Characterization of Agouti-related protein binding to melanocortin receptors.
Characterization of Agouti-related protein binding to melanocortin receptors.
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DOI：
10.1210/mend.13.1.0223
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发表时间：
1999
影响因子：
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通讯作者：
Y. Yang;Darren A. Thompson;Chris J. Dickinson;J. Wilken;G. Barsh;Stephen B. H. Kent;I. Gantz
中科院分区：
文献类型：
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作者：
Y. Yang;Darren A. Thompson;Chris J. Dickinson;J. Wilken;G. Barsh;Stephen B. H. Kent;I. Gantz
Agouti-related protein（AGRP）是一种黑皮质素的天然拮抗剂，被认为在下丘脑控制摄食行为中起重要作用。AGRP和Agglutamine蛋白作用的确切机制一直难以研究，部分原因是难以产生可用于直接结合测定的这些分子的均质形式。在这份报告中，我们描述了应用化学蛋白质合成的建设两个新的AGRP变体。对AGRP变体的生物活性的检查表明，截短的变体，人AGRP（87-132），一种基于AGRP的羧基末端富含半胱氨酸的结构域的46个氨基酸的变体，与111个氨基酸的变体，小鼠[Leu 127 Pro]AGRP是等效的（成熟AGRP减去其信号序列），其剂量依赖性地抑制在克隆的黑皮质素受体处α-MSH产生的cAMP产生的能力。此外，全长变体的氨基末端部分的缺失不改变AGRP的MCR亚型特异性（87-132）。最后，人AGRP（87-132）的碘化提供了一种有用的试剂，可以用它来分析AGRP的结合特性。在常规和感光乳剂结合研究中，观察到[125 I]AGRP（87-132）仅与表达黑皮质素受体MC 3R、MC 4 R和MC 5 R的细胞结合。这些结果表明，对受体结合、α-MSH抑制和黑皮质素受体亚型特异性至关重要的残基都位于分子的羧基末端。由于[Nle 4，D-Phe 7]（NDP）-MSH取代了[125 I]AGRP（87-132）与MCR的结合，AGRP（87 -132）取代了[125 I] NDP-MSH的结合，我们得出结论，这些分子以竞争方式与黑皮质素受体结合。
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.