Systematic analysis of the entire second extracellular loop of the V1a vasopressin receptor -: Key residues, conserved throughout a G-protein-coupled receptor family, identified
Systematic analysis of the entire second extracellular loop of the V1a vasopressin receptor -: Key residues, conserved throughout a G-protein-coupled receptor family, identified
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DOI：
10.1074/jbc.m702151200
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发表时间：
2007-06-15
影响因子：
4.8
通讯作者：
Wheatley, Mark
中科院分区：
文献类型：
--
作者：
Conner, Matthew;Hawtin, Stuart R.;Wheatley, Mark
与跨膜螺旋中的残基相比，G蛋白偶联受体（GPCRs）的胞外残基的作用还没有很好地定义。然而，已经确定肽-GPCR和胺-GPCR的胞外结构域都掺入功能上重要的残基。细胞外环2（ECL 2）引起了特别的兴趣，因为牛视紫红质的X射线结构显示，ECL 2项目到跨膜束内的结合裂缝。我们的研究首次全面研究了构成小肽GPCR整个ECL 2结构域的各个残基的作用。使用V-1a血管加压素受体，用Ala系统地取代所有的ECL 2残基，产生了30个突变体受体，其特征在于：这些突变体受体构建体中的大多数（总共24个）基本上具有野生型配体结合和细胞内信号传导特征，表明这些残基对于正常受体功能不是关键的。然而，四个芳香族残基Phe（189）、Trp（206）、Phe（209）和Tyr（218）对于激动剂结合和受体活化是重要的，并且在肽-GPCR的神经垂体激素亚家族中高度保守。Trp（206）和Phe（209）位于ECL 2的中间，与高度保守的二硫键并列，突出到结合缝隙中。实际上，Phe（209）是Cys-X-X-X-Ar（其中Ar是芳香族残基）基序的一部分，其在肽-GPCR和胺-GPCR中都是很保守的。相比之下，位于ECL 2的最末端的Phe（189）和Tyr（218）对于确定ECL 2帽在结合裂缝上的位置可能是重要的。这项研究提供了高度保守的ECL 2残基的作用机制的见解。
The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V-1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe(189), Trp(206), Phe(209), and Tyr(218) are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp(206) and Phe(209) project into the binding crevice. Indeed, Phe(209) is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe(189) and Tyr(218), located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.