A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: Implications for family B G-protein-coupled receptors
A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: Implications for family B G-protein-coupled receptors
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DOI：
10.1124/mol.67.1.20
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发表时间：
2005-01-01
影响因子：
3.6
通讯作者：
Poyner, DR
中科院分区：
文献类型：
--
作者：
Conner, AC;Hay, DL;Poyner, DR
降钙素受体样受体（Calcitonin receptor like-receptor，GPCR）是B家族G蛋白偶联受体（GPCR）。它需要受体活性修饰蛋白（RAMP）1来产生降钙素基因相关肽（CGRP）受体。人们对这个受体家族成员的功能知之甚少。脯氨酸残基通常在α-螺旋中形成重要的扭结。因此，受体的跨膜螺旋内的所有脯氨酸残基（螺旋4中的Pro 241、Pro 244、螺旋5中的Pro 275、螺旋6中的Pro 321和Pro 331）突变为丙氨酸。Pro 241、Pro 275和Pro 321在所有家族B GPCR中高度保守。用RAMP 1瞬时转染COS-7细胞后，在突变型和野生型受体中研究了CGRP的结合及其刺激cAMP产生的能力。P321 A突变显著降低CGRP的pEC（50），降低其亲和力，但不改变细胞表面表达。拮抗剂结合[CGRP（8-37）和1-哌啶甲酰胺，N-[2-[[5氨基-1-[[4-（4-吡啶基）-1-哌嗪基]羰基]戊基]氨基]-1-[（3，5-二溴-4-羟基苯基）甲基]-2-氧代乙基]-4-（1，4-二氢-2-氧代-3（2 H）-喹唑啉基）（BIBN 4096 BS）]几乎未因突变而改变。当P321 A与RAMP 1、RAMP 2或RAMP 3共表达时，肾上腺髓质素介导的信号传导被破坏。P331 A突变体产生CGRP结合和受体活化的中度减少。其他残基的突变对受体功能没有影响。因此，Pro321和Pro331是激动剂结合和受体活化所必需的。建模表明，Pro321诱导螺旋6中的弯曲，使其C末端接近螺旋3的C末端，如在许多家族A GPCR中所见。这在P321 A中被废除。P321 A-I325 P，预测恢复这种构象，显示野生型激活。建模也可以合理化的跨膜脯氨酸突变体的影响，以前报告的另一个家庭B GPCR，VPAC（1）受体。
Calcitonin receptor like-receptor is a family B G-protein coupled receptor ( GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide ( CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor ( Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241, Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide, N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl] pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2- oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P, predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.