Diverse functional motifs within the three intracellular loops of the CGRP1 receptor
Diverse functional motifs within the three intracellular loops of the CGRP1 receptor
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DOI：
10.1021/bi0615801
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发表时间：
2006-10-31
期刊：
影响因子：
2.9
通讯作者：
Poyner, David R.
中科院分区：
文献类型：
--
作者：
Conner, Alex C.;Simms, John;Poyner, David R.
CGRP(1)受体作为异二聚体复合物存在于单通跨膜辅助蛋白（RAMP1）和家族g蛋白偶联受体（GPCR）之间，称为降钙素受体样受体（CLR）。本研究调查了该受体胞内环（ICLs）中发现的结构基序。分子模型用于预测每个ICL的活性和非活性构象。保守残基通过定点诱变被改变为丙氨酸。表征了cAMP积累、细胞表面表达、激动剂亲和力和cgrp刺激受体内化。在ICL1中，L147，特别是R151对于G(s)的偶联很重要。R151可能直接与g蛋白相互作用，在涉及ICL2和ICL3的构象改变后进入g蛋白。在ICL3的近端，I290和L294可能位于R螺旋的同一面，形成了一个g蛋白偶联基序。I290A对偶联的影响最大；此外，它还降低了CGRP的亲和力和内化功能。I290可能与TM6相互作用以稳定ICL3的构象，但它也可能直接与Gs相互作用。R314在ICL3远端破坏了g蛋白偶联，并在较小程度上降低了CGRP的亲和力；它可能通过与膜磷脂的极性头基相互作用来稳定TM6-ICL3连接。ICL2中的Y215和L214是细胞表面表达所必需的；它们与具有相同功能的H216形成微畴。本研究揭示了CLR和其他gpcr在TM6和ICL3作用下的激活之间的相似性，但表明其他保守基序在其功能上存在差异。
The CGRP(1) receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein ( RAMP1) and a family B G-protein-coupled receptor ( GPCR) called the calcitonin receptor-like receptor ( CLR). This study investigated the structural motifs found in the intracellular loops ( ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to G(s). R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an R helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. I290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function.