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首页> 外文期刊>Biochemistry >Structure−Function Analysis of RAMP1 by Alanine Mutagenesis
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Structure−Function Analysis of RAMP1 by Alanine Mutagenesis

机译：丙氨酸诱变对RAMP1的结构功能分析

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Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein−protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.
机译：受体活性修饰蛋白1（RAMP1）是包括降钙素基因相关肽（CGRP）受体在内的几种受体的组成部分。它与降钙素受体样受体（CLR）形成复合物，是受体运输和配体结合所必需的。 RAMP1的N端包含三个螺旋。当前的研究调查了丙氨酸诱变对CGRP或CLR相互作用重要的RAMP1区域。建模表明第二个和第三个螺旋在蛋白质-蛋白质相互作用中很重要。 N端的大多数保守残基（M48，W56，Y66，P85，N66，H97，F101，D113，P114，P115）以及分布在三个RAMP1螺旋中的另外13个残基被突变为丙氨酸和与CLR在Cos 7细胞中共表达。这些突变均未显着降低RAMP表达。在来自螺旋1的9个突变体中，只有M48A具有任何作用，从而使CLR向细胞表面的运输适度降低。在螺旋2中，Y66A几乎完全废除了CLR贩运； L69A和T73A降低了CGRP产生cAMP的能力。在第3螺旋中，H97A废除了CLR贩运； P85A，N86A和F101A导致CLR交易量的适度减少，并且还降低了CGRP对cAMP产生的效力。 F93A仅导致CLR贩运略有减少，而L94A增加了cAMP的产生。数据与CLR识别位点一致，尤其是涉及Y66和H97，对螺旋3中相邻残基的作用较小。L69和T73可能对螺旋2中的CGRP识别位点也有贡献，也涉及附近的残基。

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