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ABSTRACT: Agouti signaling protein (ASIP), the human (h) homolog of agouti, is an endogenous melanocortin peptide antagonist. To date, characterization of this protein has been performed with recombinant protein only and without the availability of an ASIP/agouti radioligand. In this report we describe the functional characteristics of a chemically synthesized truncated ASIP variant, ASIP-[90-132 (L89Y)], and the binding characteristics of its cognate radioligand, (125)I-ASIP-[90-132 (L89Y)]. Similar to full-length recombinant ASIP/agouti, ASIP-[90-132 (L89Y)] was a potent inhibitor of alpha-melanocyte-stimulating hormone cAMP generation at the cloned human melanocortin receptor (hMCR) subtypes hMC1R and hMC4R. It also displayed a lesser degree of inhibition at the hMC3R and hMC5R. However, ASIP-[90-132 (L89Y)] was found to be less potent than full-length recombinant ASIP and, surprisingly, only exhibited weak inhibitory activity at the hMC2R. In competition binding assays with the radioligand (125)I-ASIP-[90-132 (L89Y)], ASIP-[90-132 (L89Y)] displayed a hierarchy of binding affinity that roughly paralleled its rank order of inhibitory potency at the various MCR subtypes, i.e., hMC1R approximately hMC4R > hMC3R approximately hMC5R > hMC2R. Structure-activity studies revealed that ASIP-[90-132 (L89Y)] possessed greater pharmacological potency than either the further truncated ASIP variants ASIP-(116-132) or cyclo(CRFFRSAC). Interestingly, the latter molecules were both weak agonists at the hMC1R. These studies further support the concept that ASIP/agouti inhibits melanocortin action by directly binding to target MCRs and provide additional insight into the structural requirements for maximal inhibitory potency.
AJP Regulatory Integrative and Comparative Physiology 01/2002; 281(6):R1877-86. · 3.34 Impact Factor
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ABSTRACT: To explore the role of agouti-related protein (AGRP) in diabetic hyperphagia changes in hypothalamic AGRP mRNA levels were examined in diabetic rats. Rats rendered diabetic by streptozotocin displayed marked hyperglycemia (blood glucose 456.0+/-8.4 mg/dl versus 71.8+/-1.9 mg/dl) and hyperphagia (36.9+/-1.0 g/day versus 22.0+/-0.4 g/day), that was associated with a 286.6+/-4.4% increase in hypothalamic AGRP mRNA and a 178.9+/-13.5% increase in hypothalamic NPY mRNA. Insulin treatment of diabetic rats partially corrected blood glucose (147.4+/-13.1 mg/dl) and ameliorated hyperphagia (26.6+/-2.0 g/day). Insulin replacement was also associated with a return of hypothalamic AGRP mRNA (111.7+/-8.3% of controls) and NPY mRNA (125.0+/-8.9% of controls) from the elevated levels that were observed in untreated diabetic rats. In contrast to insulin treated rats, sodium orthovanadate treated diabetic rats remained significantly hyperglycemic (361.5+/-12.5 mg/dl). However, despite their persistent hyperglycemia, orthovanadate treated diabetic rats were still observed to have a significant reduction of hypothalamic AGRP mRNA (138.7+/-11.4%) and NPY mRNA (129.9+/-9.8%). Simultaneous measurement of serum leptin revealed suppressed levels in both untreated diabetic (0.5+/-0.1 ng/ml) and sodium orthovanadate treated rats (0.5+/-0.1 ng/ml) compared to non-diabetic controls (2.1+/-0.1 ng/ml). These data indicate that AGRP is a mediator of diabetic hyperhpagia and suggest that insulin can directly influence hypothalamic AGRP and NPY mRNA expression.
Regulatory Peptides 05/2001; 98(1-2):69-75. · 2.11 Impact Factor
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ABSTRACT: To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.
Biochemistry 01/2001; 39(48):14900-11. · 3.42 Impact Factor
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ABSTRACT: A novel RIA was used to examine the release of agouti-related protein-like immunoreactivity (AGRP-LI) from perfused rat hypothalamic tissue slices and to characterize AGRP-LI in rat serum. A continuous low level basal AGRP-LI release was observed from hypothalami of rats fed ad libitum before the rats were killed. Basal AGRP-LI release was 3-fold greater in rats fasted 48 h. In fasted animals leptin dose-dependently suppressed basal AGRP-LI release. In fed animals no change in basal AGRP-LI release was detected in response to 10(-6) M alpha-MSH, orexin B, melanin-concentrating hormone, or serotonin. HPLC analysis of AGRP-LI in rat serum identified a single peak that eluted in close proximity to synthetic AGRP (87-132) and mouse [Leu127Pro]AGRP and that was identical to the peak seen in hypothalamic and adrenal tissue extracts. The serum concentration of AGRP-LI in rats fed ad libitum was 0.865+/-0.323 nmol/liter (mean +/- SE). Food deprivation resulted in a slow, but statistically significant rise in serum immunoreactivity at 48 h [1.174+/-0.118 nmol/liter (mean +/- SE)]. Bilateral adrenalectomy did not change serum levels of AGRP-LI. These studies demonstrate that in the rat there are different levels of basal hypothalamic AGRP-LI release in fed and fasted states and that in the fasted rat this release can be profoundly suppressed by leptin. These studies also suggest that AGRP is present in the systemic circulation of rats.
Endocrinology 07/2000; 141(6):1942-50. · 4.46 Impact Factor
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ABSTRACT: Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.
Journal of Biological Chemistry 06/1999; 274(20):14100-6. · 4.77 Impact Factor
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ABSTRACT: 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 46-amino 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 alpha-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, alpha-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 02/1999; 13(1):148-55. · 4.54 Impact Factor
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ABSTRACT: Expression of Agouti protein is normally limited to the skin where it affects pigmentation, but ubiquitous expression causes obesity. An expressed sequence tag was identified that encodes Agouti-related protein, whose RNA is normally expressed in the hypothalamus and whose levels were increased eightfold in ob/ob mice. Recombinant Agouti-related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin receptor subtypes implicated in weight regulation. Ubiquitous expression of human AGRP complementary DNA in transgenic mice caused obesity without altering pigmentation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control of body weight downstream of leptin signaling.
Science 11/1997; 278(5335):135-8. · 31.20 Impact Factor
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ABSTRACT: The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G-protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and alpha-, beta-, and gamma- melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent alpha-MSH analog [Nle4,D-Phe7]MSH (NDP-MSH) and the human MC1R we examined the effects of site-directed receptor mutagenesis on the binding affinity and potency of NDP-MSH. In addition, we also examined the effects of these same mutations on the binding affinity and potency of the structurally related agonists alpha-MSH, gamma-MSH, and Ac-Nle4-cyclic-[Asp5,His6,D-Phe7,Arg8,Trp9,Lys10]NH2 (MT-II). Mutagenesis of acidic receptor residues Glu94 in TM2 and Asp117 or Asp121 in TM3 significantly altered the binding affinity and potency of all four agonists suggesting that these receptor residues are important to the ligand-receptor interactions of all. A disproportionate change in agonist potency versus affinity observed with simultaneous mutation of these acidic residues (mutant constructs D117A/D121A or E94A/D117A/D121A) or introduction of a single positive charge (mutant construct D121K) also implicates these residues in receptor activation. In addition, results from the individual mutation of aromatic receptor residues Phe175, Phe196, and Phe257, and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand-receptor interactions between these melanocortins and the hMC1R.
Journal of Biological Chemistry 10/1997; 272(37):23000-10. · 4.77 Impact Factor
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ABSTRACT: Using the techniques of relaxed stringency polymerase chain reaction and genomic library screening, we have isolated homologous canine and human genes that encode a novel putative seven transmembrane G-protein-linked receptor. The gene encodes an open reading frame (ORF) of 993 bp. The sequences of the canine and human ORFs are highly conserved, sharing 89% nucleotide identity and 92% amino acid similarity between the two species. Northern blot analysis demonstrates that mRNA transcripts of the gene are abundantly expressed in testis and spleen with a lesser degree of expression observed in several other tissues associated with endocrine and immunologic/hematologic function. The gene, designated GPR18, was localized to human chromosome 13q32 using fluorescence in situ hybridization.
Genomics 07/1997; 42(3):462-6. · 3.02 Impact Factor
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ABSTRACT: Examination of conformationally constrained melanotropin peptide (Ac-Nle4-c[Asp5-His-Phe7-Arg-Trp9-Ala-Lys]-NH2) on four human melanotropin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) resulted in identifying the importance of ligand stereochemistry at positions 5, 7, and 9 for agonist binding affinity and receptor selectivity. A trend in ligand structure-activity relationships emerged for these peptides, with the hMC1R and hMC4R possessing similar tendencies, as did the hMC3R and hMC5R. alpha-MSH (Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-D-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), and MTII (Ac-Nle4-c[Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)-NH2) were also examined at each of these melanocortin receptors. Interestingly, the linear NDP-MSH possessed greater binding affinity for the hMC3R and hMC5R than did the cyclic analogue MTII. The peptide Ac-Nle-c[Asp-His-Phe-Arg-D-Trp9-Ala-Lys]-NH2 demonstrated the greatest differentiation in binding affinity between the hMC1R and hMC4R (78-fold). Analogue Ac-Nle-c[Asp-His-Phe7-Arg-Trp-Ala-Lys]-NH2 resulted in micromolar binding affinity (or greater) at the hMC3R and hMC5R, demonstrating the importance of D-Phe7 for ligand binding potency at these receptors. Ac-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2 resulted in loss of binding affinity at the hMC5R, implicating the importance of Nle4 (or a hydrophobic residue in this position) for binding to this receptor. Ac-Nle-c[D-Asp5-His-Phe-Arg-Trp-Ala-Lys]-NH2 was unable to competitively displace [125I]NDP-MSH binding at micromolar concentrations on the hMC3R and hMC5R, suggesting the importance of chirality of Asp5 either for ligand-receptor interactions or for orientation of the side chain lactam bridge and the structural integrity of the peptide conformation. Energy calculations performed for these peptides resulted in the identification of a low-energy ligand conformer family that is common to all the ligands. The differences in ligand binding affinities observed in this study are postulated to be a result of different ligand-receptor complexed interactions and not solely to the ligand structure.
Journal of Medicinal Chemistry 06/1997; 40(11):1738-48. · 5.25 Impact Factor
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ABSTRACT: 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 alpha-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 alpha-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, [125I-Nle4, D-Phe7] alpha-MSH and [125I-Phe2, Nle4]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 03/1997; 11(3):274-80. · 4.54 Impact Factor
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ABSTRACT: We report the cloning of a novel human gene, CMKLR1, which encodes a protein that has notable sequence and structural homology to the seven transmembrane G-protein linked chemokine receptors. This gene has 55% nucleotide sequence homology to the IL-8 type 1 receptor and 53% to the N-formyl peptide related receptor 1 genes. The mRNA of this receptors is expressed in a broad array of tissues associated with hematopoietic and immune function including, spleen, thymus, appendix, lymph node, bone marrow, and fetal liver. Using fluorescence in situ hybridization the gene encoding CMLKR1 (chemokine-like receptor 1) was localized to human chromosome 12q24.1.
Cytogenetics and cell genetics 02/1996; 74(4):286-90.
