Membrane Targeting Of G Protein

G 蛋白的膜靶向

• 批准号: 6821109
• 负责人: TERESA L Z JONES
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6821109
• 关键词: CHO cells; G protein; biological signal transduction; cholesterol; cyclic AMP; enzyme activity; fatty acylation; guanosine triphosphate; guanosinetriphosphatases; hydrolysis; membrane activity; membrane proteins; membrane structure; mutant; palmitates; protein localization; protein protein interaction; protein structure function; receptor coupling; receptor expression; sphingolipids; CHO cells; G protein; biological signal transduction; cholesterol; cyclic AMP; enzyme activity; fatty acylation; guanosine triphosphate; guanosinetriphosphatases; hydrolysis; membrane activity; membrane proteins; membrane structure; mutant; palmitates; protein localization; protein protein interaction; protein structure function; receptor coupling; receptor expression; sphingolipids

Regulators of G-protein Signaling (RGS) proteins downregulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the Galpha subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of [3H]palmitate and ability to turn-off Gi and Gq signaling and significantly inhibited its GTPase activating protein (GAP) activity toward a Galpha subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the nonpalmitoylated cysteine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-beta-cyclodextrin did not decrease the GAP activity of RGS16. The lipid raft fractions were enriched in protein acyl transferase activity and RGS16 incorporated [3H]palmitate into a peptide fragment containing Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessible cysteine residue that is critical for RGS16 and RGS4 GAP activity. Palmitoylation is an important post-translational modification used by cells to regulate protein activity. The rapid and reversible addition of palmitate to cellular proteins facilitates protein-protein interactions or targets proteins to a specific subcellular fraction. The regulator of G protein signaling (RGS)16 protein (RGS16) shares a number of conserved cysteine residues with RGS4 and RGS5 that undergo palmitoylation. N-terminal cysteine residues (Cys2, Cys12) in RGS4 and RGS16 regulate protein function, and a cysteine residue in the RGS box (Cys95 in RGS4) may modulate the GAP activity of RGS4 and RGS10. We investigated palmitoylation of RGS16 at residue Cys98 to determine its role in RGS16 localization and GTPase accelerating (GAP) activity. Mutation of RGS16 Cys98 to alanine diminished the ability of transfected RGS16 to promote serotonin-induced GTPase activity of a GPCR/Galpha fusion protein in mammalian cell membranes as well as its negative regulation of adenylyl cyclase inhibition induced by a Gi-linked GPCR in HEK293T cells. The C98A mutation of RGS16 had no discernable effect on the localization of RGS16 to membranes or on the GAP activity of recombinant RGS16 toward purified G protein alpha subunits. Most importantly, enzymatic palmitoylation of RGS16 by a partially purified protein acyltransferase (pPAT) resulted in internal palmitoylation on residue Cys98 and a dramatic, time-dependent increase in the GAP activity of RGS16 on membranes expressing the GPCR/Galpha fusion protein. These results suggest that palmitoylation of Cys98 is critical for the normal in vivo function of RGS16. Galpha 13 stimulates guanine nucleotide exchange factors (GEFs) for Rho, such as p115RhoGEF. Activated Rho induces numerous cellular responses including actin polymerization, serum response element (SRE)-dependent gene transcription, and transformation. P115RhoGEF contains a Regulator of G protein Signaling domain (RGS box) conferring GTPase activating protein (GAP) activity toward G alpha 12 and 13. In contrast, classical RGS proteins (such as RGS16 and RGS4)exhibit RGS domain-dependent GAP activity on Galpha i and Galpha q, but not Galpha 12/13. Here we show that RGS16 amino-terminus binds Galpha 13 directly, resulting in Galpha 13 translocation to detergent-resistant membranes (DRMs) and reduced p115RhoGEF binding. RGS4 does not bind Galpha 13 or attenuate G alpha 13-dependent responses, and neither RGS16 nor RGS4 affects Galpha 12-mediated signaling. These results elucidate a new mechanism whereby a classical RGS protein regulates Galpha 13-meidated signal transduction independently of the RGS box. Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.

G蛋白信号传导（RGS）蛋白的调节剂通过在Galpha亚基上加速GTP水解来通过异三聚体G蛋白下调信号。棕榈酰化是在几种RGS蛋白上可逆地添加到半胱氨酸残基中的棕榈酸酯，对其活性至关重要。对于RGS16，CYS-2和CYS-12的突变阻止了其纳入[3H]棕榈酸酯的纳入和关闭GI和GQ信号的能力，并显着抑制其GTPase激活蛋白（GAP）激活蛋白（GAP）的活性，该蛋白（GAP）的活性与Galpha亚基融合在一起，并未降低基于5-Hydroxyptyptamine frosron的细胞定位，但没有降低plaremantion and prination and prination and plaction fratiation and plastion and plactiation。显微镜。棕榈酰化可以靶向包括许多信号蛋白，包括称为脂质筏的膜微区。大鼠肝膜中内源性RGS16的亚群和COS细胞中过表达的RGS16的亚群，而不是RGS16的非层状半胱氨酸突变体，而不是局部为脂质筏。然而，用甲基-Beta-Cyclodextrin处理脂质筏的破坏不会降低RGS16的间隙活性。将脂质筏级分富集在蛋白酰基转移酶活性中，RGS16掺入[3H]棕榈酸酯中，含有含有Cys-98的肽片段，该肽是RGS盒中高度保守的半胱氨酸。这些结果表明，RGS蛋白的氨基末端棕榈酰化促进了其脂质筏靶向，从而允许棕榈酰化不足的半胱氨酸残基，这对于RGS16和RGS4 GAP活性至关重要。 棕榈酰化是细胞用于调节蛋白活性的重要后翻译后修饰。在细胞蛋白中快速且可逆的添加棕榈酸酯促进了蛋白质 - 蛋白质相互作用，或者将蛋白靶向特定的亚细胞分数。 G蛋白信号传导（RGS）16蛋白（RGS16）的调节剂具有许多经历棕榈酰化的RGS4和RGS5的保守半胱氨酸残基。 RGS4和RGS16中的N末端半胱氨酸残基（CYS2，CYS12）调节蛋白质功能，RGS盒中的半胱氨酸残基（RGS4中的Cys95）可能会调节RGS4和RGS10的间隙活性。我们研究了RGS16在残基Cys98处的棕榈酰化，以确定其在RGS16定位和GTPase加速（GAP）活性中的作用。 RGS16 Cys98对丙氨酸的突变降低了转染RGS16促进哺乳动物/GALPHA融合蛋白在哺乳动物细胞膜中GPCR/GALPHA融合蛋白的GTPase活性以及其对Gi-link gpcr in gi-link gpcr in hekccr中的腺苷循环抑制的负调控的GPCR/GALPHA融合蛋白的能力。 RGS16的C98A突变对RGS16对膜的定位或重组RGS16对纯化G蛋白α亚基的间隙活性没有可见的影响。最重要的是，通过部分纯化的蛋白酰基转移酶（PPAT）对RGS16的酶促棕榈酰化导致残基Cys98的内部棕榈酰化，并且在表达GPCR/Galpha fifusion Protein的膜上RGS16的间隙活性显着，依赖于时间依赖的RGS16。这些结果表明，Cys98的棕榈酰化对于RGS16的正常体内功能至关重要。 Galpha 13刺激RHO的鸟嘌呤核苷酸交换因子（GEF），例如P115Rhogef。活化的Rho诱导了许多细胞反应，包括肌动蛋白聚合，血清反应元件（SRE）依赖性基因转录和转化。 P115RHOGEF包含G蛋白信号域（RGS框）的调节剂，该调节剂赋予GTPase激活蛋白（GAP）对G alpha 12和13的活性。相反，经典的RGS蛋白（例如RGS16和RGS4）展示了RGS域域依赖性gap Active on galpha i和galpha i和galpha i和galpha i和galpha i和galpha，在这里，我们表明RGS16氨基末端直接结合Galpha 13，从而导致Galpha 13易位与耐洗涤剂的膜（DRMS）和降低的P115RHOGEF结合。 RGS4不结合Galpha 13或减弱G alpha 13依赖性响应，RGS16和RGS4都不会影响Galpha 12介导的信号传导。这些结果阐明了一种新的机制，从而使经典的RGS蛋白与RGS盒子独立于Galpha 13-筛选信号转导。 激动剂刺激了α2a-肾上腺素受体和GO1α的百日咳抗毒素耐药突变体的共表达后HEK293细胞膜中的高亲和力GTPase活性。 VMAX和KM的酶动力学分析无法通过GTPase激活蛋白来检测激动剂对激动剂的作用。但是，当细胞也转染以表达RGS4时，确实会发生这种情况。融合蛋白的两个元素与α2A-肾上腺素受体的C末端尾部有关，因为它能够提供协同的G蛋白刺激和失活。相比之下，刺激了α2A-肾上腺素pecceptor-RGS4融合蛋白，但没有增强对GO1α的停用，该蛋白具有对G蛋白信号传导（RGS）蛋白质的影响的抗性。采用此模型系统，ASN128的突变，但没有ASN88消除了可检测的GTPase激活RGS4对GO1α的蛋白活性。 RGS4中翻译后酰化位点的所有三种半胱氨酸残基的突变也消除了GTPase激活蛋白活性，但这并不能通过这些位点的协调突变而实现。这些研究表明，G蛋白偶联受体和RGS蛋白之间的融合蛋白在提供增强的鸟嘌呤核苷酸交换和GTP水解的GTP水解方面完全有效。它们还提供了一种直接的方法来评估在哺乳动物细胞中RGS蛋白突变对RGS与其靶G蛋白的空间关系和取向的情况下的功能的影响。