Effects of Recombinant Agouti- Signaling Protein on Melanocortin Action

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1 Effects of Recombinant Agouti- Signaling Protein on Melanocortin Action Ying-Kui Yang, Michael M. Ollmann, Brent D. Wilson, Chris Dickinson, Tadataka Yamada, Gregory S. Barsh, and Ira Gantz Departments of Internal Medicine (Y-K.Y., T.Y.), Pediatrics (C.D.), Physiology (T.Y.), and Surgery (I.G.) University of Michigan Ann Arbor, Michigan Howard Hughes Medical Institute and The Departments of Pediatrics and Genetics Stanford University (M.M.O., B.D.W., G.S.B.) Stanford, California /97/$3.00/0 Molecular Endocrinology Copyright 1997 by The Endocrine Society Mouse agouti protein is a paracrine signaling molecule that has previously been demonstrated to be an antagonist of melanocortin action at several cloned rodent and human melanocortin receptors. In this study we report the effects of agouti-signaling protein (ASIP), the human homolog of mouse agouti, on the action of -MSH or ACTH at the five known human melanocortin receptor subtypes (hmcr 1 5). When stably expressed in L cells (hmc1r, hmc3r, hmc4r, hmc5r) or in the adrenocortical cell line OS3 (hmc1r, hmc2r, hmc4r), purified recombinant ASIP inhibits the generation of camp stimulated by -MSH (hmc1r, hmc3r, hmc4r, hmc5r) or by ACTH (hmc2r). However, dose-response and Schild analysis indicated that the degree of ASIP inhibition varied significantly among the receptor subtypes; ASIP is a potent inhibitor of the hmc1r, hmc2r, and hmc4r, but has relatively weak effects at the hmc3r and hmc5r. These analyses also indicated that the apparent mechanism of ASIP antagonism varied among receptor subtypes, with characteristics consistent with competitive antagonism observed only at the hmc1r, and more complex behavior observed at the other receptors. ASIP inhibition at these latter receptors, nonetheless, can be classified as surmountable (hmc3r, hmc4r and hmc5r) or nonsurmountable (hmc2r). Recombinant ASIP also inhibited binding of radiolabeled melanocortins, [ 125 I-Nle 4, D-Phe 7 ] -MSH and [ 125 I-Phe 2, Nle 4 ]ACTH 1 24, to the hmcr 1 5 receptors, with a relative efficacy that paralleled the ability of ASIP to inhibit camp accumulation at the hmc1r, hmc2r, hmc3r, and hmc4r. These results provide new insight into the biochemical mechanism of ASIP action and suggest that ASIP may play an important role in modulating melanocortin signaling in humans. (Molecular Endocrinology 11: , 1997) INTRODUCTION The human homolog of the mouse agouti gene encodes an 132-amino acid paracrine factor named agouti-signaling protein (ASIP). Expression of the mrna for this protein has been observed in diverse human tissues including heart, liver, kidney, testis, ovary, adipose tissue, and foreskin (1, 2). Although there is no firmly established physiological function for this protein in humans, its role in determination of coat color in fur-bearing mammals is well recognized (3). Expression of agouti causes melanocytes to switch from the synthesis of black or brown pigment, eumelanin, to the synthesis of yellow pigment, pheomelanin, and normally controls the distribution of these pigment types in individual hairs and in different regions of the body (4). The effects of agouti on fur color require the presence of a functional melanocortin 1 receptor (MC1R), a G protein-coupled receptor in which activating or loss-of-function mutations result in constitutive synthesis of eumelanin or pheomelanin, respectively. The MC1R can be activated by several peptide hormones derived from POMC, including -MSH and ACTH. These peptides are produced at very high levels in the brain and pituitary gland, but the role of melanocortins in the normal control of pigmentation is uncertain. By contrast, activation of the adrenal-specific MC2R by circulating ACTH is the major stimulus for cortisol production, and activation of the MC3R and MC4R in the 274

2 ASIP Inhibition of Melanocortins 275 central nervous system is thought to account for effects of melanocortins on learning, behavior, and cardiovascular regulation. Insight into the mechanism by which agouti exerts its control over coat color has come from studies of Lu et al. (5), in which recombinant mouse agouti protein was shown to antagonize the effects of -MSH on heterologous cells that expressed the melanocortin-1 receptor (MC1R). Lu et al. found that mouse agouti protein had no effect on the MC3R or MC5R but was a potent antagonist of the MC4R, a brain-specific receptor that responds to ACTH as well as to -MSH (6). Additional studies by Blanchard et al. (7) have suggested that mouse agouti protein is a competitive antagonist of the endogenous MC1R expressed on B16F10 mouse melanoma cells. Potential pathophysiological action of agouti protein at one or more of the extrapigmentary melanocortin receptors has been suggested by the phenotype of mutant mice in which agouti is expressed ubiquitously, such as viable yellow (A vy ) or lethal yellow (A y ), which have pleiotropic effects including obesity, diabetes, and increased tumor susceptibility (8 10). These findings have generated additional interest in the possible role of agouti, melanocortins, and their receptors in weight homeostasis, but the relevance of ASIP in human physiological or pathophysiogical function remains to be elucidated. Here we report the biochemical properties of ASIP relative to the full spectrum of human melanocortin receptors including the adrenalspecific MC2R. Our findings indicate that ASIP is a potent inhibitor of MC1R, MC2R, and MC4R signaling, but that its mechanism of action is complex and unlikely to be explained by simple competitive antagonism. RESULTS The preparations of agouti-signaling protein (ASIP) used in these studies were purified from the conditioned media of insect cells infected with a baculovirus construct that expressed recombinant ASIP (M. M. Ollmann and G. S. Barsh, unpublished). Identical results were obtained with several preparations that ranged in purity from 90% to 99%. Cell lines that stably expressed each of the human melanocortin receptor subtypes, hmcr 1 5, were exposed to various concentrations of ASIP for 30 min before the addition of melanocortin agonist, and camp accumulation was then measured after an additional 30 min incubation. Control experiments indicated that ASIP had no effect on basal camp levels in cell lines transfected with the MCRs and also had no effect on histamine-stimulated camp levels in L cells that stably express the histamine H2 receptor (data not shown). As shown in Fig. 1, ASIP is an antagonist of melanocortin action at all the human MCRs, but the extent of this inhibition is variable. Furthermore, the apparent mechanism of ASIP inhibition at the various receptor subtypes is also different. In L cells that express the hmc1r, ASIP induces a parallel rightward shift in the dose-response curves for -MSH-stimulated camp generation and has no significant effect on maximal stimulation (Fig. 1A). To gain additional insight into the mechanism of ASIP action, we plotted the EC 50 of -MSH (dose ratio) as a function of antagonist concentration (inset, Fig. 1A). For a competitive antagonist, the agonist dose ratio is proportionate to the change in antagonist concentration, and the slope of this plot should approximate unity (Schild analysis). Our results with the hmc1r are consistent with competitive antagonism, since the slope of the Schild regression is (Fig. 1A), with an inhibitory constant (K i ) of nm (Table 1). Similar results were obtained when we expressed the hmc1r in an adrenocortical cell line, OS3 (see below), with a Schild regression slope of and a K i of nm (Fig. 2A and Table 1). The action of agouti or ASIP at the mouse or human MC2R has not been reported previously, in part due to difficulties that we and others have encountered in generating heterologous cell lines that functionally express significant levels of this receptor. To surmount these problems, we made use of OS3 cells, a receptor-deficient derivative of the adrenocortical carcinoma cell line Y1 (11). OS3 cells stably transfected with the hmc2r exhibit nearly a 100-fold increase in camp accumulation after stimulation by ACTH (Fig. 1B), in contrast to hmc2r-transfected L cells or 293 cells, in which there is no response to ACTH (data not shown). The magnitude of this response is especially striking compared with other MCRs expressed in L cells, which generally exhibit a maximal level of ligandstimulated camp accumulation less than 10-fold (Fig. 1), and is not due to differences in levels of receptor expression, because similar results were observed using hmc2r expressed in two different vectors (data not shown), and competitive binding assays indicated similar levels of expression for all five receptor subtypes (see legend to Fig. 1). When OS3 cells that expressed the hmc2r were exposed to different concentrations of ACTH and ASIP, we observed an unexpected pattern of antagonism (Fig. 1B). Similar to its effects at the hmc1r, ASIP is a potent antagonist of the hmc2r; however, the pattern of dose-response inhibition is very different in that the maximum stimulatory responses elicited by ACTH are markedly diminished and plateau well below the levels attained in the absence of ASIP. This characteristic is a hallmark of noncompetitive antagonism, but the effects we observed are not completely consistent with the textbook definition of noncompetitive antagonism because the EC 50 values for camp generation at different ASIP concentrations do not remain constant. In the discussion that follows, we describe the effects of ASIP on the hmc2r as nonsurmountable antagonism.

3 MOL ENDO 1997 Vol 11 No Fig. 1. Inhibition of Melanocortin Peptide Action on Cloned Human Melanocortin Receptors hmc1r (panel A); hmc2r (panel B); hmc3r (panel C); hmc4r (panel D); hmc5r (panel E). hmcr1, 3, 4 and 5 are Stably Expressed in L Cells. hmc2r is Stably Expressed in OS3 Cells The panels depict dose-response curves for the melanocortin agonist in the presence of increasing concentration of ASIP (n 3 for hmcr 1, 3, 4, 5, n 4 for hmc2r). Insets depict the Schild regression plots. The amount of receptor protein was determined as described in Materials and Methods and is 521 fmol/mg protein (hmc1r), 500 fmol/mg protein (hmc2r), 592 fmol/mg protein (hmc3r), 480 fmol/mg protein (hmc4r), and 524 fmol/mg protein (hmc5r).

4 ASIP Inhibition of Melanocortins 277 Table 1. Inhibitory Constants (K i ) Values for ASIP- Mediated Inhibition of -MSH Action at the Cloned Human MCRs a Receptor L cells OS3 cells hmc1r hmc3r hmc4r hmc5r a K i values are given in nm and were derived from Schild analysis of dose-response curves indicated in Figs. 1 or 2 as described in Materials and Methods. The hmc3r and hmc5r were not studied in OS3 cells. The K i values of hmc1r, hmc3r, hmc4r, and hmc5r are calculated in the same way, but the slope of the Schild regression is less than unity for the hmc3r, hmc4r, and hmc5r, and therefore the values shown do not reflect true dissociation constants. The effects of ASIP on the hmc3r, hmc4r, and hmc5r were examined in L cells (Fig. 1, C-E); for the hmc4r, the effects of ASIP were also examined in OS3 cells (Fig. 2B). At all three receptors, the effects of ASIP are surmountable at high agonist concentrations, similar to the pattern observed at the hmc1r (Fig. 1A, C-E). The Schild regression slopes for the hmc3r, hmc4r, and hmc5r are significantly less than unity; therefore, the empirical K i values for these receptors can only be used as an estimate of the dissociation constant (Table 1). Nonetheless, comparison of Ki values suggest a relative order of sensitivity to ASIP antagonism of MC4R hmc1r hmc5r hmc3r (Table I). Analysis of the hmc4r expressed in OS3 cells gave nearly identical results to those obtained in L cells, with Schild regression slopes of and and K i values of nm and nm, respectively (Fig. 2B and Table 1). To investigate whether the effects of ASIP on camp generation could be explained by effects on melanocortin binding, we used the radiolabeled melanocortin agonist [I 125 -Nle 4, D-Phe 7 ] -MSH (NDP-MSH) (12) to study the hmc1r, hmc3r, hmc4r, and hmc5r (Fig. 3A). With the exception of the hmc5r, the ability of ASIP to inhibit I 125 -NDP-MSH to these receptors parallels the ability of ASIP to inhibit camp generation, with MC4R hmc1r hmc3r hmc5r. I 125 -NDP- MSH did not bind to the hmc2r (data not shown), and therefore we used [I 125 -Phe 2, Nle 7 ]ACTH1 24 (Fig. 3B). The effects of ASIP on binding of this melanocortin to the hmc2r are approximately equivalent to that seen at the hmc1r and hmc4r and indicate that while ASIP interacts specifically with all human MCRs, it is a potent antagonist of the hmc1r, hmc2r, and hmc4r, and a relatively weak antagonist of the hmc3r and hmc5r. DISCUSSION The human ASIP gene was isolated based on its sequence similarity to the mouse agouti gene, but its role in human skin color, hair color, or regulation of body weight has been uncertain. Our results demonstrate that melanocortin signaling and binding to the hmc1r, hmc2r, and hmc4r are inhibited by nanomolar levels of ASIP and therefore suggest that variation in ASIP expression could affect the physiological endpoints controlled by these receptors in melanocytes, the adrenal gland, or the central nervous system, respectively. Low levels of ASIP expression detectable by RT-PCR have been reported in several tissues including skin and adipocytes, but expression has not been detected in the adrenal gland or the brain. Our conclusions regarding ASIP action at the hmc1r and hmc4r are consistent with previous analyses of the mouse agouti protein (5, 7). However, we found that ASIP could antagonize melanocortin signaling at the hmc3r and hmc5r, which differs from the results of Lu et al. (5), who found that mouse agouti protein had no effect on the rat MC3R or the mouse MC5R. These differences are unlikely to be explained by variation between the sequences for mouse agouti and ASIP or by impurities in the preparations used, because we have observed similar results with a preparation of mouse agouti protein that is 99% pure (data not shown). Instead, the absence of rat MC3R inhibition by mouse agouti protein, as reported by Lu et al. (5), may reflect a quantitative rather than qualitative difference from our observations with the hmc3r, because we observed significant inhibition of the hmc3r only at ASIP or mouse agouti concentrations 10 7 M. With the mouse MC5R, however, Lu et al. (5) found no inhibition of 10 7 M agouti protein; therefore, our observations that the hmc5r is sensitive to relatively low concentrations of ASIP or mouse agouti may reflect differences between the MC5R of rodents and humans. In examining the effects of ASIP on the MC2R we made the surprising observation that the ACTH response of OS3 cells transfected with the hmc2r is extremely robust, with a nearly 100-fold increase in levels of camp accumulation. Expression of a functional MC2R has not been possible in L cells or 293 cells, but recently, Cammas et al. (13) reported that a functional mouse MC2R could be expressed in HeLa cells. The basis of the difference between OS3 cells or HeLa cells and other cell types is not known, but is likely to involve both receptor-specific and cell-specific components, because several attempts at expressing the hmc1r and hmc4r receptor in OS3 cells gave results similar to those observed when these receptors were expressed in L cells, with no more than a 6-fold increase in levels of camp accumulation. The nonsurmountable nature of the hmc2r response to ASIP inhibition in OS3 cells is also surprising. This characteristic is receptor-specific and not cell-specific, since the effects of ASIP on the hmc1r or the hmc4r were nearly identical in L cells compared with OS3 cells. At the hmc3r, hmc4r, and hmc5r, -MSH can surmount the effects of ASIP, but inhibition of these receptors is not consistent with

5 MOL ENDO 1997 Vol 11 No Fig. 2. Inhibition of hmc1r and hmc4r Stably Expressed in OS3 Cells by Recombinant Human Agouti-Signaling Protein (ASIP) (n 3) Fig. 3. Competition Binding Studies Demonstrating the Effect of ASIP on Binding of (A) 125 I-Labeled NDP-MSH to hmcr 1, 3, 4, and 5 (n 3) and (B) [ 125 I-Phe 2, Nle 4 ]ACTH 1 24 Binding to hmc2r (n 3) competitive antagonism, since changes in the EC 50 of -MSH (dose ratio) are not proportionate to changes in ASIP concentration. In fact, it is only the hmc1r at which the effects of ASIP conform to those predicted for a competitive antagonist; in this regard, our findings are consistent with those studies by Blanchard et al. (7) of the endogenous MC1R of mouse B16F10 melanoma cells (7). Nonetheless, our conclusions, and those of others, are not based on direct measurement of ASIP binding, and further studies that utilize a radiolabeled or tagged form of ASIP may help determine whether or not the molecular nature of ASIP action exhibits fundamental differences among melanocortin receptor subtypes. Our data underscore the potential for ASIP or a closely related molecule to act as a physiological or pathophysiological antagonist of the hmc1r, hmc2r, or hmc4r. By analogy to the phenotypes displayed by mice that carry various agouti alleles, three hypotheses might be considered. First, inhibition of MC1R signaling in mice produces a switch from eumelanin to phaeomelanin, a cysteine-rich yellow pigment. Very high levels of phaeomelanin are found in bright red human hair, and it is possible that dominant inheritance of this phenotype through pedigrees represents normal action of ASIP in humans. If so, ASIP structure and/or expression is likely to be highly polymorphic in humans, a hypothesis that is easily testable. Second, if ASIP or a closely related molecule normally functions to inhibit hmc2r activation, loss-of-function mutations might result in adrenocortical hyperplasia with hypercortisolism and secondary suppression of ACTH production, while gain-of-function mutations would produce a relative deficiency of corticosteroids and

6 ASIP Inhibition of Melanocortins 279 increased serum ACTH levels. Closer study of ASIP expression in normal and abnormal adrenal samples may shed light on this possibility. Finally, the pleiotropic effects observed in rodents with ectopic expression of agouti protein (obesity, diabetes, predisposition to tumorigenesis) suggests that somatic or germline mutations leading to ectopic ASIP expression in humans would have pleiotropic effects similar to those observed in mice. MATERIALS AND METHODS Receptor Expression The coding regions of the genes for the human melanocortin receptors (hmcr) 1 5 were subcloned into the eukaryotic expression vectors CMVneo (14) or in pbk-cmv (Stratagene; La Jolla, CA) according to methods previously described (15). The genes encoding hmcr 1, 2, 3, and 4 were previously cloned in our laboratory (6, 16) and that encoding hmc5r was generated by PCR based on the sequence published by Dr. Vijay Chhajlani (17). For these experiments, the receptor constructs were stably expressed in L cells (fibroblast lineage) or in OS3 cells (adrenal cortical lineage). Wild type OS3 cells and OS3 cells stably expressing hmc2r in the eukaryotic expression vector pcdna I were a gift of Dr. Bernard P. Schimmer (University of Toronto, Toronto, Canada). The hmc2r pcdna I plasmid construct originated in the laboratory of Dr. Roger Cone (Vollum Institute, Portland, OR). OS3 cells expressing both the hmc2r pcdna I and hmc2r CMVneo constructs were used in our studies. Transfection of cells was accomplished using calcium phosphate coprecipitation (18), and permanently transfected clonal cell lines were selected by resistance to the neomycin analog G418. Untransfected L cells and OS3 cells exhibit no response to melanocortin stimulation, and therefore there is no significant background. The results depicted in each panel of Figs. 1 and 2 have been obtained from single clones; qualitatively similar results have been obtained with independent clones in replicate experiments. Receptor number determined by competitive binding assay with the Graphpad Prism program (Graphpad Software, San Diego, CA) was found to be similar for the different cell lines depicted in Fig. 1 and is given in the figure legend. camp Assays For our studies, we measured intracellular camp using an assay kit (TRK 432, Amersham, Arlington Heights, IL). Cells transfected with the coding regions of the human melanocortin receptor genes were grown to confluence in 12-well ( cm) tissue culture plates. L cells were maintained in DMEM ( Life Technologies; Gaithersburg, MD) containing 4.5 g/100 ml glucose, 10% fetal calf serum, 1 mm sodium pyruvate. OS3 cells were grown on Ham s F-10 nutrient mixture containing L-glutamine (Life Technologies). Media for both cell lines contained 100 U/ml penicillin and streptomycin and, in the case of transfected cells, 1 mg/ml of geneticin (G418). For assays, the media was removed and cells were washed twice with Earle s balanced salt solution (EBSS, Life Technologies) containing 10 mm HEPES (ph 7.4), 1 mm glutamine, 26.5 mm sodium bicarbonate, and 1 mg/ml BSA. Cells were preincubated for 30 min with human recombinant ASIP in 0.5 ml EBSS before the addition of melanocortin agonist and 5 l of M isobutylmethylxanthine. Cells were then incubated for another 30 min at 37 C at which time the reaction was stopped by adding ice-cold 100% ethanol (500 l/well). The cells in each well were scraped and transferred to a 1.5-ml Eppendorf tube and centrifuged for 10 min at 1900 g. The supernatant was evaporated in a 55 C water bath with prepurified nitrogen gas. camp content was measured by competitive binding assay according to the assay instructions. Human ACTH (1 39), -MSH, [Phe 2, Nle 7 ]ACTH (1 24), and [Nle 4, D-Phe 7 ] -MSH were obtained from Peninsula Laboratories, Inc. (Belmont, CA). Recombinant mouse agouti protein and human ASIP were prepared according to the method of Ollmann et al. (unpublished results). Each experiment was performed a minimum of three times with duplicate wells. The mean value of the dose-response data was fit to a sigmoid curve with a variable slope factor using the nonlinear squares regression in Graphpad Prism (Graphpad Software). Values determined from these fits were used for calculating the Schild analysis linear regression plot. pa 2 values were derived from the y 0 intercept of the Schild plot of the log of dose ratio minus one (log DR 1) as previously described (20). K i values (the negative log of the pa 2 ) presented in Table 1 were determined for relative comparison of ASIP potency. Since the slopes of the linear regression analysis of hmcr 3, 4, and 5 are not unity, by strict definition, true K i values cannot be determined. Binding Assays After removal of media the cells were washed twice with EBSS and preincubated with ASIP in 0.5 ml MEM (Life Technologies) containing 0.2% BSA for 30 min before incubation with 10 6 cpm of radioligand. [ Nle 4, D-Phe 7 ] -MSH was prepared by the chloramine-t method according to the protocol modified from Tatro and Reichlin (21), and binding experiments were performed using conditions previously described (15, 16). [I 125 -Phe 2, Nle 7 ]ACTH(1 24) was prepared as described by Hofmann et al. (22), and binding experiments using this radioligand were performed using conditions described by Rainey et al. (23). Binding reactions were terminated by removing the media and washing the cells twice with MEM containing 0.2% BSA. The cells were lysed with 1% Triton X-100, and the radioactivity in the lysate was quantified in a model

7 MOL ENDO 1997 Vol 11 No Tracor Analytic -counter. Nonspecific binding was determined by measuring the amount of 125 I- labeled melanocortin remaining bound in the presence of 10 5 M unlabeled melanocortin, and specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Acknowledgments We thank Dr. Richard R. Neubig for his assistance in data analysis. We are very grateful to Dr. Bernard P. Schimmer for providing us with access to, and information about, the OS3 cell line. Received May 15, Revision received December 13, Accepted December 18, Address requests for reprints to: Ira Gantz, 6504 MSRBI, 1150 West Medical Center Drive, Ann Arbor, Michigan This work was supported by a Veterans Administration Merit Review Award (I.G.), funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30DK-34933), and a grant from the NIH to G.S.B. (DK ). M.M.O. and B.D.W. are supported by Graduate (EY07106) and Medical Scientist Trainee (GM07365) grants. G.S.B. is an Assistant Investigator of the Howard Hughes Medical Institute. REFERENCES 1. Kwon HY, Bultman SJ, Löffler C, Chen W-J, Furdon PJ, Powell JG, Usala A-L, Wilkison W, Hansmann I, Woyhik RP 1994 Molecular structure and chromosomal mapping of the human homolog of the agouti gene. Proc Natl Acad Sci USA 91: Wilson BD, Ollmann MM, Kang L, Stoffel M, Bell GI, Barsh GS 1995 Structure and function of ASP, the human homolog of the mouse agouti gene. Hum Mol Genet 4: Silvers WK 1979 The Coat Colors of Mice. Springer- Verlag, New York, pp Vrieling H, Duhl DMJ, Millar SE, Miller KA, Barsh GS 1994 Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene. Proc Natl Acad Sci USA 91: Lu D, Willard D, Patel IR, Kadwell S, Overton L, Kost T, Luther M, Chen W, Woychik RP, Wilkison WO, Cone RD 1994 Agouti protein is an antagonist of the melanocytestimulating-hormone receptor. Nature 371: Gantz I, Miwa H, Konda Y, Shimoto Y, Tashio T, Yamada T 1993 Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J Biol Chem 268: Blanchard SG, Harris CO, Ittoop ORR, Nichols JS, Parks DJ Truesdale AT, Wilkison WO 1995 Agouti antagonism of melanocortin binding and action in the B 16 F 10 murine melanoma cell line. Biochemistry 34: Duhl DMJ, Vrieling H, Miller KA, Wolff GL, Barsh GS 1994 Neomorphic agouti mutations in obese yellow mice. Nat Genet 8: Michaud EJ, van Vugt MJ, Bultman SJ, Sweet HO, Davisson, MT, Woychik RP 1994 Differential expression of a new dominant agouti allele (A iapy ) is correlated with methylation state and is influenced by parental lineage. Genes Dev 8: Yen TT, Gill AM, Frigeri LG, Barsh GS, Wolff GL 1994 Obesity, diabetes, and neoplasia in yellow A vy /- mice: ectopic expression of the agouti gene. FASEB J 8: Schimmer BP, Kwan WK, Tsao J, Qiu R 1995 Adrenocorticotropin-resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor. J Cell Physiol 163: Hadley ME, Anderson B, Heward CB, Sawyer TK, Hruby VJ 1981 Calcium-dependent prolonged effects on melanophores of [4-norleucine, 7-D-phenylalanine]- -melanotropin. Science 213: Cammas FM, Kapas S, Barker S, Clark AJ 1995 Cloning, characterization and expression of a functional mouse ACTH receptor. Biochem Biophys Res Commun 212: Brown NA, Steofko RE, Uhler MD 1990 Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of camp-dependent protein kinase. J Biol Chem 265: Gantz I, Schaffer M, DelValle J, Logsdon C, Campbell V, Uhler M, Yamada T 1991 Molecular cloning of a gene encoding the histamine H2 receptor. Proc Natl Acad Sci USA 88: Gantz I, Konda Y, Tashiro T, Shimoto Y, Miwa H, Munzert G, Watson S, DelValle J, Yamada T 1993 Molecular cloning of a novel melanocortin receptor. J Biol Chem 268: Chhajlani V, Muceniece R, Wikberg JES 1993 Molecular cloning of a novel human melanocortin receptor. Biochem Biophys Res Commun 95: Chen CA, Okayama H 1988 Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6: Deleted in proof 20. Sue R, Toomey ML, Todisco A, Soll AH, Yamada T 1985 Pirenzepine-sensitive muscarinic receptors regulate gastric somatostatin and gastrin. Am J Physiol 248: G184 G Tatro JB, Reichlin S 1987 Specific receptors for alphamelanocyte-stimulating hormone are widely distributed in tissues of rodents. Endocrinology 121: Hofmann K, Romavacek H, Stehle CJ, Finn FM, Bothner-By AA, Mishra PK 1986 Radioactive probes for adrenocorticotropic hormone receptors. Biochemistry 25: Rainey WE, Viard I, Saez JM 1989 Transforming growth factor treatment decreases ACTH receptors on ovine adrenocortical cells. J Biol Chem 264: