NOs: 5 and 6 respectively) are annealed, the resulting double strand is an α-glycoprotein CRE (Delegean et al.) With a 5'Sst I attachment and a 3'Eco RI attachment. , Mol.Cell.Biol.7: 3994-4002,1987). Oligonucleotides were annealed according to standard procedures. A thymidine kinase TATA box was obtained as the Eco RI-Pst I fragment spanning nucleotides of the thymidine kinase gene -79 to +18 (McKnight, Cell 31: 355-366, 1982). lac The 3'sequence of the Z gene and its associated poly (A) sequence were obtained as a Pst I-Bam HI fragment from the plasmid pLacF (obtained from Jaques Peschon, Immunex Corp., Seattle, Wash.). It contains a mouse protamine terminator sequence cloned into the lacZ coding region and pUC18 vector. Sst I-Bam HI linearized Zem233, Sst I-Eco RI ZC3509 / ZC3510 adapter, Eco RI-pst I TATA box fragment and Pst I-Bam HI lac Z sequence were ligated. The plasmid containing the expression unit in the correct direction for the neomycin resistance gene expression unit of Zem233 was named KZ5. The luciferase gene and human growth hormone (hGH) terminator sequences were used to replace the lac Z coding and poly (A) sequences present within KZ5. This luciferase gene is plasmid α-168luc (Delegean et First as a 1.7 kb Xho I-Xba I fragment from al., Mol.Cell.Biol.7: 3994-4002,1987; and de Wet et al., Mol.Cell.Biol.7: 725-737,1987) Obtained. The hGH terminator was obtained as an Xba I-Sal I fragment from Zem219b (deposited under deposit number ATCC 68979 as an American Type Culture Collection (Rockville, Md.) And E. coli transformant). The luciferase gene and hGH terminator sequence were subcloned into Xho I-Sal I linearized pIC19H (Marsh et al., Ibid.) For convenience. The resulting plasmid, KZ8, was digested with Xho I and Sal I to isolate the luciferase-hGH terminator sequence. The plasmid KZ5 was digested with Sal I, vector-containing fragments were isolated, and treated with bovine alkaline phosphatase to prevent recyclization. This Xho I-Sal I luciferase-hGH terminator fragment is placed in a container Sal Reigate with I-digestion KZ5. The plusmid containing the luciferase-hGH terminator in the appropriate direction for the promoter was named KZ6. The plasmid KZ6 was subjected to calcium phosphate precipitation as essentially described by Graham and Van der Eb (Virology 52: 456,1973, which is incorporated herein by reference in its entirety). Then, it was transfected into BHK570 cells (deposited in the American Type Culture Collection under deposit number 10314). The transfected cells were subjected to growth medium (10% fetal bovine serum, 1x PSN antibiotic mixture (GIBCO-BRL)), and 2.0 mM. It was grown in Dulbecco's modified Eagle's medium (DEME) containing L-glutamine. After a few days in non-selective growth medium, the growth medium was replaced with selective medium (growth medium containing 500 μg / ml G418). The cells were then subjected to proliferation to confluence, after which they were trypsinized and plated in the wells of a 96-well plate at limiting dilution. Cells were grown in G418 selective medium for 1-2 weeks. Clones from wells containing a single colony were tested for their ability to respond to forskolin in the luciferase assay described below. Forskolin raises intracellular cAMP levels and, therefore, the associated cAMP-dependent biological response pathway in a receptor-independent manner. The clone capable of responding to Falsekin was named BHK / KZ6-19-46. Simultaneous transfection of BHK / KZ6-19-46 cells with pLJ4 and pLJ1 or with pZCEP and pLJ1 (pZCEP transfected form was used as a negative control) using calcium phosphate-mediated transfection as described above. did. Transfectants were selected in 250 nM methotrexate as described above. Transfectants were assayed in triplicate for induction of CRE-luciferase response by selected agents. Six random pLJ4 transfectants were tested, and random pZCEP transfectants were tested (negative control). Microtiter test plate 2x10 in 100 μl of selective medium for each well⁴The cells were set to contain and the cells were grown overnight. The agent was adjusted in the selective medium at the 2x final test concentrations listed below: 1 μl glucagon 200nM glucagon-like peptide (GLP) 20nM Vasoactive Intestinal Peptide (VIP) 100nM calcitonin (CT) 20 μM Forskolin (CalBiochem, San Diego, Calif.) Induction was initiated by adding 100 μl of each 2x solution in triple sample wells. Non-inducible levels were measured in triple wells to which 100 μl DMEM containing 10% fetal bovine serum was added. Plate 37 , 5% CO₂Incubated in 4 hours to allow induction of luciferase production. After induction, the medium was removed and washed once with 200 μl / well PBS. After washing, 25 μl of 1X 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. Transfer the plate to a Labsystems Luminoskan Microtiter Photometer (Labsystems Inc., Morton Grove, Ill.), Add 40 μl of Luciferase Assay Substrate (Luciferase Assay System, Promega), mix the reactants, and per well. The luciferase signal was integrated for 2 seconds. The doubling of luciferase for each agent was calculated as follows:<img file="000010.tif" id="000010" he="025" wi="116" img-format="tif" img-content="drawing" /> One pLJ4 transfect clone, KZ6 / rGR-DHFR-2, showed 6-10-fold induction of glucagon-induced luciferase, but not by GLP, VIP or CT. The cAMP response of the transfected clone KZ6 / rGR-DHFR-2 to glucagon and forskolin using the manufacturer's instructions cAMP [¹²⁵] It was also tested by radioimmunoassay using a scintillation counter-testing device (Amersham). Simply put, 2x10 per 1 ml of KZ6 / rGR-DHFR-2 cells⁵100 μl of cells were plated in wells of a multi-well culture dish and grown overnight in selective medium. Glucagon and forskolin were prepared in IBM X at DMEM, 10 μM fetal bovine serum, 0.0001-1000 nM and 25 μM, respectively. Growth medium was replaced with 50 μl / well of agent (either glucagon or forskolin). Cell cells at 37 ° C, 5% CO₂Incubated with the agent for 10 minutes. After incubation, cells were lysed by adding 200 μl of boiling water to each well. After 15 minutes, collect the supernatant and acetate buffer (cAMP [¹²⁵I] Diluted 1: 5 or 1:40 in Scintillation Proximity Assay System (Amersham). Samples were acetylated using triethylamine and acetic anhydride according to the protocol provided by the manufacturer. Place 100 μl aliquots of each acetylated sample in 75 μl in the wells of the LKB T-tray.¹²⁵I-cAMP, 75 μl anti-succinyl cAMP antiserum and 75 μl donkey anti-rabbit IgG-bound SPA beads (all cAMP [¹²⁵I] Combined with the assay solution provided in the Scintillation Proximity Assay System (Amersham). The trays were sealed and incubated overnight by continuous shaking on a rotating platform shaker at 200 rpm. Samples were counted in a 1205 BETAPLATE liquid scintillation counter (Pharmacia LKB Instruments Inc., Gaithersburg, Md.). A standard curve of 2-128 f mol of acetylated cAMP was also run. all¹²⁵I-cAMP binding and non-specific binding were also measured. KZ6 / rGR-DHFR-2 induces 140 times the cAMP level in saturated glucagon (10-100 nM), and ED of 0.25 nM.₅₀showed that. E. Measurement of intracellular calcium concentration Essential to the intracellular calcium response of plJ4 transfectants to glucagon by Grynkiewicz et al. (J. Biol. Chem. 260: 3440-3450, 1985 (collectively incorporated herein by reference)). Tested using a method as described in. 5x10 plasmid pLJ4 BHK transfects per chamber⁴The cells were inoculated into a 2-well cover glass chamber (NUNC). Cells were grown between 1-3 under normal culture conditions in methotrexate selective medium. The medium was removed by suction and the chamber was rinsed twice with 1 ml of imaging buffer (Table 3). Cells were incubated with 0.5 ml Fura-2 AM solution (Table 3) in the dark at room temperature for 30 minutes. After incubation, Fura-2 AM solution was removed and cells were rinsed 3 times with 1 ml imaging buffer. After the final rinse, 0.5 ml of buffer was left in each chamber. The cells were kept in the dark for 30-120 minutes at room temperature. Image formation was performed on a Nikon Diaphot inverted fluorescence microscope equipped with a mercury discharge lamp and 10X and 40X Nikon Flour lenses to be dried. Experiments were controlled and analyzed using Sun SPARC II workstations and Inovision (Research Triangle Park, NC) PATIOTOOL software. Radiation image is Genesis by dichroic mirror (380nm cutoff) II Projected onto a Dage-MTI 72 CCD camera equipped with an image forming sensitizer and digitally recorded by the above software. The intracellular calcium concentration is monitored by calculating the ratio of radiant intensity at each of the two excitation wavelengths (340/380) for each pixel in the digital microscope image field of view (512 x 480 pixels). Grynkiewicz et al. (Ibid.) Show that this ratio is due to the intracellular fura-2 dye that has taken up and deesterified the acetylmethoxy derivative (fura-2 AM) used to load the cells. It has been shown to be related to calcium concentration. The RATIOTOOL software presents the above information as a false color image that can be converted to calcium concentration. Images were acquired and this calculation was performed at 5 second intervals between each experiment. The cells were monitored for at least 60 seconds (12 images) to establish a pre-stimulation baseline, and the stimulation was applied to a 0.5 ml image-forming buffer containing 200 nM glucagon in a 0.5 ml image-forming buffer in a coverglass chamber. Was added and thus achieved a final concentration of 100 nM. Cells were monitored and images were recorded for at least 3 minutes after stimulation. It was observed that the ratio image corresponding to the number of cells in each visual field changed dramatically in a short time after the addition of glucagon. This was quantified using RATIOTOOL software to calculate the mean for a particular region of the ratio image corresponding to each of the responding cells. Cells with an average rest ratio of about 1.4 rapidly rose to values of 3-4. They remained at high values for 40-50 seconds and then gradually attenuated to baseline. Independent conversion experiments showed that this reflex changed the intracellular calcium concentration from a resting value of about 150 nM to a peak of about 400 nM. F. Inositol / phosphate measurement BHK 570 cells expressing the glucagon receptor from pLJ4 or simulated-transfected BHK 570 cells were plated in 24-well tissue culture dishes at approximately 200,000 cells per well. After 24 hours, the cells in each well were 2.0 μCi myo (2-³H) Labeled by incubation in 0.5 ml MTX selective medium containing inositol (specific activity -20 Ci / m mol; Amersham). After 24-hour incubation, cells were buffered in 20 mM Hepes, pH 7.0 buffer (Sigma Chemical Co.) containing 10 mM LiCl, and 1 ml of preheated DMEM (Dulbecco's Modified Eagles Medium; JRH Biosciences, Lenexa, Kan.) Was washed with. The wash medium was removed by suction and replaced with 900 μl of fresh buffered medium. Cells were incubated at 37 ° C for 5 minutes. After incubation, each agonist or antagonist was added to triple wells and incubated according to the volume and conditions described in Table 8.<img file="000011.tif" id="000011" he="115" wi="145" img-format="tif" img-content="drawing" /> The reaction was terminated by placing the cells on ice. After aspiration of the medium, cells were lysed by addition of 1 ml cold DMEM and 1 ml ice-cold 10% perchloroic acid. After 10 minutes, the cell lysates were transferred to tubes on ice, each containing 500 μl of 10 mM EDTA, pH 7.0. Samples were neutralized by adding 1.5 M KOH in 60 mM Hepes Buffer and dropping KOH-HEPES solution in 900 μl until a pH between 7 and 7.5 was achieved. The frozen sample was thawed and the precipitate was allowed to stand from the sample. The supernatant is 5 ml of methanol and 1 M KHCO, respectively.₃And then applied to AMPREP mini-columns (Amicon) washed with 15 ml of water. After applying the sample, the eluate was collected. The column was washed 4 times with 1 ml of water, and 1 ml of sample was collected after each wash. 4 consecutive 0.25M KHCO with inositol phosphate₃1 ml of the sample was eluted from the column by application of 1 ml and 1 ml of sample was collected after each application. 10 ml of OPTIFLUOR (Packard Instrument Co., Menden, Conn.) Was added to each sample and the samples were counted. Stimulation of the inositol-phosphate pathway was indicated by an increase in radiolabeled inositol-phosphate levels. No irritation in inositol phosphate production was observed in any of the samples. Expression of human glucagon receptor in G. COS 7 cells The plasmid pLJ6'was transfected into COS 7 cells using the DEAE-dextran procedure as described above. Cells were grown on glass chamber slides (as described in Example 3) for 72 hours after transfection.¹²⁵In-situ glucagon binding assay using I-glucagon Subsequent emulsion autoradiography was performed as described in Example 3. Over 50% of cells transfected with pLJ6' binding glucagon in a specific manner. H. Expression of human glucagon receptor in BHK570 cells BHK570 cells (deposited in the American Type Culture Collection under deposit number 10314) were transfected with the plasmid pLJ6'using the calcium phosphate transfection method (Example 8). Transfected cells were selected in the presence of G418 until a single colony was visible. Suitable colonies were cloned using a cloning cylinder. The clones were shown to bind to glucagon using in-situ binding to iodinated glucagon as described above. PLJ6'-transfected cells were assayed for cAMP accumulation as essentially described in Example 8.C. Transfects were later tested for stimulation with glucagon, secretin, VIP or GLP-I. The assay showed that pLJ6'-transfected cells accumulated increased cAMP levels after glucagon stimulation than control cells. Stimulation of the transfectants with secretin, VIP or GLP-I showed no increased accumulation of cAMP. The intracellular calcium response was measured as essentially described in Example 8.E. Glucagon-stimulated pLJ6'-transfected cells demonstrated an increase in intracellular calcium levels as indicated by a rapid increase in fluorescence of the calcium indicator Fura-2. These results indicate that pLJ6'encodes a functional human glucagon receptor that can bind to glucagon and facilitate signal transduction. As described above, although specific aspects of the invention have been described herein for purposes of explanation, various modifications can be made without departing from the essence and scope of the invention. That will be understood. Therefore, the present invention is not limited to anything other than the accompanying claims. Sequence listing (1) General information: (I) Applicant: Name: Zymo Gynetics, Inc. Address: 4225 Roosevelt Way North East City: Seattle, Washington Country: United States Zip code: 98105 Phone: (206) 547-80808 (Ii) Title of invention: Glucagon receptor (Iii) Number of sequences: 25 (Iv) Communication destination: (A) Address: SEED AND BERRY (B) Address: 6300 COLUMBIA CENTER (C) City: SEATTLE (D) State: WA (E) Country: USA (F) Postal code: 99104-7092 (V) Computer reading mode: (A) Medium type: Proppy disc (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS (D) Software: Patent In Release # 1.0, Version # 1.25 (Vi) Latest application data: (A) Applicant number: 08 / 086,631 (B) Filing date: July 1, 1993 (C) Classification: (Vii) Previous application data: (A) Applicant number: US 07 / 938,331 (B) Filing date: August 28, 1992 (Viii) Atony / Agent Information: (A) Name: McMasters, David D. (B) Registration number: 33,963 (C) Reference / Document number: 990008.424C1 (Ix) Long-distance communication information: (A) Telephone: 206-622-4900 (B) Telefax: 206-682-6031 (2) Information about SEQ ID NO: 1: (I) Characteristics of the array: (A) Length: 17 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC447 (Xi) Sequence: SEQ ID NO: 1:<img file="000012.tif" id="000012" he="010" wi="157" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 2: (I) Characteristics of the array: (A) Length: 18 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC976 (Xi) Sequence: SEQ ID NO: 2:<img file="000013.tif" id="000013" he="010" wi="157" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 3: (I) Characteristics of the array: (A) Length: 71 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC982 (Xi) Sequence: SEQ ID NO: 3:<img file="000014.tif" id="000014" he="020" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 4: (I) Characteristics of the array: (A) Length: 63 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC983 (Xi) Sequence: SEQ ID NO: 4:<img file="000015.tif" id="000015" he="020" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 5: (I) Characteristics of the array: (A) Length: 38 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC3509 (Xi) Sequence: SEQ ID NO: 5:<img file="000016.tif" id="000016" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 6: (I) Characteristics of the array: (A) Length: 46 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC3510 (Xi) Sequence: SEQ ID NO: 6:<img file="000017.tif" id="000017" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 7: (I) Characteristics of the array: (A) Length: 42 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC3747 (Xi) Sequence: SEQ ID NO: 7:<img file="000018.tif" id="000018" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 8: (I) Characteristics of the array: (A) Length: 25 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC4701 (Xi) Sequence: SEQ ID NO: 8:<img file="000019.tif" id="000019" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 9: (I) Characteristics of the array: (A) Length: 26 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC4715 (Xi) Sequence: SEQ ID NO: 9:<img file="000020.tif" id="000020" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 10: (I) Characteristics of the array: (A) Length: 26 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC4758 (Xi) Sequence: SEQ ID NO: 10:<img file="000021.tif" id="000021" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 11: (I) Characteristics of the array: (A) Length: 26 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC4778 (Xi) Sequence: SEQ ID NO: 11:<img file="000022.tif" id="000022" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 12: (I) Characteristics of the array: (A) Length: 21 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC5432 (Xi) Sequence: SEQ ID NO: 12:<img file="000023.tif" id="000023" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 13: (I) Characteristics of the array: (A) Length: 21 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC5433 (Xi) Sequence: SEQ ID NO: 13:<img file="000024.tif" id="000024" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 14: (I) Characteristics of the array: (A) Length: 1875 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Ii) Molecular type: cDNA (Vi) Biological source: (F) Tissue type: Liver (Vii) Direct source: (B) Clone: pLJ4 (Ix) Features: (A) NAME / KEY: CDS (B) Position: 145..1599 (Xi) Sequence: SEQ ID NO: 14:<img file="000025.tif" id="000025" he="075" wi="159" img-format="tif" img-content="drawing" /><img file="000026.tif" id="000026" he="230" wi="161" img-format="tif" img-content="drawing" /><img file="000027.tif" id="000027" he="230" wi="161" img-format="tif" img-content="drawing" /><img file="000028.tif" id="000028" he="230" wi="159" img-format="tif" img-content="drawing" /><img file="000029.tif" id="000029" he="115" wi="157" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 15: (I) Characteristics of the array: (A) Length: 485 amino acids (B) Sequence type: Amino acid (D) Topology: Linear (Ii) Molecular type: protein (Xi) Sequence: SEQ ID NO: 15:<img file="000030.tif" id="000030" he="035" wi="137" img-format="tif" img-content="drawing" /><img file="000031.tif" id="000031" he="230" wi="139" img-format="tif" img-content="drawing" /><img file="000032.tif" id="000032" he="230" wi="139" img-format="tif" img-content="drawing" /><img file="000033.tif" id="000033" he="095" wi="137" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 16: (I) Characteristics of the array: (A) Length: 576 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: G30 (Ix) Features: (A) NAME / KEY: Intron (B) Position: 225..314 (Ix) Features: (A) NAME / KEY: Exxon (B) Position: 1..225 (Ix) Features: (A) NAME / KEY: Exxon (B) Position: 315..576 (Xi) Sequence: SEQ ID NO: 16:<img file="000034.tif" id="000034" he="125" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 17: (I) Characteristics of the array: (A) Length: 487 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: 40-2-2 (Ix) Features: (A) NAME / KEY: CDS (B) Position: 1..486 (Xi) Sequence: SEQ ID NO: 17:<img file="000035.tif" id="000035" he="190" wi="161" img-format="tif" img-content="drawing" /><img file="000036.tif" id="000036" he="085" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 18: (I) Characteristics of the array: (A) Length: 162 amino acids (B) Sequence type: Amino acid (D) Topology: Linear (Ii) Molecular type: protein (Xi) Sequence: SEQ ID NO: 18:<img file="000037.tif" id="000037" he="075" wi="139" img-format="tif" img-content="drawing" /><img file="000038.tif" id="000038" he="125" wi="139" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 19: (I) Characteristics of the array: (A) Length: 21 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC4812 (Xi) Sequence: SEQ ID NO: 19:<img file="000039.tif" id="000039" he="010" wi="157" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 20: (I) Characteristics of the array: (A) Length: 34 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC4814 (Xi) Sequence: SEQ ID NO: 20:<img file="000040.tif" id="000040" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 21: (I) Characteristics of the array: (A) Length: 20 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC5624 (Xi) Sequence: SEQ ID NO: 21:<img file="000041.tif" id="000041" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 22: (I) Characteristics of the array: (A) Length: 41 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC5763 (Xi) Sequence: SEQ ID NO: 22:<img file="000042.tif" id="000042" he="010" wi="157" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 23: (I) Characteristics of the array: (A) Length: 44 base pairs (B) Sequence type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (Vii) Direct source: (B) Clone: ZC5849 (Xi) Sequence: SEQ ID NO: 23:<img file="000043.tif" id="000043" he="010" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 24: (I) Characteristics of the array: (A) Length: 1809 base pairs (B) Sequence type: Nucleic acid (C) Chain: Double chain (D) Topology: Linear (Ii) Molecular type: cDNA (Vi) Biological source: (A) Organism: Homo sapiens (Vii) Direct source: (B) Clone: pLJ6' (Ix) Features: (A) NAME / KEY: CDS (B) Position: 53..1486 (Xi) Sequence: SEQ ID NO: 24:<img file="000044.tif" id="000044" he="075" wi="159" img-format="tif" img-content="drawing" /><img file="000045.tif" id="000045" he="230" wi="159" img-format="tif" img-content="drawing" /><img file="000046.tif" id="000046" he="230" wi="159" img-format="tif" img-content="drawing" /><img file="000047.tif" id="000047" he="230" wi="159" img-format="tif" img-content="drawing" /><img file="000048.tif" id="000048" he="100" wi="159" img-format="tif" img-content="drawing" />(2) Information about SEQ ID NO: 25: (I) Characteristics of the array: (A) Length: 477 amino acids (B) Sequence type: Amino acid (D) Topology: Linear (Ii) Molecular type: protein (Xi) Sequence: SEQ ID NO: 25:<img file="000049.tif" id="000049" he="075" wi="137" img-format="tif" img-content="drawing" /><img file="000050.tif" id="000050" he="210" wi="137" img-format="tif" img-content="drawing" /><img file="000051.tif" id="000051" he="230" wi="139" img-format="tif" img-content="drawing" /><img file="000052.tif" id="000052" he="055" wi="137" img-format="tif" img-content="drawing" />

Continuation of front page (51) Int.Cl.⁷ Identification code FI C12P 21/08 G01N 33/566 C12Q 1/68 C12N 15/00 ZNAA G01N 33/566 5/00 B (72) Inventor Shepherd, Paul O. United States, Washington 98053, Redmond, Northeast Second De 20717 (72) Inventor Grant, Francis Jay. United States, Washington 98115, Seattle, Thirty Seventh Aveni North East 7714 (72) Inventor Kaiper, Joseph El. United States, Washington 98011, Bothell, Northeast One Handle Dfifty Force 6640 (72) Inventor Foster, Donald Sea. United States, Washington 98155, Seattle Northeast One Hand Red Eighty First Story 3002 (72) Inventor Roku, Shi United States, Washington 98107, Seattle, Northwest Fifty -Second Street 806 (72) Inventor O'Hara, Patrick Jay. United States, Washington 98103, Seattle, North Sixty Fo S Street 515 (56) References International Publication 92/012998 (WO, A1) J. Biol. Chem. , Vol. 265, No. 34 (1990) p. 21302 21308 J. Cell Biol. , Vol. 107, No. 6, Pt. 3 (1988) p. 65A J. Biol. Chem. , Vol. 259, No. 14 (1984) p. 9285-9294 J. Med. Chem. , Vol. 30, No. 8 (1987) p. 1409-1415 (58) Surveyed field (Int.Cl.⁷, DB name) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / G eneSeq BIOSIS / WPI (DIALOG) PubMed