Nicole P, Du K, Couvineau A, Laburthe M
Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier Bichat, 75018 Paris, France.
J Pharmacol Exp Ther. 1998 Feb;284(2):744-50.
Vasoactive intestinal peptide (VIP1 and VIP2) receptors belong to the new class II subfamily of G protein-coupled receptors. We investigated here human VIP1 and VIP2 receptors by mutating in their extracellular domains all amino acid residues that are conserved in VIP receptors but are different in other members of their subfamily. They are present in 1) the N-terminal domain, i.e., E36, I43, S64, D132 and F138 in the VIP1 receptor and E24, I31, S53, D116 and F122 in the VIP2 receptor; 2) the second extracellular loop, i.e., T288 and S292 in the VIP1 receptor and T274 and S278 in the VIP2 receptor. These residues were changed to alanine (A), and cDNAs were transfected into Cos cells. For the VIP1 receptor, no specific 125I-VIP binding could be detected in cells transfected with the E36A mutant, whereas other mutants exhibited Kd values similar to that of the wild-type receptor, with the exception of S64A, for which a 3-fold increase of Kd was observed. For the VIP2 receptor, no specific 125I-VIP binding could be observed with the E24A mutant, whereas other mutants exhibited dissociation constants similar to that of the wild-type receptor, with the exception of I31A and T274A mutants, for which a 11- and 5-fold increase of Kd was observed, respectively. cAMP production experiments provided evidence that the E36A VIP1 receptor and the E24A VIP2 receptor mutants mediated almost no response upon VIP exposure. For the I31A and T274A mutants of the VIP2 receptor and the S64A mutant of the VIP1 receptor, the EC50 values of VIP for stimulating cAMP production were increased 35, 8 and 3 times as compared with that observed for the wild-type receptor, respectively. Immunofluorescence studies indicated that all mutants were normally expressed by Cos cells. These data provide the first evidence for differences in the structure-function relationship of VIP1 and VIP2 receptors.
血管活性肠肽（VIP1和VIP2）受体属于G蛋白偶联受体新的II类亚家族。我们在此通过将VIP受体中保守但在其亚家族其他成员中不同的所有细胞外结构域氨基酸残基进行突变，对人VIP1和VIP2受体展开研究。这些残基存在于：1）N端结构域，即VIP1受体中的E36、I43、S64、D132和F138以及VIP2受体中的E24、I31、S53、D116和F122；2）第二个细胞外环，即VIP1受体中的T288和S292以及VIP2受体中的T274和S278。将这些残基突变为丙氨酸（A），并将cDNA转染到Cos细胞中。对于VIP1受体，在用E36A突变体转染的细胞中未检测到特异性的125I-VIP结合，而其他突变体表现出与野生型受体相似的Kd值，但S64A除外，其Kd值增加了3倍。对于VIP2受体，用E24A突变体未观察到特异性的125I-VIP结合，而其他突变体表现出与野生型受体相似的解离常数，但I31A和T274A突变体除外，其Kd值分别增加了11倍和5倍。cAMP生成实验提供了证据，表明E36A VIP1受体和E24A VIP2受体突变体在暴露于VIP时几乎不介导反应。对于VIP2受体的I31A和T274A突变体以及VIP1受体的S64A突变体，与野生型受体相比，VIP刺激cAMP生成的EC50值分别增加了35倍、8倍和3倍。免疫荧光研究表明，所有突变体均由Cos细胞正常表达。这些数据首次为VIP1和VIP2受体结构-功能关系的差异提供了证据。
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