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Characterization of Agouti-Related Protein Binding to Melanocortin Receptors Ying-kui Yang, Darren A. Thompson, Chris J. Dickinson, Jill Wilken, Greg S. Barsh, Stephen B. H. Kent, and Ira Gantz Departments of Surgery (Y-k.Y., I.G.) and Pediatrics (C.J.D.) University of Michigan Medical Center Ann Arbor, Michigan 48109-0682 Howard Hughes Medical Institute (G.S.B.) Stanford University School of Medicine Stanford, California 94305 Gryphon Sciences (D.A.T., J.W., S.B.H.K.), South San Francisco, California 94080 Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action that is thought to play an important role in the hypothalamic control of feeding behavior. The exact mechanism of AGRP and Agouti protein action has been difficult to examine, in part because of difficulties in producing homogeneous forms of these molecules that can be used for direct binding assays. In this report we describe the application of chemical protein synthesis to the construction of two novel AGRP variants. Examination of the biological activity of the AGRP variants demonstrates that a truncated variant, human AGRP(87–132), a 46amino acid variant based on the carboxyl-terminal cysteine-rich domain of AGRP, is equipotent to an 111-amino acid variant, mouse [Leu127Pro]AGRP (mature AGRP minus its signal sequence), in its ability to dose dependently inhibit a-MSH-generated cAMP generation at the cloned melanocortin receptors. Furthermore, deletion of the amino-terminal portion of the full-length variant did not alter the MCR subtype specificity of AGRP(87–132). Finally, iodination of human AGRP(87–132) provided a useful reagent with which the binding properties of AGRP could be analyzed. In both conventional and photoemulsion binding studies [125I]AGRP(87– 132) was observed only to bind to cells expressing melanocortin receptors MC3R, MC4R, and MC5R. These results demonstrate that the residues critical for receptor binding, a-MSH inhibition, and melanocortin receptor subtype specificity are all located in the carboxyl terminus of the molecule. Because [Nle4, D-Phe7] (NDP)-MSH displaces the binding of [125I]AGRP(87–132) to MCRs and AGRP(87–132) displaces the binding of [125I]NDP- MSH, we conclude that these molecules bind in a competitive fashion to melanocortin receptors. (Molecular Endocrinology 13: 148–155, 1999) INTRODUCTION Agouti-related protein (AGRP) is a recently discovered neuropeptide that has generated intense interest because a growing body of evidence indicates it has a major role in the regulation of mammalian feeding behavior (1, 2). AGRP was identified by virtue of its sequence similarity to the product of the Agouti coat color gene, a paracrine signaling molecule normally expressed in skin whose transient expression during hair growth leads to the barring of coat fur in rodents (e.g. dark hair with a subapical yellow band) (3). Ubiquitous expression of Agouti, which occurs in mice that carry mutations in the 59-flanking region of the Agouti gene, gives rise to pleiotropic effects including a yellow coat, obesity, insulin resistance, increased body length, and premature infertility (4). The recent identification of AGRP indicates that the obesity and diabetes caused by ectopic Agouti expression are likely explained by its ability to mimic AGRP, since ubiquitous expression of AGRP in transgenic mice causes an increased weight gain and body length phenotype identical to that produced by ubiquitous expression of Agouti (2). Structurally, however, the similarity between Agouti and AGRP is confined almost entirely to their 40-residue carboxyl termini where a total of 20 residues, including 10 cysteine residues, are identical (Fig. 1). Both Agouti and AGRP have been shown to antagonize the action of melanocortin peptides such as a-MSH and ACTH at specific melanocortin receptor subtypes. Agouti potently antagonizes the action of melanocortins at the melanocyte melanocortin recep- 0888-8809/99/$3.00/0 Molecular Endocrinology Copyright © 1999 by The Endocrine Society 148 AGRP Binding Fig. 1. Amino Acid Sequence Alignments of Mouse and Human AGRP and Mouse and Human Agouti Protein Conserved C-terminal cysteine residues are enclosed in boxes. The presumed signal sequence cleavage position is denoted by , which also denotes the N terminus of mouse [Leu127Pro]AGRP. 2 Denotes the start of the C-terminal sequence that is AGRP(87–132). Note that the C-terminal portion of mouse and human AGRP are identical except for position 127 in the human sequence (126 of the mouse sequence) where Pro is present in the human sequence and Leu in the mouse sequence. m, Mouse; h, human. tor (MC1R), adrenocortical ACTH receptor (MC2R), and the MC4R (5, 6). In contrast, we recently demonstrated that AGRP primarily antagonizes the MC3R and MC4R (2), each of which is expressed in areas of the hypothalamus implicated in feeding behavior (7, 8). Some or all of the growth and weight gain phenotypes caused by Agouti or AGRP are likely mediated via the MC4R, since mice carrying an MC4R knockout mutation display obesity and metabolic abnormalities similar to those caused by ubiquitous expression of Agouti or AGRP (2, 9). Several explanations have been proposed to explain the mechanism of Agouti or AGRP action, including simple competitive antagonism (10), inverse agonism (11), or activation of an effector other than adenylate cyclase (12). Distinguishing among these alternatives has been complicated, in part, by the absence of direct assays for Agouti or AGRP binding, since a hallmark of competitive antagonism is the ability of agonist to displace labeled antagonist. We have recently demonstrated that an epitope-tagged form of Agouti protein interacts directly with the MC1R in an overlay experiment, but these assays are not quantitative and so do not allow a comparison among different receptors or antagonists (13). Our previous studies were carried out with preparations of recombinant AGRP that were partially purified and heterogeneous in length. In contrast to recombinant techniques, chemical protein synthesis can be used to make a homogeneous preparation of defined molecular structure that can be chemically labeled and derivatized and that can serve as a substrate for structure-function analyses. Here we describe the application of two novel chemically synthesized AGRP variants to studies directed at understanding the mechanism of AGRP action. The first, AGRP(87–132), is a 46-residue peptide containing five disulfide bonds formed by folding the C-terminal portion of human 149 AGRP. We demonstrate that radioiodinated AGRP(87– 132) can be used as a high-affinity tracer to directly quantitate cell surface AGRP binding. The second variant, mouse [Leu127Pro]AGRP, is a 111-amino acid AGRP molecule (mature AGRP minus its 20-amino acid signal sequence) that was made by joining the N-terminal residues 21–85 of mouse AGRP to human AGRP(87–132) by native chemical ligation (14). In these studies we address three important issues: 1) the bioactivity of the synthetic AGRPs; 2) the binding properties of AGRP; and 3) the demonstration that AGRP(87–132) maintains the activity of full-length AGRP. RESULTS Effect of Mouse [Leu127Pro]AGRP and Human AGRP(87–132) on a-MSH-Stimulated cAMP Generation in Cell Lines Transfected with Melanocortin Receptors To help verify the biological activity of the chemically synthesized proteins, we examined the ability of the proteins to inhibit a-MSH-stimulated cAMP generation. We have shown previously that partially purified recombinant human AGRP is a potent antagonist of the hMC3R and hMC4R, but has little or no effect on the hMC1R, hMC2R, or hMC5R. Figure 2, A–E, demonstrates that chemically synthesized mouse [Leu127Pro]AGRP potently inhibits the action of a-MSH at the hMC3R and hMC4R. With increasing concentrations of mouse [Leu127Pro]AGRP, a progressive rightward shift of the a-MSH dose-response curves is observed. Mouse [Leu127Pro]AGRP was completely devoid of activity at the hMC1R and hMC2R. However, at higher concentrations, mouse [Leu127Pro]AGRP had a modest inhibitory effect on a-MSH action at the hMC5R. Schild analysis performed by plotting a linear regression of the log concentration of AGRP (x-axis) and log (DR-1) (y-axis) revealed a slope of 0.94 and 0.96 for mouse [Leu127Pro]AGRP at the hMC3R and hMC4R, respectively (Fig. 2, D and E, insets) (15). Slopes approaching unity indicate that mouse [Leu127Pro]AGRP has the characteristics of a competitive antagonist of a-MSH action at the hMC3R and hMC4R. Inhibitory constants (Ki) for mouse [Leu127Pro]AGRP derived from this Schild analysis revealed a Ki of 4.3 6 0.6 nM at the hMC3R and a Ki of 2.5 6 0.25 nM at the hMC4R (Table 1). We next compared the pharmacological effects of the 111-residue mouse [Leu127Pro]AGRP to those displayed by the 46-residue AGRP(87–132) at the various hMCR subtypes. AGRP(87–132) had no effect at hMC1R or hMC2R and only a minimal effect at the hMC5R. Schild analysis revealed that AGRP(87–132), like mouse [Leu127Pro]AGRP, is a competitive antagonist (data not shown) with Ki values for inhibition of MOL ENDO · 1999 150 Vol 13 No. 1 Fig. 2. Mouse [Leu127Pro]AGRP Inhibition of a-MSH-Stimulated cAMP Generation at the Five Melanocortin Receptor Subtypes (A–E) Shown in F are the effects of recombinant AGRP Form A 1 B on a-MSH-stimulated cAMP generation at the hMC4R. Inset graphs in D, E, and F represent Schild analysis linear regression plots. f, No AGRP; Œ, AGRP 1029 M; , AGRP 1028 M; l, AGRP 1027 M; F, AGRP 1026 M. Table 1. Comparison of AGRP Ki and IC50 Values on hMC3R and hMC4R hMC3R Ki (nM) Mouse [Leu 127Pro] AGRP Human AGRP(87–132) Recombinant Form A 1 B IC50 (nM) [125I]NDP-MSHa [125I]AGRP(87–132)b 4.3 6 0.6 3.3 6 0.28 ND 17.4 6 3.7 11.2 6 3.1 hMC4R 2.5 6 0.25 2.6 6 0.21 1.2 6 0.17 15.7 6 4.1 9.0 6 1.7 ND, Not done. a 125 [ I]NDP-MSH was displaced by mouse [Leu 127Pro] AGRP. b 125 [ I]AGRP(87–132) was displaced by unlabeled AGRP(87–132). a-MSH at the hMC3R and hMC4R of 3.3 6 0.28 nM and 2.6 6 0.21 nM, respectively. We also examined the effect of recombinant human AGRP Form A 1 B on a-MSH-stimulated cAMP generation at the hMC4R (Fig. 2F). Although the dose-response curves for recombinant human AGRP Form A 1 B were not parallel, a linear regression of the data revealed a slope of 0.94 and Ki of 1.2 6 0.17 nM (Fig. 2F, inset). In contrast to chemically synthesized mouse [Leu127Pro]AGRP, the Emax of a-MSH in the presence of recombinant human AGRP Form A 1 B was about 10% below that observed in the absence of this antagonist. Neither chemically synthesized nor recombinant AGRP had an effect on cAMP accumulation when applied to cells in the absence of agonist. [125I][Nle4, D-Phe7] (NDP)-MSH Binding to Cell Lines Transfected with Melanocortin Receptors Because the effects of AGRP on cAMP generation are an indirect measure of antagonism, we measured the ability of chemically synthesized or recombinant AGRP to inhibit the binding of NDP-MSH to hMCRexpressing cells. Figure 3A reveals that chemically synthesized mouse [Leu127Pro]AGRP dose dependently displaces [125I]NDP-MSH from the hMC3R, hMC4R, and hMC5R. The displacement curve of [125I]NDP-MSH from the hMC5R was shifted to the right as compared with the hMC3R and hMC4R. No significant displacement was observed at the hMC1R (data not shown). These binding studies are consistent AGRP Binding 151 Fig. 3. Displacement of Radioligand Binding from the Human Melanocortin Receptors Stably Expressed in HEK-293 Cells A, Displacement of [125I]NDP-MSH binding from the hMCR 3, 4, and 5 by chemically synthesized mouse [Leu127Pro]AGRP. B, Comparison of the displacement of [125I]NDP-MSH binding from the hMC4R by recombinant human AGRP Form A 1 B, chemically synthesized mouse [Leu127Pro]AGRP, and AGRP(87–132). C, Displacement of [125I]AGRP(87–132) binding from the hMCR 3, 4, and 5 by AGRP(87–132). D, Displacement of [125I]AGRP(87–132) binding from the hMCR 3, 4, and 5 by NDP-MSH. [125I]AGRP(87–132) does not bind the hMC1R or hMC2R. with the actions of mouse [Leu127Pro]AGRP and AGRP(87–132) in the cAMP assays. IC50 values for [125I]NDP-MSH displacement are hMC1R.1026 M, hMC3R 5 17.4 6 3.7 nM, hMC4R 5 15.7 6 4.1 nM, hMC5R 5 310.6 6 18.7 nM. Figure 3B compares the ability of mouse [Leu127Pro]AGRP, AGRP(87–132), and recombinant human AGRP Form A 1 B to displace [125I]NDP-MSH from the hMC4R. The displacement curves of baculovirus-produced human AGRP Form A 1 B and chemically synthesized mouse [Leu127Pro]AGRP are identical, while the curve of chemically synthesized AGRP(87–132) is slightly shifted to the left (3 times more potent). The IC50 for recombinant human AGRP Form A 1 B was 13.4 6 2.9 nM. [125I]AGRP(87–132) Binding to Cell Lines Transfected with Melanocortin Receptors Previous biochemical studies of Agouti protein have been complicated by the lack of a radiolabeled derivative. In contrast, AGRP has two tyrosine residues, both of which are present in its carboxyl-terminal sequence. We were therefore able to take advantage of the purity of chemically synthesized human AGRP(87– 132) to use standard oxidative chemistries to generate a radiolabeled molecule that exhibited specific binding to hMCR-expressing cells. In initial autoradiographic experiments using slides coated with photoemulsion, we asked whether tracer amounts of [125I]AGRP(87– 132) would bind to HEK 293 cells that expressed equivalent levels of the different melanocortin receptors (;2.5 3 105 per well). As shown in Fig. 4, [125I]AGRP(87–132) only binds to cells expressing the hMC3R, hMC4R, and hMC5R. No specific radioligand binding was observed in wild-type cells or at the hMC1R or hMC2R (Fig. 4 and data not shown). The intensity of the binding studies is consistent with the rank order of AGRP inhibition noted in our functional studies (hMC4R 5 hMC3R . hMC5R). As a quantitative measure of binding, we examined the ability of unlabeled AGRP(87–132) to displace [125I]AGRP(87–132) from the hMC3R, hMC4R, and hMC5R-expressing cells (Fig. 3C). Notably, the displacement curves of [125I]AGRP(87–132) from the hMC3R and hMC4R are overlapping, which is consistent with our previous functional and photoemulsion studies and indicate that AGRP(87–132) is essentially MOL ENDO · 1999 152 Vol 13 No. 1 Fig. 4. Representative Photomicrographs of [125I]AGRP(87–132) Binding to HEK 293 Cells Transfected with hMCR 3, 4, and 5 Under bright field illumination (right) cells are seen as outlines on a light background. Under dark field illumination (left) the identical cells are seen. Under dark field, cells binding [125I]AGRP(87–132) are seen as white elements in a surrounding dark background. Because of the absence of binding, wild-type cells only appear as faint outlines. No binding was observed in similar experiments with cells expressing the hMC1R and hMC2R. equipotent at the two receptor subtypes. The IC50 values of [125I]AGRP(87–132) displacement by AGRP(87–132) at the hMC3R 5 11.2 6 3.1 nM, hMC4R 5 9.0 6 1.7 nM, and hMC5R 5 25.6 6 4.3 nM. A hallmark of competitive binding is the ability of one ligand to displace the other, and vice versa. As indicated in Fig. 3A, AGRP(87–132) can displace [125I]NDP-MSH from the hMC3R, hMC4R, and hMC5R. The converse is also true as shown in Fig. 3D, which examines the ability of NDP-MSH to displace [125I]AGRP(87–132). The IC50 values of [125I]AGRP(87–132) displacement by NDPMSH are as follows: at the hMC3R 5 1.9 6 0.15 nM; hMC4R 5 3.75 6 0.1 nM; and hMC5R 5 11.2 6 2.1 nM. The [125I]NDP-MSH and [125I]AGRP(87–132) displacement data and the cAMP data reveal a hierarchy of melanocortin receptor subtype sensitivity to AGRP such that hMC3R 5 hMC4R . hMC5R. DISCUSSION The identification of AGRP has added additional complexity to our attempts to understand weight ho- meostasis. Recent pharmacological and anatomical data have further strengthened the link between melanocortins and weight control and indicate that melanocortins act downstream of the fat hormone leptin (16–19). Levels of AGRP mRNA exhibit up to 10-fold alterations in different obesity models (1, 2) and, therefore, as a naturally occurring orexigenic agent that antagonizes melanocortins, AGRP may represent a unique target for antiobesity drug development. While our previous studies of the action of baculovirus-produced AGRP allowed us to determine some aspects of this regulatory protein’s action, our inability to produce highly purified product led us to consider an alternative approach. Chemical protein synthesis uses native chemical ligation of unprotected synthetic peptide segments in aqueous solution, followed by folding/disulfide formation to give the functional protein molecule (14). Our present experiments demonstrate that the techniques of chemical protein synthesis can be used to rapidly produce highly purified, biologically active AGRP molecules in amounts of tens of milligrams in a convenient and straightforward fashion. In the present correspondence we capitalized on AGRP Binding the use of these synthetic techniques to build upon our previous observations of AGRP. The availability of highly purified AGRP protein variants allowed us not only to readily develop an AGRP radioligand with which the site of AGRP action could be explored, but also enabled us to perform more detailed pharmacological analysis of this molecule. Both chemically synthesized mouse [Leu127Pro] AGRP and AGRP(87–132) have similar inhibitory potency and efficacy as baculovirus-produced human AGRP Form A 1 B. In cAMP assays [Leu127Pro] AGRP, AGRP(87–132), and recombinant human AGRP Form A 1 B are essentially equipotent at inhibiting the hMC4R. Both chemically synthesized variant AGRP molecules were also found to display a similar nanomolar range of activity as previously observed for human recombinant Form A 1 B at the hMC3R (2). Like recombinant human Form A 1 B, both chemically synthesized AGRP variants had only minimal activity at the hMC5R, and neither displayed any inhibitory activity at the hMC1R or the hMC2R. AGRP(87–132) was only slightly more potent than either longer synthetic or recombinant forms of AGRP in displacing [125I]NDP-MSH from the hMC4R. Having demonstrated the biological activity of the chemically synthesized AGRP variants, we used AGRP(87–132) to further study the actions of this protein. Our ability to radiolabel AGRP(87–132) with 125I allowed us to directly study AGRP(87–132) binding. [125I]AGRP(87–132) bound only to those heterologous cell lines expressing melanocortin receptor subtypes susceptible to AGRP inhibition in cAMP assays and at which [125I]NDP-MSH was displaced by mouse [Leu127Pro]AGRP (Fig. 3). Typical displacement curves appear to indicate that the iodination process did not alter the biological activity of AGRP(87–132). The finding that the displacement of [125I]AGRP(87– 132) from the hMC5R was shifted to the right is consistent with the decreased potency of AGRP at this receptor subtype observed in cAMP assays. A persistent controversy that has existed regarding the action of Agouti protein is whether it has effects independent of its antagonism of a-MSH (11–13). Much of this speculation is based on the sequence similarity between agouti protein, and cone snail (conotoxins) and spider (plectoxins) toxins. These toxins, which affect calcium channels, contain a cysteinerich motif that can be closely aligned against 10 cysteine residues present in the C terminus of both Agouti and AGRP (Fig. 1). While some of the effects of Agouti in the absence of a-MSH may be explained by its ability to act as inverse agonist, it has been suggested that a separate agouti receptor may exist (20, 21). This controversy has been approached by examining the action of Agouti on melanoma cell lines lacking the MC1R (13). More recently, epitope-tagged Agouti has been used (22). However, this matter has been somewhat difficult to study since a radiolabeled Agouti has not been developed. Because of this controversy we used the novel radioligand [125I]AGRP(87–132) to examine the binding sites of AGRP. Both conventional 153 binding studies and photoemulsion studies indicate that [125I]AGRP(87–132) only binds to melanocortin receptors demonstrated to be susceptible to AGRP inhibition in cAMP assays. This does not, however, exclude the possibility that an endogenous cell type that expresses a native hMC3R, hMC4R, or hMC5R may also possess additional binding sites. The competitive pattern of AGRP inhibition of melanocortins binding to the MC3R and MC4R observed in the present studies does not necessarily imply that AGRP and melanocortin agonist occupy the same site on the receptor. It is possible that the two ligands simply influence each other’s binding through an allosteric mechanism. In fact, there is no significant sequence similarity between melanocortins and AGRP, although this does not exclude some similarity on the basis of three-dimensional structure. Future receptor mutagenesis studies using our novel radioligand [125I]AGRP(87–132) and the radioligand [125I]NDPMSH should be helpful in this respect. Although AGRP(87–132) was approximately 3-fold more potent than mouse [Lue127Pro] AGRP in its ability to displace [125I]MSH-MSH from hMC4R-expressing cells, the antagonists were equipotent in their ability to inhibit a-MSH-induced cAMP accumulation mediated by the hMC4R. Regardless of the reasons for this apparent difference, these results indicate that the structural determinants for both MCR binding and melanocortin antagonism are located within the cysteine-rich C-terminal domain. Furthermore, AGRP(87–132) retains the pattern of melanocortin receptor selectivity displayed by the fulllength molecule. Further truncation and other manipulations of human AGRP(87–132) will help identify its minimally active form, and modification of residues within this fragment should provide insight into the determinants of receptor subtype selectivity. In summary, these studies demonstrate the ability to chemically synthesize biologically active AGRP variants. These studies also demonstrate that AGRP(87– 132), a variant lacking the N terminus of AGRP and consisting of only the C-terminal cysteine-rich AGRP module, retains the biological activity of full-length AGRP. Finally, these studies describe the AGRP radioligand, [125I]AGRP(87–132), and demonstrate the binding of this radioligand directly to melanocortin receptor protein. [125I]AGRP(87–132) should be a helpful tool for anatomical studies of the natural sites of AGRP binding, development of an AGRP RIA, and identification of small molecule antagonists of AGRP interaction with the melanocortin receptors. The latter compounds could have potential applications as regulators of human feeding behavior. MATERIALS AND METHODS Mouse [Leu127Pro]AGRP and AGRP(87–132) Synthesis Peptides were synthesized by Boc chemistry using manual stepwise solid-phase peptide synthesis as previously de- MOL ENDO · 1999 154 scribed (23). The 46-amino acid polypeptide corresponding to the C-terminal module, human AGRP(87–132), was assembled on Thr-OCH2-Pam-resin (Perkin-Elmer Applied Biosystems, Foster City, CA). The N-terminal basic segment, mouse AGRP 21–85, was assembled on a thioester resin. Peptides were cleaved from the resin with hydrogen fluoride containing 5–10% p-cresol (Fluka, Buchs, Switzerland) for 1 h at 0 C, lyophilized, and then purified using reversed phase HPLC on C4 columns (Vydac, Murrieta, CA) with water (0.1% trifluoroacetic acid)/acetonitrile (0.1% trifluoroacetic acid) gradients. The molecular weights of these peptides were confirmed by electrospray ionization mass spectrometry (Perkin-Elmer SCIEX, Foster City, CA). To generate the fulllength construct, purified mouse AGRP(21–85) thioester and human AGRP(87–132) were dissolved in 6 M guanidine hydrochloride and 200 mM phosphate (pH 7.0) containing 1% thiophenol at a concentration of 2–4 mM and stirred overnight. Under these conditions native chemical ligation joined the two peptides to form full-length mouse [Leu127Pro]AGRP(21–131), appearing as a new peak on analytical HPLC with mol wt indicative of segment condensation by peptide bond formation [observed: 12,397.4 6 1.50; calculated: 12,394.5 (average isotopes)]. Ligated peptides were then fully reduced by incubating 1 h with 20% b-mercaptoethanol, purified by HPLC, and lyophylized [24 mg mouse AGRP(21–85)] thioester 1 16.9 mg human AGRP(87– 132) yielded 14.3 mg mouse [Leu127Pro]AGRP. Protein folding of human AGRP(87–132) and mouse [Leu127Pro]AGRP was initiated by dissolving the lyophilized peptide in a solution of 2 M guanidine hydrochloride and 100 mM Tris (pH 8.0) containing 8 mM cysteine and 1 mM cystine (Fluka), and stirring overnight. The folded proteins were then purified by HPLC and lyophilized. Human AGRP(87–132) (138.2 mg) (reduced) yielded 52.5 mg AGRP(87–132) (oxidized); 14.3 mg [Leu127Pro]AGRP (reduced) yielded 4.7 mg [Leu127Pro]AGRP (oxidized). Two-dimensional nuclear magnetic resonance studies of AGRP(87–132) confirmed the existence of a single homogeneous folded state (K. Bolin, J. Trulson, and G. L. Millhauser, unpublished results). This observation was supported by the formation of a sharp peak on analytical reverse phase HPLC eluting earlier than the reduced state, and the loss of 10 mass units by electrospray ionization mass spectrometry, which is consistent with the formation of five disulfides in the oxidized form (AGRP(87– 132) observed: 5,191.1 6 1.05; calculated: 5,191.2 (average isotopes); mouse [Leu127Pro]AGRP observed: 12,384.9 6 1.11; calculated: 12,383.5 (average isotopes). Baculovirus-produced recombinant human AGRP Form A 1 B was produced and partially purified as previously described (2, 24). Form A 1 B refers to nonhomogeneous fractions of recombinant AGRP that run closely together on Western blot (2). Form A consists of mature AGRP minus its signal sequence of 20 amino acids, and Form B contains several fragments cleaved after residues 46, 48, or 50. cAMP Assays cAMP generation was measured using a competitive binding assay kit (TRK 432, Amersham, Arlington Heights, IL) according to a standardized protocol (6). Heterologous cell lines stably expressing the human (h) melanocortin receptors that have been previously described were used in these assays (6). For assays, culture media were removed and cells were incubated with 0.5 ml Earle’s balanced salt solution that contained AGRP and melanocortin agonist for 30 min at 37 C in the presence of 1023 M isobutylmethylxanthine. The reaction was stopped by adding ice-cold 100% ethanol (500 ml/well). The cells in each well were scraped and transferred to a 1.5-ml tube and centrifuged for 10 min at 1900 3 g, and the supernatant was evaporated in a 55 C water bath with prepurified nitrogen gas. cAMP content was measured according to instructions accompanying the assay kit. a-MSH and human ACTH 1–39 were obtained from Peninsula Lab- Vol 13 No. 1 oratories, Inc. (Belmont, CA). Each experiment was performed a minimum of three times with duplicate wells. The mean value of the dose-response data were fit to a sigmoid curve with a variable slope factor using the nonlinear squares regression in Graphpad Prism (Graphpad Software, San Diego, CA). EC50 values determined from these fits were used for plotting Schild analysis linear regressions. pA2 values were derived from the y 5 0 intercept of the Schild plot of the log of dose ratio minus one (log DR-1) as previously described (6). Ki values were determined as the negative log of the pA2. All statistical analyses represent the mean of the data 6 SE. Radioiodination NDP-MSH, a long acting superpotent melanocortin agonist, was obtained from Peninsula Laboratories, Inc. (Belmont, CA) (25). [125I]NDP-MSH and [125I]AGRP(87–132) were prepared by a modification of a chloramine-T method previously described (26). 125I-labeled Na (0.5 mCi) (Amersham) was added to 20 mg of either NDP-MSH or AGRP(87–132) in 5 ml of 50 mM sodium phosphate buffer (pH 7.4). Ten microliters of a 2.4 mg/ml solution of chloramine T (Sigma Chemical Co., St. Louis, MO) in 50 mM sodium phosphate (pH 7.4) were added for 15 sec, and the reaction was stopped with 50 ml of a 4.8 mg/ml solution of sodium metabisulfite (Sigma). The reaction mixture was then diluted in 800 ml of 50 mM ammonium acetate (pH 5.8) and purified by reverse phase chromatography. BSA (100 ml of a 2% solution) was added to all fractions containing radioactivity. Binding Experiments After removal of media the cells were washed twice with MEM and then preincubated with AGRP in 0.5 ml MEM (Life Technologies, Gaithersburg, MD) containing 0.2% BSA for 30 min before incubation with radioligand. Binding experiments were performed using conditions previously described (6). [125I]NDP-MSH (3 3 105 cpm; ;61 fmol) or 3 3 105 cpm 125 I-AGRP(87–132) (;55 fmol) were used. Binding reactions were terminated by removing the media and washing the cells twice with MEM containing 0.2% BSA. The cells were lysed with 0.1 N NaOH 1% Triton X-100, and the radioactivity in the lysate was quantified in an analytical g-counter. Nonspecific binding was determined by measuring the amount of 125 I-label remaining bound in the presence of 1025 M unlabeled ligand, and specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Typically, total binding of [125I]AGRP(87–132) was about 13.5 6 1.3 3 104 cpm, and nonspecific binding was 3.0 6 0.4 3 103 cpm. For photoemulsion studies, the binding assays were performed directly on a chambered microscope slides (SlideFlask, NUNC, Roskilde, Denmark). Approximately 105 cells were placed on each slide and allowed to grow for 12 h. After binding experiments were performed, slides were fixed with gluteraldehyde and dried. Slides were then dipped in Kodak NTB2 photoemulsion (Eastman Kodak Co., New Haven, CT) and exposed for 3 days before being developed, examined, and photographed using a Leica DMRB microscope (Leica, Inc., Deerfield, IL). Acknowledgments Received July 7, 1998. Revision received September 15, 1998. Accepted September 18, 1998. Address requests for reprints to: Ira Gantz, M.D., 6504 MSRB I, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0682. E-mail: [email protected]. 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 AGRP Binding P30 DK-34933), RO1 DK-47398 (C.J.D.), and RO1 DK-28506 (G.S.B). G.S.B is an Associate Investigator of the Howard Hughes Medical Institute. REFERENCES 1. Shutter JR, Graham M, Kinsey AC, Scully S, Lüthy R, Stark KL 1997 Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated on obese and diabetic mutant mice. Gene Dev 11:593–602 2. Ollmann MM, Wilson BD, Yang Y-K, Kerns JA, Chen Y, Gantz I, Barsh GS 1997 Agouti-related protein is an endogenous antagonist of the melanocortin-4 receptor in vitro and in vivo. Science 278:135–138 3. Bultman SJ, Michaud EJ, Woychik RP 1992 Molecular characterization of the mouse agouti locus. Cell 71:1195–1204 4. Leibel RL, Chung WK, Chua SC 1997 The molecular genetics of rodent single gene obesities. J Biol Chem 272:31937–31940 5. 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 371:799–802 6. Yang Y-K, Ollmann MM, Wilson BD, Dickinson C, Yamada T, Barsh GS, Gantz I 1997 Effects of recombinant ASIP on melanocortin action. Mol Endocrinol 11:274–280 7. Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Watson SJ, DelValle J, Yamada T 1993 Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J Biol Chem 268:15174–15179 8. Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD 1994 Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8:1298–1308 9. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the MC4R results in obesity in mice. Cell 88:131–141 10. Willard DH, Bodnar W, Harris C, Kiefer L, Nichols JS, Blanchard S, Hoffman C, Moyer M, Burkhart W, Weiel J, Luther MA, Wilkison WO, Rocque WJ 1995 Agouti structure and function: characterization of a potent a-melanocyte stimulating hormone receptor antagonist. Biochemistry 34:12341–12346 11. Siegrist W, Drozdz R, Cotti R, Willard DH, Wilkison WO, Eberle AN 1997 Interactions of a-melanotropin and agouti on B16 melanoma cells: evidence for inverse ago- 155 nism of agouti. J Recept Signal Transduct Res 17:75–98 12. Kim JH, Kiefer LL, Woychik RP, Wilkison WO, Truesdale A, Ittoop O, Willard D, Nichols J, Zemel MB 1997 Agouti regulation of intracellular calcium: role of melanocortin receptors. Am J Physiol 272:E379–E384 13. Ollmann MM, Lamoreux ML, Wilson BD, Barsh GS 1998 Interaction of agouti protein with the melanocortin 1 receptor in vitro and in vivo. Genes Dev 12:316–330 14. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH 1994 Synthesis of proteins by native chemical ligation. Science 266:776–779 15. Kenakin TP (ed) 1987 Drug antagonism. In: Pharmacologic Analysis of Drug-Receptor Interaction. Raven Press, New York, pp 221–236 16. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168 17. Seeley RJ, Yagaloff KA, Fisher SL, Burn P, Thiele TE, Van Dijk G, Baskin DG, Schwartz MW 1997 Melanocortin receptors in leptin effects. Nature 390:349 18. Thronton JE, Cheung CC, Clifton DK, Steiner RA 1997 Regulation of hypothalamic POMC mRNA by leptin in ob/ob mice. Endocrinology 138:5063–5066 19. Håkansson M-L, Brown H, Ghilardi N, Skoda RC, Meister B 1998 Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 18:559–572 20. Sakai C, Ollmann M, Kobayashi T, Abdel-Malek Z, Muller J, Vierira WD, Imokawa G, Barsh GS, Hearing VJ 1997 Modulation of murine melanocyte function in vitro by agouti signal protein. EMBO J 16:3544–3552 21. Conklin BR, Bourne HR 1993 Muse coat colour reconsidered. Nature 364:110 22. Siegrist W, Willard DH, Wilkison WO, Eberle AN 1996 Agouti protein inhibits growth of B16 melanocma cells in vitro by acting through melanocortin receptors. Biochem Biophys Res Commun 218:171–175 23. Schnolzer M, Alewood P, Jones A, Alewood D, Kent SBH 1992 In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int J Pept Protein Res 40:180–193 24. Potenza MN, Lerner MR 1992 A rapid quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line. Pigment Cell Res 5:372–378 25. Hadley ME, Anderson B, Heward CB, Sawyer TK, Hruby VJ 1981 Calcium-dependent prolonged effects on melanophores of [4-norleucine, 7-D-phenylalanine]-a-melanotropin. Science 213:1025–1027 26. Tatro JB, Reichlin S 1987 Specific receptors for alphamelanocyte-stimulating hormone are widely distributed in tissues of rodents. Endocrinology 121:1900–1907