Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity
Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity
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DOI：
10.1074/jbc.m302571200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Eto, T
中科院分区：
文献类型：
--
作者：
Kuwasako, K;Kitamura, K;Eto, T
当与受体活性修饰蛋白（RAMP）1共表达时，降钙素受体样受体（CRLR）可以作为降钙素基因相关肽（CGRP）和肾上腺髓质素（AM）的受体发挥作用。为了研究配体结合特异性的结构决定因素，我们使用各种缺失突变体检测了人（h）RAMP 1的胞外结构域。在HEK-293细胞中hRAMP 1突变体与hCRLR的共表达揭示了残基91-94、96-100或101-103的缺失阻断了[I-125] CGRP结合并完全消除了通常由CGRP或AM引起的细胞内cAMP积累。另一方面，残基78-80或88-90的缺失仅显著减弱AM诱发的反应。在所有这些情况下，受体异源二聚体在细胞表面完全表达。用丙氨酸一次一个地取代残基91-103对CGRP诱导的反应几乎没有影响，表明尽管该区段对于高亲和力激动剂与受体的结合是必需的，但没有一个残基直接与CGRP或AM相互作用。这一发现表明，RAMPs可能决定配体的特异性，通过促进配体结合口袋的结构或通过受体构象的变构调节。有趣的是，L94 A突变体上调受体异源二聚体的表面表达的程度大于野生型hRAMP 1，从而增加CGRP结合和信号传导。当与hCRLR共转染时，L94 A还显著增加hRAMP 1缺失突变体D101-103的细胞表面表达，并且L94 A/D101-103双突变体的表达显著减弱HEK-293 T细胞中内源性RAMP 1的活性。
When co-expressed with receptor activity-modifying protein (RAMP) 1, calcitonin receptor-like receptor (CRLR) can function as a receptor for both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). To investigate the structural determinants of ligand binding specificity, we examined the extracellular domain of human (h) RAMP1 using various deletion mutants. Co-expression of the hRAMP1 mutants with hCRLR in HEK-293 cells revealed that deletion of residues 91-94, 96-100, or 101-103 blocked [I-125] CGRP binding and completely abolished intracellular cAMP accumulation normally elicited by CGRP or AM. On the other hand, the deletion of residues 78-80 or 88-90 significantly attenuated only AM-evoked responses. In all of these cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine for residues 91-103 one at a time had little effect on CGRP-induced responses, indicating that although this segment is essential for high affinity agonist binding to the receptors, none of the residues directly interacts with either CGRP or AM. This finding suggests that RAMPs probably determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Interestingly, the L94A mutant up-regulated surface expression of the receptor heterodimer to a greater degree than wild-type hRAMP1, thereby increasing CGRP binding and signaling. L94A also significantly increased cell surface expression of the hRAMP1 deletion mutant D101-103 when co-transfected with hCRLR, and expression of a L94A/D101-103 double mutant markedly attenuated the activity of endogenous RAMP1 in HEK-293T cells.