Gaudin P, Couvineau A, Maoret J J, Rouyer-Fessard C, Laburthe M
Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, INSERM U 410, Faculté de Médecine Xavier Bichat, Paris, France.
Biochem Biophys Res Commun. 1995 Jun 26;211(3):901-8. doi: 10.1006/bbrc.1995.1897.
We have used site-directed mutagenesis to investigate the requirement of cysteine residues in the extracellular domains of the human VIP 1 receptor for binding VIP. Cys37, Cys50, Cys63, Cys72, Cys86, Cys105 and Cys122 (N-terminal extracellular domain), Cys208 and Cys215 (second extracellular loop) and Cys288 (third extracellular loop) were mutated into glycine, and mutated cDNAs transfected into Cos-7 cells. It appeared that mutants C50G, C63G, C72G, C86G, C105G, C122G, and C288G did not bind VIP whereas mutants C37G, C208G and C215G bound VIP with the same dissociation constant (#0.5 nM) as the wild-type receptor. All mutated receptor proteins were synthesized by Cos-7 cells (Western blot) and delivered at the plasma membrane level (confocal microscopy). The fact that C208G and C215G mutants retained a complete binding activity while the C288G mutant was inactive does not suggest the presence of a functionally relevant disulfide bond between the second and third extracellular loop of the human VIP receptor contrary to what has been shown in several other heptahelical receptors. It is also concluded that the six crucial cysteine residues, e.g., Cys50, Cys63, Cys72, Cys86, Cys105 and Cys122 in the N-terminal extracellular domain, may be functionally important by forming intramolecular disulfide bonds which help to maintain the topology for ligand binding in human VIP 1 receptors.
我们利用定点诱变技术研究了人血管活性肠肽1型（VIP 1）受体胞外域中半胱氨酸残基对结合血管活性肠肽（VIP）的必要性。将第37、50、63、72、86、105和122位半胱氨酸（N端胞外域）、第208和215位半胱氨酸（第二个胞外环）以及第288位半胱氨酸（第三个胞外环）突变为甘氨酸，并将突变的cDNA转染到Cos-7细胞中。结果显示，突变体C50G、C63G、C72G、C86G、C105G、C122G和C288G不结合VIP，而突变体C37G、C208G和C215G以与野生型受体相同的解离常数（约0.5 nM）结合VIP。所有突变的受体蛋白均由Cos-7细胞合成（蛋白质免疫印迹法）并定位于质膜水平（共聚焦显微镜观察）。与其他几种七螺旋受体的情况相反，C208G和C215G突变体保留了完整的结合活性，而C288G突变体无活性，这一事实并不表明人VIP受体的第二个和第三个胞外环之间存在功能相关的二硫键。研究还得出结论，N端胞外域中的六个关键半胱氨酸残基，如Cys50、Cys63、Cys72、Cys86、Cys105和Cys122，可能通过形成分子内二硫键在功能上具有重要意义，这些二硫键有助于维持人VIP 1受体中配体结合的拓扑结构。
