STRUCTURE OF ANGIOTENSIN II RECEPTOR AND THE SIGNAL TRANSDUCTION MECHANISM

为了确定大鼠血管紧张素II受体1A型（AT_<1A>）羧基末端末端g_q蛋白偶联所必需的结构域，通过在cDNA中引入停止密码子的位点定向诱变截断了假定的细胞质区域的受体，并确定了GTPgammaS对血管紧张素II （Ang II）结合和肌醇三磷酸（IP_3）产生的影响。为了确定G_q激活的结构域，我们研究了代表AT_<1A>的羧基末端结构域（残基306-320）的合成肽（P-5）及其类似物（用AAA代替Y^<312>-F^<314>-L^<314>: Mut P-5）对^<35> s标记的GTPgammaS与G_q结合的影响。GTPgammaS对缺失突变体1-317 AT_<1A>的Ang II结合（AT_<1A>由高亲和状态向低亲和状态转变）和IP_3产生的影响与野生型AT_<1A>相似，而突变体1-309、1-311、1-312和1-313 AT_<1A>的影响明显降低。P-5能促进^<35> s标记的GTPgammaS与G_q的结合，而Mut P-5则不能。这些结果表明，大鼠AT_<1A>的胞质羧基末端结构域Tyr^<312>, Phe^<313>和Leu^<314>对G_q的偶联和激活至关重要。

To delineate domains essential for G_q-protein coupling in the carboxyl-terminal tail of rat angiotensin II receptor type 1A (AT_<1A>), the receptor in the putative cytosolic regions was truncated by site-derected mutagenesis introducing stop codons in the cDNA and determined consequent changes in the effect of GTPgammaS on angiotensin II (Ang II) binding and in inositol trisphosphate (IP_3) production in response to Ang II.To identify the domains responsible for the activation of G_q, we studied the effect of synthetic peptide (P-5) representing domains of carboxyl-terminal segment (residues 306-320) of AT_<1A> and its analog (substituting AAA for Y^<312>-F^<314>-L^<314> : Mut P-5) on the binding of the ^<35>S-labeled GTPgammaS to G_q. The effects of GTPgammaS on Ang II binding (shift of AT_<1A> from a high affinity state to a low affinity form) and IP_3 production in deletional mutant receptor 1-317 AT_<1A> were similar to wild type AT_<1A>, whereas those of mutants 1-309,1-311,1-312 and 1-313 AT_<1A> were markedly reduced. The binding of ^<35>S-labeled GTPgammaS to G_q was promoted by P-5, but not by Mut P-5. These results indicate that cytosolic carboxyl-terminal domains Tyr^<312>, Phe^<313> and Leu^<314> of rat AT_<1A> are essential for coupling and activation of G_q.