Identification of cytoplasmic domains of hVPAC1 receptor required for activation of adenylyl cyclase -: Crucial role of two charged amino acids strictly conserved in class II G protein-coupled receptors
Identification of cytoplasmic domains of hVPAC1 receptor required for activation of adenylyl cyclase -: Crucial role of two charged amino acids strictly conserved in class II G protein-coupled receptors
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DOI：
10.1074/jbc.m301916200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Laburthe, M
中科院分区：
文献类型：
--
作者：
Couvineau, A;Lacapère, JJ;Laburthe, M
VPAC 1受体介导两种神经肽的作用，即血管活性肠肽（VIP）和垂体腺苷酸环化酶激活肽。是一种II类G蛋白偶联受体激活腺苷酸环化酶（AC）。hVPAC 1受体的N-末端胞外结构域对于VIP结合的作用现已确立（Laburthe，M.，Couvineau，A.和玛丽，J.C.（2002）Recept. Channels 8，137-153），但关于负责AC激活的胞质结构域还一无所知。在这里，我们构建了一个大系列的突变体取代氨基酸与丙氨酸的细胞内循环（IL）1，2和3和近端C-末端尾的受体。在中国仓鼠卵巢细胞中40个氨基酸的突变和突变体的表达显示如下。(i)突变IL 1导致突变体表达的缺失，表明该环在受体折叠中的作用。(ii)IL 2的所有残基都可以突变而不改变受体表达和AC对VIP的反应。(iii)残基IL 3的突变指出赖氨酸322在VIP刺激AC活性的功效中的特定作用。这种功效在K322 A突变体中降低了50%。(iv)近端C末端尾部配备了另一个重要的氨基酸，因为谷氨酸394的突变使AC反应降低了50%。双突变体K322 A/E394 A在刺激膜中AC活性和完整细胞中cAMP反应的VIP功效方面表现出>85%的急剧降低，而不改变受体表达或对VIP的亲和力。这些数据突出了IL 3中的带电残基和hVPAC 1受体的近端C-末端尾对于激动剂诱导的AC活化的作用。因为这些带电残基在肽的II类受体中是绝对保守的，它们都介导AC活化，所以它们可能在II类受体与G（s）蛋白的偶联中发挥一般作用。
The VPAC1 receptor mediates the action of two neuropeptides, vasoactive intestinal peptide ( VIP) and pituitary adenylate cyclase-activating peptide. It is a class II G protein-coupled receptor-activating adenylyl cyclase (AC). The role of the N-terminal extracellular domain of hVPAC1 receptor for VIP binding is now established (Laburthe, M., Couvineau, A. and Marie, J. C. (2002) Recept. Channels 8, 137-153), but nothing is known regarding the cytoplasmic domains responsible for AC activation. Here, we constructed a large series of mutants by substituting amino acids with alanine in the intracellular loops (IL) 1, 2, and 3 and proximal C-terminal tail of the receptor. The mutation of 40 amino acids followed by expression of mutants in chinese hamster ovary cells showed the following. (i) Mutations IL1 result in the absence of expression of mutants, suggesting a role of this loop in receptor folding. (ii) All residues of IL2 can be mutated without alteration of receptor expression and AC response to VIP. (iii) Mutation of residues IL3 points to the specific role of lysine 322 in the efficacy of the stimulation of AC activity by VIP. This efficacy is reduced by 50% in the K322A mutant. (iv) The proximal C-terminal tail is equipped with another important amino acid since mutation of glutamic acid 394 reduces AC response by 50%. The double mutant K322A/E394A exhibits a drastic reduction of >85% in the efficacy of VIP in stimulating AC activity in membranes and cAMP response in intact cells without alteration of receptor expression or affinity for VIP. These data highlight the role of charged residues in IL3 and the proximal C-terminal tail of hVPAC1 receptor for agonist-induced AC activation. Because these charged residues are absolutely conserved in class II receptors for peptides, which are all mediating AC activation, they may play a general role in coupling of class II receptors with the G(s) protein.