MYOTONIN RECEPTORS AND SCREENING METHODS

Abstract: Purified myotonin receptors, nucleic acid encoding such receptors, and methods for identifying or screening or characterizing or assaying or isolating known or candidate compounds useful for treatment of a hypoglycemic condition, or a disease characterized by insulin resistance, including binding assays utilizing preparations containing specific myotonin receptors are described. Membranes from leiomyosarcoma cells that contain myotonin receptors are particularly useful for the methods of this invention, and as a source of myotonin receptor protein.

Description: DESCRIPTION Myotonin Receptors and Screening- Methods Field of the Invention The present invention relates to methods for identifying physiologically active materials,' such as chemical compounds, by assessing their ability to interact with naturally occurring or isolated or cloned receptor sites. Description of Related Art The following discussion is provided as a basic background to the claimed invention. Resistance to insulin may be present in several serious disorders, including Type 2 diabetes mellitus, obesity and hyper¬ tension. Resistance to insulin is manifest by a reduction in the effectiveness of a given dose of insulin compared to that obtained in a non-resistant state. Thus, in an insulin-resistant patient with Type 2 diabetes mellitus, the ability of both endogenous insulin and insulin administered exogenously to control the chronic hyper- glycemia suffered by such patients is seriously compromised. Consequently, the complications that result from uncontrolled diabetes mellitus, such as premature atherosclerosis, intercapillary glomerulosclerosis, retinopathy, neuropathy and kidney failure, are more likely to occur in insulin-resistant diabetics than in insulin-sensitive diabetics. In clinical terms, insulin resistance is present when normal or elevated glucose concentrations persist in the face of normal or elevated concentrations of insulin in the general circulation. Insulin resistance in skeletal muscle represents, in essence, inhibition of glycogen synthesis from metabolic precursors due to a reduction to subnormal levels of either basal or insulin-stimulated glycogenesis, or both. There are at least two causes of hyperglycemia in Type 2 diabetes mellitus: (1) failure of glucose storage to be activated (Lillioja, J. Clin. Endocr. Metab.. 62:922-927, 1986); and (2) a defect in insulin release from the pancreas (Waingot et al. r Proc. Nat'1 Acad. Sci. USA. 79:4432-4436, 1982) . Treatment of this disease has focused on attempts to reverse either or both of these defects. Recently, a novel protein hormone isolated from the pancreas has been shown to modulate certain of the actions of insulin. The hormone, termed amylin (initially referred to as diabetes associated peptide or DAP) , was recently purified to homogeneity from pancreatic amyloid from human Type 2 diabetes mellitus patients. E.g. , Cooper et al.. Proc. Νat'l Acad. Sci. USA. 84:8628-8632, 1987. Amylin is the subject of United Kingdom patent application Serial No. 8709871, filed April 27, 1987, and corresponding U.S. applications filed April 27, 1988, November 23, 1988 and May 2, 1989. The use of amylin for the treatment of diabetes mellitus is the subject of United Kingdom patent application Serial No. 8720115 filed on August 26, 1987, by G.J.S. Cooper et al. , and filed in the United States on August 26, 1988. Native amylin is a 37 amino acid protein characterized by a disulfide bridge between the Cys residues at positions 2 and 7 and an amide group at the C- terminal tyrosine. The amylin subpeptide 18-27 is amyloidogenic, that is, it possesses the tendency to form amyloid. The structure of amylin shows a 43% homology to calcitonin gene related peptide-1 (CGRP-1) , a 46% homology to CGRP-2, and some similarity to insulin. Amylin may be one member of a family of related peptides which include CGRP, insulin, insulin-like growth factors, and the relaxins and which share common genetic heritage. Cooper et al. , Prog * . Growth Factor Research 1:99-105, 1989. See Amara et al. , Science. 229:1094-1097, 1985; Rosenfeld et al. , Nature, 304:129-135, 1983. The two peptides calcitonin and CGRP-1 share common parentage in the calcitonin gene where alternative processing of the primary mRNA transcript leads to the generation of the two distinct peptides, which share only limited sequence homology (about 30%). Amara et al.. Science, 229:1094- 1097, 1985. It has been disclosed in commonly-owned copending U.S. patent application Serial No. 275,475, filed November 23, 1988 by Cooper, G.J.S. et al. , the contents of which are incorporated herein by reference, that amylin causes reduction in both basal and insulin-stimulated incorporation of labelled glucose into glycogen in skeletal muscle. The latter effect was also disclosed to be shared by CGRP. See also Leighton and Cooper, Nature, 335:632-635, 1988. Amylin and CGRP were approximately equipotent, showing marked activity at 1 to 10 nM. Amylin is also reported to reduce insulin-stimulated uptake of glucose into skeletal muscle and reduce glycogen content. Young et al.. A er. J. Phvsiol. 259:457-461, 1990. It is believed that the amylin family of peptide hormones acts through receptors present in plasma membranes. Young et al. have shown that amylin works in skeletal muscle via a receptor-mediated mechanism that promotes glycogenolysis, by activating the rate-limiting enzyme for glycogen breakdown, phosphorylase a (Young et al. 281 FEBS Lett. 149, 1991). The major metabolic effects of amylin reported in vivo are: (1) a reduction in insulin action observed under "euglycemic clamp" conditions whereby infusion of amylin reduces insulin mediated glucose clearance (Molina et al. Diabetes 39:260-265, 1990, and Young et al. , Am. J. Phvsiol. 259:457-461, 1990) and partly reverses insulin- mediated suppression of hepatic glucose output (Molina et al. supra; Koopmans et al. Diabetes 39:101A, 1990); (2) In lightly anaesthetized, 18-hour fasted rats, bolus injections of amylin evoke first an increase in plasma lactate, and second a sustained increase in plasma glucose. The increase in plasma lactate is thought to reflect lactate production in skeletal muscle, consequent upon amylin stimulation of glycogenolysis. This action of amylin is observed in the fed as well as the 18-hour fasted rat, and also during somatostatin infusion given to limit secretion of pancreatic hormones including insulin and glucagon. These data substantiate the determination that amylin acts independent of other hormonal regulators to promote lactate release from skeletal muscle in intact animals. The increase in plasma glucose is associated with a dilution of infused trace glucose, indicating increased hepatic glucose output. It is not presently known whether the actions of amylin infusion in "euglycemic clamped" rats, and amylin injections, to increase hepatic glucose output is a result of direct actions of amylin on liver, or solely an indirect effect of the amylin actions, such as the release of lactate from muscle. For instance, it was reported that amylin increases gluconeogenesis and glycogenolysis in cultured Hep G2 cells (a cell line derived from a human liver tumor) . Ciaraldi et al. , Diabetes 39: Supp. 1, 145A, 1990. On the other hand, it was reported that amylin had no observable effects on glucose metabolism in isolated rat hepatoyctyes or in perfused rat liver (Stephens et al. Diabetes in press, 1991) . It is also reported that amylin can exert certain other actions in vivo, including vasodilatation. Brain et al. Am. J. Pathol. 136:487-490, 1990. Amylin was 100 to 1000 fold less potent as vasodilator than the related peptide CGRP. This could reflect a weak action of amylin on CGRP receptors although no evidence was provided in support of this. Amylin is also reported to lower plasma calcium in rabbits and rats (Datta et al. Biochem. Biophys. Res. Commun. 162 :876-881, 1989) . This action resembles that of calcitonin. Human calcitonin was more effective than amylin in adducing hypocalcaemia and it is possible, though also unproven, that amylin acts less potently than calcitonin at calcitonin receptors on bone cells. It is believed that CGRP and calcitonin act via membrane receptors at least some of which serve to activate adenylate cyclase and generate cyclic AMP as an intracellular second messenger. In this regard, high affinity binding sites (receptors) for CGRP on liver membrane were reported; at these sites CGRP is said to potently activate adenylate cyclase. E.g. Morishita, et al. Diabetes 3_9:875-877, 1990. Amylin was reported to displace labelled CGRP from these binding sites but with much lower affinity, approximately 200 nM, than CGRP, approximately 20 pM. Somewhat similar findings are reported by Chantry et al.. Biochem J. , in press, 1991) ; these authors report apparent affinities for CGRP and amylin on liver membranes of approximately 100 to 300 pM and lOnM respectively. Chantry et al. remark that liver contains very little CGRP, that amylin is secreted from the portal vein directly supplying the liver, and they propose that the glucose regulatory role of these peptides in hepatic metabolism may be mediated primarily by amylin. However, Chantry, et al. were not able to measure amylin binding to liver membranes. Stephens et al. , supra, were able to measure CGRP and amylin binding to a preparation of liver cell membranes; however, from studies of separated parenchymal and interstitial liver cells they concluded that the main binding was to interstitial cells and that the lack of binding to parenchymal cells was consistent with a lack of amylin action on hepatocyte metabolism. Thus, the question of the nature and even existence of functionally relevant amylin receptors on liver is confused and unresolved. Furthermore, the very existence of an amylin receptor or receptors has been questioned by some, while still others perceive that such a receptor will someday be identified (Banks, Journal of NIH Research 2:34-35, 1990; Diabetes Forecast, March 1991 , 27-31) . Thus far, there is only one report of studies seeking amylin receptors in skeletal muscle. Chantry et al. report that CGRP and amylin compete for 125 I-CGRP binding to crude membrane preparations, CGRP with an IC 50 of 300 pM and amylin with an IC 50 of 10 nM. They indicate that these data may be indicative of a common system for mediation of the action of both peptides on skeletal muscle. However, this appears inconsistent with the fact that in functional studies of skeletal muscle, amylin is somewhat more potent than CGRP, not 30 times less potent as found in the reported binding studies. It will be apparent then, that while several investigators have postulated and/or sought receptors that could mediate the metabolic actions of amylin, none have been identified. From the available binding data and the cardiovascular responses reported by Brain et al. one might speculate only that the high affinity receptor reported in liver membranes may be that which mediates the potent vasodilator effects of CGRP. This receptor is appropriately designated a CGRP receptor, and may be type of receptor in skeletal muscle membranes that was investigated by Chantry et al. Binding sites for calcitonin and CGRP are widely distributed in the central nervous system. However, the two peptides act at their own distinct high affinity receptors with distinct biochemical specificities and little interaction at the alternate receptor site. Sexton et al. , 1988, supra. For example, it has been reported that in both human (Tschopp et al. , Proc. Nat. Acad. Sci. USA 82:248-252, 1985) and rat (Sexton et al. , Neuroscience 9.:1235-1245, 1986) cerebral cortex, calcitonin was 500- 1000 fold less potent than either rat or human CGRP in competition for CGRP binding sites. Similarly, CGRP was 500-1000 fold less potent in competing for calcitonin sites in both brain and renal membranes (Goltzman and Mitchell, Science 227:1343-1345, 1985) , as well as in whole kidney sections (Sexton et al . , Kidney Int . 32:862- 868, 1987) . Some data regarding atypical CGRP binding sites in regions of the rat brain have been reported. Dennis et al . , Soc. Neurosci . Abs . 16:514, Abstract 220.7, 1990; Sexton et al.. Neurochem. Int. .12:323-335, 1988. High densities of binding sites with high affinity for the two otherwise biochemically distinct peptides salmon calcitonin and CGRP were reported to have been identified by the use of autoradiographic techniques in parts of the ventral striatum, including posterior nucleus accumbens (except for a small medial band associated with the shell of the accumbens) and its caudal continuation with the posteriocaudal caudate putamen and posterior fundus striati, as well as the lateral border of the lateral bed nucleus of the stria terminalis and the medial nucleus of the amygdala. Sexton et al.. 1988, supra. Outside the basal ganglia related nuclei, the organum vasculosum of the lamina terminalis, wings of the dorsal raphe, and area postrema were the only reported regions where calcitonin-sensitive CGRP binding was identified by Sexton et al. Currently, amylin agonist activity is assessed by measuring the inhibition of insulin-stimulated glycogen synthesis in intact rat soleus muscle. Leighton et al. (1988) supra. While effective as a quantitative biological assay, this technique is relatively slow, labor-intensive and is sensitive to the effects of cellular proteolytic enzymes on the peptides being tested. The soleus assay is effective in quantitating relative potencies of agonists but the affinity of a ligand for its receptor cannot accurately be determined from agonist dose-response relations in whole tissues or organs. For instance, different molecular sizes, or solubility, or propensity to bind to tissue components can influence the defined potencies. The soleus assay, furthermore, has little or no value as an effective, high-throughput primary screening assay for compounds active at amylin receptors. Further methods for screening potential amylin and related peptide hormone agonists and antagonists for clinical use that are inexpensive, rapid and based on physiological principles would be highly desirable. Such methods have been discovered and are disclosed below. These methods include use of amylin receptors, described by Beaumont et al., U.S. Serial No. 07/670,231, filed March 15, 1991, entitled "Receptor-Based Screening Methods For Amylin Agonists and Antagonists," the whole of which (including drawings) is hereby incorporated by reference. Summary of the Invention Applicant has discovered a novel peptide receptor, herein termed the myotonin receptor. This receptor is useful in methods for identifying compounds useful in treatment of hypoglycemic conditions, or insulin resistance. It is also useful for other screening procedures as detailed below, and described (with respect to amylin receptors) in Beaumont et al., supra. For example, the invention features rapid, inexpensive and physiological methods for identifying, screening and characterizing potential compounds useful for treatment of a hypoglycemic condition, or a disease characterized by insulin resistance. The methods include assessing the ability of candidate molecules to compete against tracer concentrations of certain labeled peptides, including certain labeled peptide hormones and fragments and analogs thereof, for binding to specific myotonin receptor binding sites in cells or membranes prepared or isolated from said cells or from tissues containing cells with membrane receptors for myotonin. Myotonin receptors can be identified by the binding characteristics described below for one example of a myotonin receptor. Thus, in one aspect, the invention provides an assay method for identifying or screening compounds useful for treatment of a hypoglycemic condition or a disease characterized by insulin resistance. The method includes bringing together a test sample and a myotonin receptor preparation. The test sample contains one or more test compounds, and the myotonin receptor preparation contains a myotonin receptor protein capable of binding to calcitonin. The test sample is incubated with the receptor preparation under conditions that allow the binding of calcitonin or related radioligand to the myotonin receptor protein. Those test samples containing one or more test compounds which detectably bind to the myotonin receptor protein are then identified. In preferred embodiments, this method further includes the steps of screening test samples which detectably bind to the myotonin receptor for in vitro or in vivo stimulation or inhibition of myotonin receptor- mediated activity, and identifying those test samples which act as receptor agonists or antagonists at this receptor. In other preferred embodiments, the test samples which detectably bind to the myotonin receptor protein are identified by measuring the displacement of a labeled first ligand from the receptor preparation by the test sample, and comparing the measured displacement of the first labeled ligand from the receptor preparation by the test sample with the measured displacement of the labeled first ligand from the receptor preparation by one or more known second ligands. Labeled first ligands and second ligands include salmon calcitonin, an amylin agonist, or an amylin antagonist. Useful receptor preparations include isolated cells bearing the myotonin receptor, isolated membrane preparations bearing the myotonin receptor and isolated myotonin receptor protein. Test samples used in any of the above methods that contain more than one test compound and which yield positive results can then be divided and retested as many times as necessary, and as appropriate, to identify the compound or compounds in the test sample which are responsible for yielding the positive result. In particularly preferred embodiments, the first ligand is labelled with a member selected from the group consisting of radioactive isotopes, nonradioactive isotopes, fluorescent molecules, chemiluminescent molecules, and biotinylated molecules; the known second ligand or ligands are selected from the group consisting of amylin, calcitonin, cu-CGRP, and β-CGRP, e.g. , human amylin, dog amylin, rat amylin, human calcitonin, rat calcitonin, eel calcitonin, salmon calcitonin, human or- CGRP, human 0-CGRP, rat α-CGRP, and rat β-CGRP; the test sample comprises one or more known or unknown test compounds; and one or more non-specific myotonin binding sites on cells which do not comprise a myotonin receptor are blocked, e.g. , one binding site includes a calcitonin or CGRP receptor, and the assay method includes the step of providing an antagonist for the calcitonin or CGRP receptor in the assay method to block the calcitonin or CGRP receptor. In other preferred embodiments, the cells bearing the myotonin receptor are smooth muscle cells, e.g. , from a vas deferens, or the cell line DDT-MF-2 leiomyosarcoma. In another aspect, the invention provides an assay method for evaluating one or more receptor binding characteristics sought to be determined for a known or a candidate amylin agonist or antagonist compound. The method includes the steps of assessing or measuring the ability of the compound to compete with a labeled ligand for binding to a myotonin receptor preparation described above; assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a CGRP receptor preparation, or assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a calcitonin receptor, or assessing or measuring the ability of the compound to compete against the labeled ligand for binding to a CGRP receptor preparation and to a calcitonin receptor preparation; and, determining the receptor binding characteristic sought to be determined for the compound. Receptor binding characteristics which may be determined include binding affinity and binding specificity. CGRP receptor preparations include hepatocyte preparations, including primary cell cultures or established cell lines e.g. , the Hep G2 cell line. Calcitonin receptor preparations include cell or membrane preparations, e.g. , from osteoclasts or equivalent calcitonin receptor-bearing cell lines, such as the LLC-PK1 cell line. In still another aspect, the invention provides an assay method for determining the presence or amount of a myotonin receptor binding compound in a test sample to be assayed. The method includes the steps of bringing together the test sample and a myotonin receptor preparation described above; measuring the ability of the test sample to compete against a labeled ligand for binding to the myotonin receptor preparation; and, optionally, relating the amount of myotonin receptor binding compound in the test sample with the amount of myotonin receptor binding compound measured for a negative control sample, the negative control sample being known to be free of any myotonin receptor binding compound, and/or relating the amount of myotonin receptor binding compound in the test sample with the amounts of myotonin receptor binding compound measured for positive control samples which contain known amounts of myotonin receptor binding compound, in order to determine the presence or amount of myotonin receptor binding compound present in the test sample. This assay method, in still further embodiments, can be utilized to evaluate the stability of an amylin preparation, to evaluate the potency of an amylin preparation, and to evaluate the solubility characteristics of an amylin preparation. In preferred embodiments, the myotonin receptor binding compound and/or labelled ligand is an amylin agonist such as salmon calcitonin, or an amylin antagonist; and the test sample is a biological fluid, selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, lymph fluid, or an amylin preparation; and the assay method includes evaluation of the stability, potency or the solubility of an amylin preparation. In another aspect, the receptor preparations of the invention can be utilized to prepare anti-myotonin receptor antibodies, including polyclonal antisera and monoclonal antibodies, utilizing art-known methods. In another aspect, the invention is used to screen cell lines, cells desegregated from tissue, and cells from human or animal blood in order to identify those which carry myotonin receptors. The myotonin receptor preparations of the invention may also be bound to a solid phase and used in various affinity chromatography methods and used, for example, for the purification of peptides such as amylin, or the evaluation of samples known or suspected to contain amylin, amylin agonists or amylin antagonists. In other aspects, the invention features purified myotonin receptor, e.g. , isolated from DDT-MF-2 leiomyosarcoma cell line cells; a method for isolating a myotonin receptor by purifying cell membrane from DDT-MF-2 cell line cells, and separating components of the cell membrane from the myotonin receptor; purified nucleic acid encoding a myotonin receptor; and a method for purifying nucleic acid encoding a myotonin receptor, e.g. , by standard techniques, such as by forming a library of clones from nucleic acid from cells of the DDT-MF-2 cell line, and screening that library for a clone able to express the myotonin receptor. By "purified" is meant that the myotonin, myotonin receptor or nucleic acid encoding either of these is separated from its natural environment, preferably as a homogeneous preparation having at least 60-90% by weight of the desired product. It is thus an object of this invention to identify receptor preparations suitable for the screening methods of this invention. It is another object of this invention to provide details of the screening methods of the invention as applied to potential agonists and antagonists of amylin. It is still another object of this invention to teach the method for assessing the relative potencies and specificities of the candidate agonists and antagonists. It is still another object of the invention to provide a method, using myotonin receptor preparations, for determining the presence or amount of amylin and other molecules that bind to related receptors, e.g. , the amylin receptor. These and other objects will become readily apparent by reference to the specification and the appended claims. Legends to Figures The drawings will first briefly be described. Drawings Figure 1 is a graphical representation of a saturation isotherm of 125 I-salmon-calcitonin binding to rat hindlimb muscle membranes; Figure 2 is a graphical representation of a Scatchard plot of data from Fig. 1 (Kd=llpM) . Figure 3 is a graphical representation of competition of salmon-calcitonin with amylin and CGRP-1 in DDT-MF-2 cells. Figure 4 is a graphical representation of AC66 Antagonism of sCT-Induced cAMP production by DDT-MF-2 cells. Figure 5 is a Schild Plot of AC66 Antagonism of sCT- Induced cAMP Production in DDT-MF-2 cells. Figure 6 is a graphical representation of amylin, calcitonin, and CGRP-induced cAMP production in DDT-MF-2 cells. Figure 7 is a graphical representation of the effects of heterologous calcitonins on cAMP production in DDT-MF2 cells. Detailed Description of the Invention The present invention provides novel inexpensive, rapid and physiological methods for screening, identifying, and characterizing potential agonists and antagonists at a novel adenylate-cyclase-coupled receptor, the myotonin receptor, as well as the use of this receptor to identify agonists and antagonists at the related amylin receptor. This includes assessing the relative abilities of candidate agonists and antagonists to compete against relevant peptides for binding to specific myotonin receptor sites. The receptor sites used for these and other purposes may be present as isolated receptor-bearing tissues, cells prepared from said receptor bearing tissues, membrane preparations derived from said cells, and isolated receptor protein preparations including cloned receptor preparations using recombinant DNA techniques. There is general agreement that the term "receptor" or "specific receptor" refers to a macromolecule capable of recognizing and selectively binding with some ligand, and which, after ligand binding, is capable of generating some physical or chemical signal that initiates the chain of events leading to the physiological response. Blecher et al. , "Receptors and Human Disease", Williams & Wilkins, Baltimore, 1981, Chapter 1. It is thus an important part of this invention that the tissue, cells, membrane, or receptor preparations used in the competition-based screening methods of the invention exhibit binding characteristics identical to or equivalent to a natural receptor. Tissue preparations or cell or membrane preparations derived from tissues bearing the myotonin receptor can be identified for use in methods for screening myotonin receptor antagonists and agonists as described below. We have found that cells or membranes bearing the myotonin receptor, or myotonin receptor protein preparations, are preferably isolated from rat skeletal muscle or cells such as DDT-MF-2 cells. The results of the Examples below establish that the method described herein for measuring the ability of peptides to compete against 125 I labeled salmon calcitonin for binding to specific receptors in membranes represents a particularly useful means for identifying peptides and other chemical compounds that interact with these receptors. The myotonin receptor assay can be used to determine the concentration of myotonin receptor- or amylin receptor-active compounds in unknown solutions or mixtures. Myotonin receptors are assayed as described below. For example, a membrane or cell preparation containing a high density of myotonin receptors is incubated with, for example, radiolabelled calcitonin and unlabelled amylin. In this manner, a competition curve is generated relating the amount of amylin in the assay tube to the inhibition of radiolabelled calcitonin binding produced. In additional tubes, unlabelled peptide is replaced by a solution containing an unknown amount of amylin to be quantified. This solution may be plasma, serum or other fluid, or solid mixture dissolved in assay buffers. The unknown solution is preferably added in a volume of less than or equal to about 10% of the final assay volume, so as not to significantly alter the ionic content of the solution. If larger volumes of unknown are used, a solution containing an equivalent salt content is included as a control for effect's of altered ionic content on binding. Nonspecific binding, i.e.. binding of radiolabelled calcitonin in the presence of a high concentration (10"°M) of unlabelled amylin or salmon calcitonin, is subtracted from total binding for each sample to yield specific binding. The amount of inhibition of specific binding of radiolabelled calcitonin produced by the unknown is compared to the inhibition curve produced by amylin in order to determine the content of amylin or amylin receptor-active substances in the unknown sample. Methods for performing these calculations are described in several sources, such as in Neurotransmitter Receptor Binding, eds H. Yamamura, S.J. Enna, and M. . Kuhar (Raven Press, New York, 1991) . This method is used to quantitate the amount of myotonin-receptor active compounds in a known or an unknown sample, and may be used to quantitate myotonin- receptor active compounds in plasma or other body fluids and tissues, for use in identifying active metabolites, pharmacokinetics, stability, solubility, or distribution of myotonin receptor agonists and antagonists, amylin agonists and amylin antagonists. It may also be used to identify, isolate and purify peptides having a high affinity for the myotonin receptor. In order to increase the specificity of the assay for amylin where this is necessary, the quantity of CGRP in the unknown sample can be determined through a radio- receptor assay for CGRP. Such a radio-receptor assay can be performed using 125 I-hCGRP and rat liver membranes according to the methods described for the myotonin radio- receptor assay. With this assay, the CGRP content of the unknown sample can be determined. Since the myotonin radio-receptor assay can identify all compounds active on myotonin receptors, including CGRP, it is useful to subtract CGRP content, as determined by radio-receptor or other assay (e.g.. radioimmunoassay) from total content of myotonin-receptor active compounds to yield the amount of amylin in samples (e.g. , serum) which may contain both amylin and CGRP. The myotonin receptor can also be used in a high throughput screen, optionally utilizing robotic systems such as those known in the art, for identifying compounds which displace myotonin or amylin from this receptor and, thus, identifying candidate myotonin or amylin agonists or antagonists. The assay can be used to screen, for example, libraries of synthetic compounds, extracts of plants, extracts of marine organisms, or bacterial or fungal fermentation broths. In one embodiment, . an initial step brings together the myotonin receptor preparation, pre-incubated with radiolabelled calcitonin and a solution of test compound. For organic extracts, the final concentration of solvent should generally not exceed that which displaces the standard displacement curve of labelled calcitonin by cold calcitonin by 25%, i.e. , shifts the measured IC 50 by less than 25%. This can be evaluated for each selected solvent. For identified compounds from synthetic libraries, the test concentration will be about lOOnM, lμM, or lOμM depending on the frequency with which positive tests occur. A positive will typically be represented by at least about a 20% reduction of specific binding of labelled amylin. With broths and extracts, a positive test will be denoted by at least about 20%, 50% or 80% reduction in specific calcitonin binding, according to the frequency of positive tests. It is useful in high throughput screening to check compounds or mixtures giving a positive test in an initial screen for non-specific interference with ligand binding. In a preferred embodiment, all positive testing compounds or extracts are exposed to a binding assay for another ligand in the same membrane preparation. A suitable assay for evaluating non-specific effects will be a radiolabelled standard reagent for determination of binding to a standard receptor in the vas deferens or tissue being used. Those receptors which are relatively abundant in the tissue and readily assayed should be chosen. Any compound, broth, or extract that tests positive in the myotonin receptor screen, and which also tests positive by the same quantitative criteria in the standard receptor screen is rejected as non-selectively interfering with ligand binding to membrane receptors. The invention also includes determination of the interaction with CGRP receptors of compounds, broths or extracts which selectively reduce amylin binding to myotonin receptors. Thus, steps similar to those described above are undertaken with a binding assay consisting of cells or membranes having myotonin receptors pre-incubated with I25 I-his 10 _human α-CGRP. Using the quantitative criteria specified above, compounds, broths or extracts are identified which selectively act at the myotonin receptor but not at the CGRP receptor. For compounds meeting described criteria, the potency of interaction with the myotonin receptor and, if relevant, the amylin, CGRP or calcitonin receptors, are determined by measuring the displacement of ligand from the membrane preparations by a range of concentrations of the test compound. With mixtures of unknown compounds, as in broths and extracts, the desired activity is isolated and purified by art-known methods including HPLC, followed by testing the separated materials to determine which retain the desired activity. When pure or relatively pure active material is obtained, its potency at the myotonin, amylin, and CGRP receptor can also be determined. Art- known methods including NMR, mass spectroscopy, and elemental analysis may be used to make a chemical identification of any isolated material having the desired receptor binding activities. At any desired stage following identification of selective displacement of myotonin or amylin from its receptors, a positive testing material can be assessed in a functional assay to assess amylin agonist activity through, for example, inhibition of insulin-stimulated incorporation of labelled glucose into glycogen in rat soleus muscle. The material can also be tested for antagonist activity in this assay by assessing its ability to restore insulin-stimulated incorporation of labelled glucose into glycogen in rat soleus muscle incubated with 10, 20, 50 or 100 nM rat amylin. Also, by applying different concentrations of the test material in these assays, the potency of myotonin or amylin agonist or antagonist action can be determined. Another test of myotonin receptor agonist action uses the measurement of elevation of plasma lactate and/or glucose in, for example, halothane-anaesthetized, 18-hour fasted rats following intravenous bolus injections of the test material. By using a series of concentrations, the potency of the material as an amylin agonist can be determined. In a related assay for antagonist activity the test material is infused intravenously into 18-hour fasted, anaesthetized rats. The reduction (compared to control conditions) of the hyperlactemic and/or hyperglycemic response to intravenous injections of a known amount of amylin agonist is then measured or otherwise evaluated. The antagonist potency of such materials can be determined by repeating the test at different infusion rates of the test material. In other embodiments, for assessment of whether materials testing positive in the myotonin receptor binding assay are agonist or antagonists, the test materials are brought together with myotonin-responsive membrane or cell systems in which amylin changes rates of synthesis of cyclic AMP (cAMP) . Such preparations include membranes prepared from cultured cell lines with abundant myotonin or amylin receptors, or the cells themselves. Changes in cAMP levels are measured by radioimmunoassay following exposure of the membrane or cell preparations, incubated according to art-known methods. Materials testing positive in displacing amylin from its receptors or myotonin receptors and having no effect on cAMP production can be expected to be amylin receptor antagonists. Antagonist action can be further evaluated by incubating various concentrations of the material analog with amylin or an amylin agonist and measuring the degree of inhibition of the changes in cAMP evoked by amylin or an amylin agonist. The invention can also be used to screen cell lines, cells disaggregated from tissue, and cells from human or animal blood for amylin receptors. These cells will be used as a readily available source for additional myotonin receptor preparations for development of agonists and antagonists of amylin. Membranes from cells are obtained by homogenization of cells with an instrument such as Polytron (Brinkman Instruments) followed by centrifugation. Membranes so obtained are combined with, e.g. , 125 I-salmon calcitonin in a buffer system such as that described in the examples below, and are incubated and collected as described in those examples. Specific binding of 125 I-salmon calcitonin to the cell membrane is identified by measuring the decrease in binding obtained in the presence of, for example, 10" 7 M rat amylin or 10" 7 M salmon calcitonin. Cells in which there is a significant difference between total binding (triplicate tubes) and nonspecific binding (triplicate tubes) at the P<0.05 level will be used for further study of myotonin receptor function. Subcellular membrane fractions obtained by differential or density gradient centrifugation are assayed for specific binding of radiolabeled calcitonin in order to identify the membrane fraction containing the highest density of myotonin receptors per milligram protein (as assayed by Bradford or Lowry protein assays) . The membrane fraction with highest receptor density is preferably used for further purification. This membrane fraction is collected and treated in a buffered solution with several membrane solubilizing agents, including triton, digitonin, octyl glucoside, deoxycholate, and cholate, at concentrations of from 0.001% to 1% detergent at reduced temperature (4°C) for about 1 hour. Protease inhibitors (including phenylmethylsulfonyl fluoride, EDTA, aprotinin) are included in the buffer system to prevent receptor degradation during or after solubilization. After treatment of membranes with detergents, unsolubilized membranes are sedimented by centrifugation at high speed (100,000 x g for 1 hour) and resulting supernatants containing solubilized receptors are assayed for binding of radiolabeled calcitonin as described above. Solubilized receptors can be collected by filtration on polyethyleneimine-coated filters (Bruns et al. Anal. Biochem. 132:74-81. 1983. Alternatively, solubilized receptors are collected by methods such as precipitation with polyethyleneglycol, gel filtration, or equilibrium dialysis. Binding characteristics (such as affinity for amylin, CGRP and calcitonins) of solubilized receptors are assessed and should match the characteristics of membrane- localized receptors. After determining conditions suitable for solu¬ bilizing myotonin receptors and for assaying solubilized receptors, these solubilized receptors are purified away from other solubilized membrane proteins by chromato- graphic procedures, such as affinity chromatography on supports to which myotonin, amylin or calcitonin has been coupled, ion exchange chromatography, lectin agarose chromatography, gel filtration, and hydrophobic inter¬ action chromatography. Chromatography column eluates are tested for specific myotonin receptor binding to protein content, in order to identify peaks containing receptors and the extent of purification. Before inclusion in the final purification protocol, each chromatographic step is tested to determine the extent to which it contributes to receptor purification, as measured by an increase in specific radiolabeled myotonin, amylin, or calcitonin binding per milligram protein. Desired chromatography steps are combined sequentially, using large quantities of starting material, in order to obtain partially or com¬ pletely purified amylin receptors, as desired. Receptors which have been partially or completely purified by this method are used to generate myotonin receptor-specific antibodies for use in diagnosis (disease states with altered receptor density, distribution, or antigenicity) and for use in screening recombinant libraries for myotonin receptor expression. Purified receptor preparations can also be used to obtain partial sequence information, which is useful in preparing oligonucleotide probes for screening recombinant libraries for myotonin receptor-encoding gene sequences. Specific embodiments of the receptor binding assay screening method of this invention are exemplified in the following Examples. These Examples are not to be inter¬ preted as limiting the scope of the invention in any way, the scope being disclosed in the entire specification and claims. Example 1: Identification of Myotonin Receptor It was discovered that salmon calcitonin has a high affinity for amylin receptors in rat nucleus accumbens, and that salmon calcitonin is more potent than amylin at inhibiting insulin-stimulated glycogen synthesis in isolated soleus muscle. To determine whether radioiodinated salmon calcitonin might be a useful ligand for labeling amylin receptors in rat soleus, salmon calcitonin was radioiodinated using chloramine-T procedures. Binding experiments were done to test the ability of 125 I-salmon calcitonin to bind to membranes prepared from skeletal muscle and other tissues according to the following procedure: Sprague-Dawley rats (200-250 gm) were sacrificed by decapitation, and skeletal muscles (hindlimb or soleus) removed to cold phosphate-buffered saline (PBS) and weighed. Muscles were transferred to approximately 10 volumes cold HEPES buffer (20 mM HEPES, pH brought to 7.4 with NaOH) , and homogenized with a Polytron. Homogenates were centrifuged 10 min at 200 x g and the pellet discarded. The supernatant was centrifuged 15 minutes at 48,000 x g to obtain muscle membranes. Membranes were washed three times in fresh buffer by centrifugation for 15 min at 48,000 x g, followed each time by rehomogeni- zation of the membrane pellet in 40 ml fresh buffer. The final membrane pellet was resuspended in 20 mM HEPES buffer containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF) at a concentration of 50 mg original wet weight/ml. To measure binding of 125 I-salmon calcitonin to skeletal muscle membranes, the following were combined in 12 x 75 polypropylene assay tubes the following: 0.3 ml HBBP buffer (20 mM HEPES; 1 mg/ml bacitracin, 2.5 mg/ml BSA, 0.2 mM PMSF); 0.05 ml 125 I- salmon calcitonin (specific activity 1000 - 2000 Ci/mmol, final concentration - 2-10 pM) 0.05 ml HBBP or test peptide dissolved in HBBP; and 0.1 ml muscle membrane in HEPES/PMSF buffer. The muscle membranes were added last to start the incubation, which was carried out for 90 minutes at ambient temperature. Incubations were terminated by filtration with a har¬ vesting apparatus (Brandel cell harvester) through glass fiber filters (Whatman, GF/B filters) which were presoaked for at least 4 hours in 0.3% polyethyleneimine. Filters were washed with 15 ml cold phosphate-buffered saline (PBS) and radioactivity on individual filters determined with a gamma counter. Competition curves were generated by measuring binding of 125 I-salmon calcitonin in the presence of 10" 12 to 10 "6 M unlabeled peptide and were analyzed by non linear regression using a 4-parameter logistic equation (Inplot program; GraphPAD Software, San Diego) . Referring to Fig. 1, to determine binding constants, saturation curves were generated and analyzed according to modifications of the method of Scatchard, as described in detail by Bylund & Yamamura. For saturation experiments, binding of 12S I-salm©n calcitonin was measured at concentrations varying from 1 - 100 pM to obtain total binding, and again in the presence of 100 nM unlabeled salmon calcitonin to obtain nonspecific binding. Referring to Fig. 2, Scatchard plots of saturation binding curves indicate that a high affinity salmon calcitonin binding site is present in rat hindlimb muscle membranes with a dissociation constant (Kd) of approximately 11 pM. In a competitive binding experiment, several peptides were tested for their ability to inhibit 125 I-salmon calcitonin binding to rat skeletal muscle, using the procedure described above. The following IC50s were obtained: IC50(nM) salmon calcitonin 0.003 rat αCGRP 21 rat amylin 30 AC66 (sCalcitonin 8 . 32 ) 0.79 hCGRP 8 . 37 180 This binding profile is quite different from that of the amylin receptor -- for which salmon calcitonin and rat amylin have nearly equal potency. At the rat amylin receptor, AC66 was approximately 4-fold more potent than CGRP 8 _ 37 , in contrast to the markedly difference in potency of these compounds for the skeletal muscle 12S I-salmon calcitonin binding site. This binding site is referred to for convenience as the myotonin receptor (mvocyte + calcitonin) to distinguish it from other binding sites. Example 2: Cell lines with myotonin receptor While binding assays demonstrate the affinity of compounds for receptors, functional assays are needed to determine whether compounds active in binding assays are agonists or antagonists, or indeed whether they interfere with binding through nonspecific actions (e.g. , membrane effects) . For this reason, and also in order to investigate post-receptor actions of amylin, binding assays were used to screen several cell lines for the presence of 125 I-rat amylin, 125 l-human CGRP-1, and 125 l-salmon calcitonin binding sites. Cell lines were obtained from ATCC, grown in tissue culture flasks in the recommended media, and harvested by scraping with a cell scraper into cold PBS. Cells were collected by centrifugation and homogenized with a polytron in 20 mM HEPES buffer. Membranes were washed 3x by homogenization in fresh buffer and centrifugation (15 min at 48,000 x g) . Membranes were resuspended in HEPES/PMSF buffer at a concentration of 2 mg membrane protein/ml. Binding was measured as in Example 1 with the following modifications: final volume was 0.2 ml containing 0.1 ml HBBP, 0.02 ml HBBP or competing peptide in HBBP, 0.03 ml radiolabeled ligand, and 0.05 ml cell membrane. Nonspecific binding was determined in the presence of 10 "6 M unlabeled peptide (rat amylin, hCGRP, or salmon calcitonin for their respective binding sites) . Numerous cell lines were found not to contain specific 125 I- salmon calcitonin binding sites, including L6 myoblast, HepG2, RBA mammary carcinoma, GH 3 pituitary, L8 skeletal muscle, BC 3 H1 smooth muscle, C 2 C 12 myoblast, Hs729T Rhabdomyosarcoma, SK-N-MC neuroblastoma, C6 glioma, G7 myoblast, G8 myoblast and Nor 10 muscle cells lines. Hamster leiomyosarcoma cells (DDT-MF-2) , derived from vas deferens smooth muscle and reported (Trevor et. al., 2 JPET 718-725) to contain a CGRP receptor variant (designated CGRP 2 by these authors) that responded to agonists and antagonists differently than the more widespread CGRP* receptor were also assayed. Referring to Fig. 3, DDT-MF-2 cells contained very high densities of 125 I-salmon calcitonin binding sites, with the following IC50s: sCal (8 pM) ; rat amylin (233 nM) ; rat CGRP-1 (69 nM) . The high affinity for salmon calcitonin, and relative potencies of amylin and CGRP, indicated that these binding sites were similar to the rat myotonin binding sites. Example 3 : Functional assay of myotonin receptor To determine functional relevance of these sites, the ability of salmon calcitonin to stimulate cyclic AMP accumulation in DDT t -MF2 cells was studied. DDT-MF-2 cells were grown to confluence in 24-well tissue culture clusters, media was removed and replaced with Dulbecco's PBS containing 0.1 mg/ml BSA + 0.1 mg/ml isobutylmethy- Ixanthine. After 15 minutes incubation at 37°C, cells were stimulated with indicated peptide for 15 minutes. Reaction was stopped by addition of trichloroacetic acid, and cycli AMP was measured in neutralized supernatants using a commercially available radioimmunoassay (RIA) kit. Referring to Fig. 4, Salmon calcitonin (1 nM) strongly stimulated cyclic AMP accumulation (greater than 3Ox baseline) and this effect was blocked by coincubation with AC66 (4 μtΛ) . To adapt this assay for use as a high throughput screen for functional activity and to explore the pharmacology of the identified activity, the assay was adapted to 96-well plate format. DDT-MF-2 cells are plated at 10 4 cells per well. On day 4 after plating, cells are washed once with Delbecco's Phosphate Buffered Saline (DBPS, Sigma Chemicals) containing 0.1 mg/ml isobu- tylmethylxanthine and test compound at 37°C. Following 15 minutes incubation at 37°C, the reaction is stopped by addition of trichloroacetic acid, followed by neutra¬ lization with TRIS base. Neutralized reactants are assayed for cAMP by RIA. Referring to Fig. 5, Schild analysis was performed using a single 96-well plate, demonstrating that AC66 has a Ki = 13 nM. Example 4: Agonist Assay Referring to Figs. 6 and 7, a study of agonist peptides demonstrated that salmon calcitonin is more than lOOOOx more potent than amylin and CGRP in stimulating cyclic AMP generation. These results are consistent with binding results for high affinity salmon calcitonin binding sites in DDT-MF-2 cells and rat skeletal muscle, indicating that these binding sites represent functional receptors, are coupled to stimulation of adenylate cyclase, and that AC66 is an antagonist at these receptors. Example 5: Antagonist Assay The ability of antagonists to block salmon calcitonin-mediated effects on glucose metabolism in soleus muscle was measured to determine whether myotonin receptors contributed to these effects. For these assays, insulin-stimulated glycogen synthesis was measured in isolated soleus muscle in the presence of 1 nM salmon calcitonin, which suppresses the effect of insulin. The IC 50 s for reversing the effects of salmon calcitonin were: AC66 (0.151 μ ) and hCGRP 8 . 37 (48 μM) . This is a 320-fold difference in potency, which compares favorably with the 230-fold difference in potency at the myotonin receptor. In contrast, there was only a 5-fold difference in potency of these two antagonists in their binding affinities for the amylin receptor (AC66 = 1.06 nM, CGRP 8 . 37 - 4.6 nM) . IC 50 s of these antagonists at blocking the effects of amylin in the soleus muscle are: AC66 = 0.9 μM and hCGRP g-37 = 6.6 μM. This 6-fold difference in potency correlates well with the 5-fold potency ratio at amylin receptors. These results indicate that salmon calcitonin antagonizes insulin actions upon skeletal muscle glycogenesis and that this action is at least partially mediated by myotonin receptors -- a receptor population distinct from the amylin receptors that mediate amylin*s actions. Studies in DDT-MF-2 cells indicate that myotonin receptors are linked to stimulation of adenylate cyclase activity. Stimulation of cyclic AMP generation in skeletal muscle would be likely to produce glycogenolysis epinephrine produces such effects via adenylate cyclase-coupled beta noradrenergic receptors located in skeletal muscle. The results indicate that AC66 may block insulin resistance produced by the endogenous compound that activates myotonin receptors. Other embodiments are within the following claims.

Claims: Claims 1. An assay method for use in identifying or screening for myotonin receptor binding compounds which comprises the steps of, (a) bringing together a test sample and a myotonin receptor preparation, said test sample containing one or more test compounds, and said myotonin receptor preparation containing a myotonin receptor protein which binds calcitonin or amylin agonist or antagonist; (b) incubating said test sample and said myotonin receptor preparation under conditions which permit the binding of calcitonin or amylin agonist or antagonist to said myotonin receptor protein; and, (c) identifying those test samples containing one or more test compounds which detectably bind to said myotonin receptor. 2. The assay method of claim 1 which further comprises, (d) screening said test samples which detectably bind to said receptor for in vitro or in vivo stimulation or inhibition of myotonin receptor-mediated activity; and, (e) identifying those test samples which act as agonists or antagonists of calcitonin or amylin. 3. The assay method of claim 1 wherein said myotonin receptor preparation comprises isolated cells bearing said myotonin receptor. 4. The assay method of claim 1 wherein said myotonin receptor preparation comprises isolated membranes bearing said myotonin receptor. 5. The assay method of claim 1 wherein said myotonin receptor preparation comprises isolated myotonin receptor protein. 6. The assay method of claim 1 wherein said test samples which detectably bind to said myotonin receptor protein are identified by measuring the displacement of a labelled first ligand from said myotonin receptor preparation by said test sample, and comparing the measured displacement of said first labelled ligand from said myotonin receptor preparation by said test sample with the measured displacement of said first labelled ligand from said myotonin receptor preparation by one or more known second ligands. 7. The assay method of claim 6 wherein said labelled first ligand is calcitonin. 8. The assay method of claim 6 wherein said labelled first ligand is an amylin agonist. 9. The assay method of claim 6 wherein said labelled first ligand is an amylin antagonist. 10. The assay method of claim 6 wherein said first ligand is labelled with a member selected from the group consisting of radioactive isotopes, nonradioactive isotopes, fluorescent molecules, chemiluminescent molecules, and biotinylated molecules. 11. The assay method of claim 7 wherein said calcitonin is salmon calcitonin. 12. The assay method of claim 11 wherein said salmon calcitonin is 125 I-salmon calcitonin. 13. The assay method of any of claims 6, 7, 8, 9 or 10 wherein said known second ligand or ligands are selected from the group consisting of amylin, calcitonin, α-CGRP, and 0-CGRP. 14. The assay method of any of claims 6, 7, 8, 9 or 10 wherein said known second ligand or ligands are selected from the group consisting of human amylin, dog amylin, rat amylin, human calcitonin, rat calcitonin, eel calcitonin, salmon calcitonin, human α-CGRP, human β-CGRP, rat α-CGRP, and rat β-CGRP. 15. The assay method of claim 6, wherein said test sample contains more than one test compound, which further comprises the steps of, (d) preparing two or more additional test samples from said test sample, said additional test samples being characterized in that they contain a lesser number of test compounds than said test sample from which they were prepared; and, (e) repeating steps (a) - (d) as many times as required until the test compound or compounds which bind to said myotonin receptor have been identified. 16. The assay method of claim 2 wherein said test samples which detectably bind to said myotonin receptor protein are identified by measuring the displacement of a labelled first ligand from said myotonin receptor preparation by said test sample, and comparing the measured displacement of said first labelled ligand from said myotonin receptor preparation by said test sample with the measured displacement of said first labelled ligand from said myotonin receptor preparation by one or more known second ligands. 17. The assay method of claim 16, wherein said test sample contains more than one test compound, which further comprises the steps of, (f) preparing two or more additional test samples from said test sample, said additional test samples being characterized in that they contain a lesser number of test compounds than said test sample from which they were prepared; and, (g) repeating steps (a) - (f) as many times as required until the test compound or compounds which bind to said myotonin receptor have been identified. 18. The assay method of any of claims 1, 2 or 6 wherein said test sample comprises one or more known test compounds. 19. The assay method of any of claims 1, 2 or 6 wherein said test sample comprises one or more unknown compounds. 20. The assay method of claim 3 wherein said cells bearing said myotonin receptor are smooth muscle cells. 21. The assay method of claim 20 wherein said smooth muscle cells are from a vas deferens. 22. The assay method of claim 4 wherein said membranes bearing said myotonin receptor are isolated from smooth muscle cells. 23. The method of claim 22 wherein said smooth muscle cells are from a vas deferens. 24. The method of claim 5 wherein said myotonin receptor protein is isolated from smooth muscle cells. 25. The method of claim 24 wherein said smooth muscle cells are from a vas deferens. 26. The method of claim 8 wherein said amylin agonist is selected from the group consisting of CGRP, salmon calcitonin, and eel calcitonin. 27. The method of claim 9 wherein said amylin antagonist is CGRP g . 37 28. An assay method for evaluating one or more receptor binding characteristics sought to be determined for a known or candidate compounds useful for treatment of a hypoglycemic condition or a disease characterized by insulin resistance, which comprises the steps of, (a) assessing or measuring the ability of said compound to compete against a labelled ligand for binding to a myotonin receptor preparation, said myotonin receptor preparation containing a myotonin receptor protein which binds calcitonin or an amylin agonist or antagonist; (b) assessing or measuring the ability of said compound to compete against said labelled ligand for binding to a CGRP receptor preparation, said CGRP receptor preparation containing a CGRP receptor protein which binds CGRP; or, (c) assessing or measuring the ability of said compound to compete against said labelled ligand for binding to a calcitonin receptor, said calcitonin receptor preparation containing a CGRP receptor protein which binds calcitonin; or, (d) carrying out both steps (b) and (c) ; and, (e) determining the receptor binding characteristic sought to be determined for said compound. 29. The assay method of claim 28 wherein said binding characteristic sought to be determined for said compound is myotonin receptor binding affinity. 30. The assay method of claim 28 wherein said binding characteristic sought to be determined for said compound is myotonin receptor binding specificity. 31. The assay method of claim 28 wherein said CGRP receptor preparation comprises hepatocytes. 32. The assay method of claim 28 wherein said CGRP receptor preparation comprises hepatocyte membranes. 33. The assay method of claim 31 wherein said hepatocytes are comprised of a primary cell culture or an established cell line. 34. The assay method of claim 33 wherein said hepatocytes comprise an established cell line, and said established hepatocyte cell line is the Hep G2 cell line. 35. The assay method of claim 28 wherein said CGRP receptor preparation comprises myocytes . 36. The assay method of claim 28 wherein said CGRP receptor preparation comprises myocyte membranes. 37. The assay method of claim 35 wherein said myocytes are comprised of a primary cell culture or an established cell line. 38. The assay method of claim 36 wherein said myocytes comprise an established cell line, and said established cell line is the L6 cell line. 39. An assay method for determining the presence or amount of a myotonin receptor binding compound in a test sample to be assayed for said compound, which comprises the steps of, (a) bringing together said test sample to be assayed and a myotonin receptor preparation, said myotonin receptor preparation containing a myotonin receptor protein which binds an amylin agonist or antagonist; (b) measuring the ability of said test sample to compete against a labelled ligand for binding to said myotonin receptor preparation; or, (c) relating the amount of myotonin receptor binding compound in said test sample with the amount of myotonin receptor binding compound measured for a control sample in accordance with steps (a) and (b) , said control sample being known to be free of any myotonin receptor binding compound, or (d) relating the amount of myotonin receptor binding compound in said test sample with the amounts of myotonin receptor binding compound measured for control samples containing known amounts of myotonin receptor binding compound in accordance with steps (a) and (b) , to determine the presence or amount of myotonin receptor binding compound in said test sample, or (e) performing both steps (b) and (c) to determine the presence or amount of myotonin receptor binding compound in said test sample, or (f) performing both steps (b) and (d) to determine the presence or amount of myotonin receptor binding compound in said test sample, or (g) performing steps (c) and (d) to determine the presence or amount of myotonin receptor binding compound in said test sample, or (h) performing steps (b) , (c) and (d) to determine the presence or amount of myotonin receptor binding compound in said test sample. 40. The assay method of claim 39 wherein said myotonin receptor binding compound is amylin. 41. The assay method of claim 39 wherein said myotonin receptor binding compound is an amylin agonist. 42. The assay method of claim 39 wherein said myotonin receptor binding compound is an amylin antagonist. 43. The assay method of claim 39 wherein said labelled ligand is amylin. 44. The assay method of claim 39 wherein said labelled ligand is an amylin agonist. 45. The assay method of claim 39 wherein said labelled ligand is an amylin antagonist. 46. The assay method of claim 39 wherein said myotonin receptor binding compound is amylin, and said labelled ligand is amylin. 47. The assay method of claim 39 wherein said myotonin receptor binding compound is an amylin agonist and said labelled ligand is an amylin agonist. 48. The assay method of claim 39 wherein said myotonin receptor binding compound is an amylin antagonist and the labelled ligand is an amylin antagonist. 49. The assay method of claim 39 wherein said test sample is a biological fluid. 50. The assay method of claim 49 wherein said biological fluid is a member selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, and lymph fluid. 51. The assay method of claim 39 wherein said test sample is an amylin preparation. 52. The assay method of claim 39, further comprising evaluating the stability of an amylin preparation. 53. The assay method of claim 39, further comprising evaluating the potency of an amylin preparation. 54. The assay method of claim 39, further comprising evaluating the solubility of an amylin preparation. 55. A method of producing monoclonal antibodies that bind to a myotonin receptor, which comprises the steps of, (a) immunizing an animal with a myotonin receptor preparation; (b) recovering B lymphocytes from said immunized animals; (c) fusing said recovered B lymphocytes with malignant cells to produce hybridomas; (d) recovering hybridomas that produce antibodies that bind the myotonin receptor; and, (e) recovering antibodies from one or more hybridomas selected in step (d) . 56. A method of producing antibodies against a myotonin receptor, which comprises the steps of, (a) immunizing an animal with an myotonin receptor preparation; (b) selecting those animals whose sera contain anti-myotonin receptor antibodies; and, (c) recovering sera containing anti-myotonin receptor antibodies from said selected animals. 57. The method of any of claims 55 or 56 wherein said myotonin receptor preparation is a member selected from the group consisting of a vas deferens membrane preparation, the cell line DDT-MF-2 leiomyosarcoma, and an isolated myotonin receptor protein preparation. 58. A method for separating myotonin receptor binding compounds from a sample, which comprises the steps of, (a) bringing together said sample and an myotonin receptor preparation, said myotonin receptor preparation comprising myotonin receptor protein molecules bound to a solid carrier; and (b) separating any myotonin receptor binding compound which is bound to said myotonin receptor preparation from the remainder of said test sample which is unbound. 59. A method for screening a biological substance for the present of myotonin receptors, which comprises the steps of, (a) bringing together said biological substance with first myotonin receptor binding compound; (b) bringing together said biological substance with a second myotonin receptor binding compound; (c) optionally bringing together said biological substance with one or more additional myotonin receptor binding compounds; and, (d) determining the relative binding affinities of said myotonin receptor binding compounds for receptors in said biological substance. 60. The method of claim 59 wherein said biological substance is a cell line. 61. The method of claim 59 wherein said biological substance comprises isolated cells. 62. The method of claim 59 wherein said myotonin receptor binding compounds are selected from the group consisting of the amylins, the calcitonins, the α-CGRPs, and the j -CGRPs. 63. The method of claim 60 wherein said cell line is the DDT-MF-2 leiomyosarcoma cell line. 64. The method of claim 3, 21, or 24 wherein said cells are DDT-MF-2 leiomyosarcoma cell line. 65. Purified myotonin receptor. 66. Purified nucleic acid encoding a myotonin receptor.

Inventors: BEAUMONT KEVIN, BEAUMONT, KEVIN
Applicants: AMYLIN PHARMACEUTICALS INC, AMYLIN PHARMACEUTICALS, INC.
Application year: 1993
Main classification: G01N33
IPC codes: C07K14, C07K16, C12N15, C12P21, C12R1, G01N33