Ishii K, Chen J, Ishii M, Koch W J, Freedman N J, Lefkowitz R J, Coughlin S R
Cardiovascular Research Institute, University of California, San Francisco 94143-0524.
J Biol Chem. 1994 Jan 14;269(2):1125-30.
The thrombin receptor, a member of the seven membrane-spanning superfamily of G-protein coupled receptors, is activated by an irreversible proteolytic mechanism, but signaling by activated thrombin receptors shuts off soon after receptor activation. This shut-off mechanism is thought to be required for concentration-dependent responses to thrombin and an important determinant of the cell's sensitivity to thrombin. We report that the thrombin receptor is rapidly phosphorylated upon activation, consistent with the action of a G-protein-coupled receptor kinase. Moreover, the G-protein coupled receptor kinase BARK2 (beta-adrenergic receptor kinase 2) blocked signaling by thrombin receptors coexpressed in Xenopus oocytes. In this system, rhodopsin kinase was inactive and BARK1 was markedly less effective than BARK2. Thrombin receptor mutants which lacked potential serine and threonine phosphorylation sites in the receptor's cytoplasmic tail were insensitive to inhibition by exogenous BARK2 but did confer concentration-dependent responses to thrombin. Our studies demonstrate that a G-protein coupled receptor kinase can shut off thrombin receptor signaling but that additional mechanism(s) for terminating signaling exist. These studies also reveal functional specificity among G-protein coupled receptor kinases in a novel in vivo reconstitution system and show that heterologous expression of these kinases can be used to manipulate cellular responsiveness.
凝血酶受体是G蛋白偶联受体的七跨膜超家族成员，通过不可逆的蛋白水解机制被激活，但激活后的凝血酶受体信号在受体激活后很快就会关闭。这种关闭机制被认为是凝血酶浓度依赖性反应所必需的，也是细胞对凝血酶敏感性的重要决定因素。我们报告，凝血酶受体在激活后迅速磷酸化，这与G蛋白偶联受体激酶的作用一致。此外，G蛋白偶联受体激酶BARK2（β-肾上腺素能受体激酶2）阻断了非洲爪蟾卵母细胞中共表达的凝血酶受体的信号传导。在这个系统中，视紫红质激酶没有活性，BARK1的效果明显不如BARK2。在受体胞质尾部缺乏潜在丝氨酸和苏氨酸磷酸化位点的凝血酶受体突变体对外源BARK2的抑制不敏感，但确实能产生对凝血酶的浓度依赖性反应。我们的研究表明，G蛋白偶联受体激酶可以关闭凝血酶受体信号，但存在其他终止信号的机制。这些研究还揭示了新型体内重组系统中G蛋白偶联受体激酶之间的功能特异性，并表明这些激酶的异源表达可用于操纵细胞反应性。
