Effects of intracellular tyrosine residue mutation and carboxyl terminus truncation on signal transduction and internalization of the rat bradykinin B2 receptor
Effects of intracellular tyrosine residue mutation and carboxyl terminus truncation on signal transduction and internalization of the rat bradykinin B2 receptor
复制标题
DOI：
10.1074/jbc.272.23.14638
复制
发表时间：
1997-06-06
影响因子：
4.8
通讯作者：
Polgar, P
中科院分区：
文献类型：
--
作者：
Prado, GN;Taylor, L;Polgar, P
目前，很少有人知道的氨基酸基序（S）参与缓激肽B2受体介导的信号转导过程。在本报告中，我们研究了细胞内区域和羧基末端中两个现有的酪氨酸（Tyr）残基在该受体调节功能中的潜在作用。将不含可检测缓激肽B2受体的大鼠-1细胞用野生型和突变型受体cDNA转染，Tyr-131和Tyr-321各自突变为相应的含丙氨酸、丝氨酸和苯丙氨酸的序列。羧基末端的最后34个氨基酸残基被截短。用包括截短的COOH-末端cDNA的受体cDNA的突变形式转染的Rat-1细胞都以与用野生型缓激肽B2受体转染的细胞基本相同的约2.2nM的K-d结合[H-3]缓激肽。然而，突变Tyr-131导致缓激肽刺激的磷酸肌醇周转和花生四烯酸释放的重要变化。例如，将Tyr-131交换为丙氨酸导致花生四烯酸释放减少80（p < 0.005），磷酸肌醇（IP）积累减少90%（p < 0.001），在15分钟时受体摄取基本保持不变。将相同的Tyr突变为苯丙氨酸导致不变的缓激肽刺激的TP积累，仅稍微降低的花生四烯酸释放，而在15分钟时受体摄取不变，Tyr-321突变为丙氨酸导致非常不同的模式，花生四烯酸释放有小但显著的减少，（p < 0.03）和IP蓄积（p < 0.008），在15分钟时受体摄取增加30%，（p < 0.010）。羧基尾的一部分的截短也证明是有意义的，花生四烯酸释放减少60%，IP积累减少80%，截短还导致15分钟时受体摄取大幅减少130%（p < 0.023）。总而言之，这些结果表明Tyr-131是决定缓激肽刺激的花生四烯酸释放和IP积累的重要因素。在该位点的酪氨酸磷酸化显然不起主要作用，Tyr-131、Tyr-321和羧基尾似乎在决定受体摄取方面很重要。
Presently, little is known of the amino acid motif(s) participating in bradykinin B2 receptor-mediated signal transduction processes. In this report we investigate the potential role of the two existing tyrosine (Tyr) residues in the intracellular regions and the carboxyl terminus in the regulatory function of this receptor. Rat-1 cells, which do not contain detectable bradykinin B2 receptor, were transfected with wild type and mutant receptor cDNAs, Tyr-131 and Tyr-321 were each mutated to corresponding alanine-, serine-, and phenylalanine-containing sequences. The last 34 amino acid residues of the carboxyl terminus were truncated, Rat-1 cells transfected with the mutant forms of the receptor cDNA including the truncated COOH-terminal cDNA all bound [H-3]bradykinin with essentially the same K-d of approximately 2.2 nM as cells transfected with the wild type bradykinin B2 receptor, However, mutating Tyr-131 resulted in important changes in bradykinin-stimulated phosphoinositide turnover and arachidonate release. For example, exchanging Tyr-131 for alanine led to an 80% decreased arachidonate release (p < 0.005), 90% decrease in inositol phosphate (IP) accumulation (p < 0.001), with receptor uptake at 15 min remaining essentially unchanged, Mutating the same Tyr to phenylalanine resulted in unchanged bradykinin-stimulated TP accumulation, only a slightly lowered arachidonate release, and unchanged receptor uptake at 15 min, Mutating Tyr-321 to alanine resulted in a very different pattern, There was a small but significant reduction in arachidonate release (p < 0.03) and IP accumulation (p < 0.008) with a large, 30%, increase in receptor uptake at 15 min (p < 0.010), Truncation of a portion of the carboxyl tail also proved meaningful, with a 60% decrease in arachidonate release and an 80% decrease in IP accumulation, The truncation also resulted in a large, 130%, decrease in receptor uptake at 15 min (p < 0.023), Taken together, these results point to Tyr-131 as an important element in determining bradykinin-stimulated arachidonate release and IP accumulation. Tyrosine phosphorylation at this site apparently does not play a major role, Tyr-131, Tyr-321, and the carboxyl tail appear to be important in determining receptor uptake.