Receptor-Antagonist Interactions in the Complexes of Agouti and Agouti-Related Protein with Human Melanocortin 1 and 4 Receptors

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• 作者：Biao-Xin Chai ; Irina D. Pogozheva ; Yu-Mei Lai ; Ji-Yao Li ; Richard R. Neubig ; Henry I. Mosberg ; and Ira Gantz
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 8, 2005
• 年：2005
• 卷：44
• 期：9
• 页码：3418 - 3431
• 全文大小：560K
• 年卷期：v.44,no.9(March 8, 2005)
• ISSN：1520-4995

文摘

The molecular interactions between human melanocortin receptor-1 and -4 (hMC1R andhMC4R) and their endogenous antagonists, agouti signaling protein (ASIP) and agouti-related protein(AGRP), were assessed by studying the effects of site-directed mutations on the binding affinity of ¹²⁵I-ASIP[90-132(L89Y)] and ¹²⁵I-AGRP(86-132). Mutations of homologous residues from transmembranehelices (TMHs) 3 and 6 and extracellular loop (EL) 3 (D121A, T124A, F257A, and F277M in hMC1Rand D126A, I129A F261A, and M281F in hMC4R) impaired binding of both antagonists to hMC4R andbinding of the ASIP fragment to hMC1R. However, the mutations in TMH2 (E94A in hMC1R and E100Ain hMC4R), TMH7 (F280A in hMC1R and F284A in hMC4R), and EL2 (Y183S, H184S, and D184H inhMC1R) only significantly affected binding of the ASIP fragment. The dependence of agonist binding onthe dithiothreitol concentration followed a monophasic curve for wild-type hMC4R and its C40A, C271A,and C279A mutants and a biphasic curve for hMC1R, suggesting the presence of at least one structurallyand functionally essential disulfide bond in both wild-type receptors and the hMC4R mutants. Models ofcomplexes of both receptors with the ASIP fragment and hMC4R with the AGRP fragment were calculatedusing constraints from the experimental structures of rhodopsin and AGRP fragments, a set of deducedhydrogen bonds, supplemented by two proposed disulfide bridges and receptor-ligand contacts, derivedfrom our mutagenesis data. In the models of the ASIP fragment complexed with both receptors, the coreligand tripeptide, Arg-Phe-Phe, positioned between TMHs 3 and 6, is shifted toward TMHs 2 and 7relative to its position in the AGRP-hMC4R model, while the N-terminal loop and two central disulfidesof the antagonists interact with EL2 of the receptors.