Mao Helen, Zhao Qingshi, Daigle Mireille, Ghahremani Mohammad H, Chidiac Peter, Albert Paul R
Department of Neuroscience, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
J Biol Chem. 2004 Jun 18;279(25):26314-22. doi: 10.1074/jbc.M401800200. Epub 2004 Apr 19.
To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.
为了鉴定脑中最丰富的G蛋白——Gα(o)的新型调节因子，我们以组成型活性Gα(o)为诱饵进行酵母双杂交筛选，鉴定出一种新的G蛋白信号调节（RGS）蛋白RGS17（RGSZ2），它是RGS蛋白RZ（或A）亚家族的一个新的人类成员。RGS17包含一个富含半胱氨酸的氨基末端基序和一个羧基末端RGS结构域，与hRGSZ1和hRGS-Gα相互作用蛋白具有最高的同源性。RGS17 RNA在小脑和其他脑区以多种形式强烈表达。通过酵母双杂交分析、体外下拉分析和共免疫沉淀研究检测到hRGS17与Gα(i1-3)、Gα(o)、Gα(z)或Gα(q)的活性形式之间的相互作用，但未检测到与Gα(s)的相互作用。在预稳态条件下，重组RGS17对游离的Gα(i2)和Gα(o)起GTP酶激活蛋白（GAP）的作用，在体外稳态GTP酶分析中，对M2-毒蕈碱受体激活的Gα(i1)、Gα(i2)、Gα(i3)、Gα(z)和Gα(o)起作用。与对G(z)具有高度选择性的RGSZ1不同，RGS17在G(i)/G(o)蛋白中对G(o)的选择性有限。所有RZ家族成员都降低了多巴胺-D2/Gα(i)介导的cAMP形成抑制作用，并消除了促甲状腺激素释放激素受体/Gα(q)介导的钙动员。RGS17是一个新的RZ成员，它优先通过G(i/o)、G(z)和G(q)而非G(s)抑制受体信号传导，以增强cAMP依赖性信号传导并抑制钙信号传导。体外GAP分析和全细胞信号传导之间观察到的差异表明，RGS GAP效应的G蛋白特异性可能还存在其他决定因素，其中可能包括受体和效应器。
