Nachtergael Ingrid, Gaspard Nathalie, Langlet Christelle, Robberecht Patrick, Langer Ingrid
Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Belgium.
Cell Signal. 2006 Dec;18(12):2121-30. doi: 10.1016/j.cellsig.2006.03.006. Epub 2006 Mar 27.
After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation. We evaluated in CHO cells expressing the VPAC(1) receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y(224), N(229), F(230), W(232), E(236), G(237), Y(239), L(240). N(229)A VPAC(1) mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated Ca(2+) increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N(229)D mutant was not expressed at the membrane, and the N(229)Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N(229)A and N(229)Q VPAC(1) were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking. Mutation of that conserved amino acid in VPAC(2) could be investigated only by a conservative mutation (N(216)Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.
在用激动剂刺激后，G蛋白偶联受体（GPCR）会发生构象变化，从而激活G蛋白以转导信号，随后通过激酶磷酸化以及与抑制蛋白结合来促进受体内化。基于对GPCR - A/视紫红质家族的研究，实际范例表明位于跨膜（TM）结构域（主要是TM3、TM6和TM7）的保守残基之间的相互作用网络参与了导致GPCR激活的分子开关。我们在表达VPAC(1)受体的CHO细胞中，通过将受体分泌素家族中高度保守的残基突变为丙氨酸来评估第三跨膜螺旋在激动剂信号传导中的作用：Y(224)、N(229)、F(230)、W(232)、E(236)、G(237)、Y(239)、L(240)。N(229)A VPAC(1)突变体的特征在于VIP刺激的腺苷酸环化酶活性的效力和功效均降低，激动剂刺激的[Ca(2+)]i增加缺失，激动剂和拮抗剂的受体识别得以保留，对GTP的敏感性得以保留，这表明该残基对有效的G蛋白激活很重要。N(229)D突变体未在细胞膜上表达，具有保守突变的N(229)Q比A突变体受影响更小。激动剂刺激的N(229)A和N(229)Q VPAC(1)的磷酸化和内化未受影响。然而，内化的突变体受体重新表达，但野生型受体未重新表达，在VIP洗脱后迅速逆转。因此，受体磷酸化、内化和重新表达可能与G蛋白激活解离，并与另一种可能影响其转运的活性构象相关联。VPAC(2)中该保守氨基酸的突变只能通过保守突变（N(216)Q）进行研究，结果产生了一种对腺苷酸环化酶、受体磷酸化和内化的VIP刺激较低的受体。这表明该受体家族TM3中保守的N残基很重要。
