Carman C V, Parent J L, Day P W, Pronin A N, Sternweis P M, Wedegaertner P B, Gilman A G, Benovic J L, Kozasa T
Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1999 Nov 26;274(48):34483-92. doi: 10.1074/jbc.274.48.34483.
G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
G蛋白偶联受体激酶（GRKs）是G蛋白偶联受体的特征明确的调节因子，而G蛋白信号调节（RGS）蛋白直接控制G蛋白α亚基的活性。有趣的是，最近一份报告（西德罗夫斯基，D.P.，赫塞尔，A.，钟，S.，马克，T.W.，和泰尔斯，M.（1996年）《当代生物学》6，211 - 212）在GRKs的N端鉴定出一个与RGS结构域具有同源性的区域。鉴于RGS结构域显示出对G蛋白α亚基的AlF₄⁻依赖性结合，我们测试了来自粗制牛脑提取物的G蛋白在不存在或存在AlF₄⁻的情况下与GRK亲和柱结合的能力。这揭示了牛脑Gαq/11以AlF₄⁻依赖性方式与GRK2和GRK3两者结合的特异性能力。相比之下，Gαs、Gαi和Gα12/13尽管存在于提取物中，但并未与GRK2或GRK3结合。进一步的研究表明，牛脑Gαq/11也能与GRK2的N端构建体结合，而未观察到Gαq/11、Gαs、Gαi或Gα12/13与GRK5或GRK6的类似构建体结合。使用纯化的Gαq进行的实验表明，Gαq GDP/AlF₄⁻和Gαq（GTPγS）两者都能与GRK2显著结合，但Gαq（GDP）则不能。在COS - 1细胞和HEK293细胞中也观察到了激活依赖性结合，因为GRK2能显著共免疫沉淀组成型活性Gαq（R183C），但不能沉淀野生型Gαq。体外分析表明，GRK2对Gαq具有微弱的GAP活性，该活性依赖于G蛋白偶联受体的存在。然而，GRK2在体外和细胞内均能有效抑制Gαq介导的磷脂酶C - β的激活，可能是通过隔离活化的Gαq来实现的。这些数据表明，GRKs的一个亚家族可能是G蛋白偶联受体信号传导的双功能调节因子，可直接作用于受体和G蛋白。
