Membrane Targeting Of G Protein

G 蛋白的膜靶向

• 批准号: 6673560
• 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/6673560
• 关键词: CHO cells; G protein; biological signal transduction; chemical association; chemical kinetics; 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

Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors and effectors. We investigated whether signaling through Gs was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low cholesterol conditions, and then treated with the cholesterol chelator, methyl-b-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line unable to synthesize sphingolipids, were incubated under low sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, of Galpha s from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha s. The cAMP accumulation in response to a receptor agonist was intact and slightly increased in cells depleted of cholesterol or sphingolipids compared to control cells. These results indicate that localization of Galpha s to detergent-resistant membranes was not required for Gs signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentation in lipid rafts may be more effective in inhibiting protein interactions and depending on the pathway, ultimately inhibit or promote signaling. 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 blocked its incorporation of [3H]palmitate, significantly inhibited its GTPase activating protein (GAP) activity toward a Galpha subunit fused to the 5HT1A receptor, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoperoxidase electron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts, that can be isolated by their resistance to detergents and buoyancy on gradient centrifugation. 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-b-cyclodextrin did not change 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 within the RGS box. These results suggest that amino-terminal palmitoylation promotes the lipid raft targeting of RGS16 that allows palmitoylation of a poorly accessible cysteine residue. The heterotrimeric G proteins, G12 and G13, are closely related in their sequences, signaling partners and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet, G12 and G13 can act through different pathways, bind different proteins and show opposing actions on some effectors. We investigated the compartmentalization of G12 and G13 at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Galpha 12 was in the lipid raft fractions, whereas Galpha 13 was not associated with lipid rafts. Mutation of C11 on Galpha 12, which prevents its palmitoylation, partially shifted Galpha 12 from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Galpha 12 and a complete loss of the C11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Galpha 12 in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Galpha 12 to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Galpha 12 only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G12 and G13 and control of these potent cell regulators. Most heterotrimeric G-protein alpha subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Galpha s palmitoylation for its intracellular distribution and ability to couple to the beta-adrenergic receptor (BAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc- cells, which lack endogenous Galpha s, showed that wild-type Galpha s was predominantly localized at the plasma membrane, but the mutant C3A-Galpha s, which does not incorporate [3H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Galpha s showed neither isoproterenol- or GTPgammaS-stimulated adenylyl cyclase activation nor GTPgammaS-sensitive high affinity agonist binding, all of which were present in the wild-type Galpha s expressing cells. Fusion of C3A-Galpha s with the BAR (BAR-(C3A)Galpha s), partially rescued its ability to induce high affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPgammaS treatment. In comparison to results with the WT-Galpha s and BAR (BAR-Galpha s) fusion protein, the BAR-(C3A)Galpha s fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing BAR-Galpha s reduced the high affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in BAR-(C3A)Galpha s expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Galpha s and facilitated bimolecular interactions of Galpha s with the BAR and adenylyl cyclase.

富含胆固醇和鞘脂的膜微域可调节许多信号转导途径，并为异三聚体G蛋白，其受体和效应子提供住宅。我们调查了通过GS信号传导是否取决于这些膜结构域，其特征是它们对洗涤剂的抗性，通过消耗胆固醇和鞘脂的细胞来取决于对洗涤剂的耐药性。对于胆固醇消耗，将大鼠唾液上皮A5细胞在低胆固醇条件下培养，然后用胆固醇螯合剂甲基-B-氯麦克塞德蛋白治疗。对于鞘脂耗竭，在低鞘脂条件下孵育了无法合成鞘脂的突变CHO细胞系LY-B细胞。胆固醇或鞘脂的消耗分别导致耐洗涤剂的膜的Galpha S损失或减少，而细胞或膜结合的Galpha s则没有任何变化。与对照细胞相比，对胆固醇或鞘脂的细胞的响应响应于受体激动剂的心积累是完整的，并且略有增加。这些结果表明，GS信号传导不需要将Galpha S定位到耐洗涤剂的膜上。分析脂质筏对膜中蛋白质关联动力学的作用表明，脂质筏中的隔室在抑制蛋白质相互作用方面可能更有效，并取决于途径，最终抑制或促进信号传导。 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的突变阻断了其[3H]棕榈酸酯的掺入，显着抑制了其GTPase激活蛋白（GAP）激活蛋白（GAP）的活性，该活性对Galpha亚基融合到5HT1A受体融合，但并未基于细胞分流研究和免疫氧化酶氧化酶氧化酶的质膜定位。棕榈酰化可以靶向包括许多信号蛋白的蛋白质，用于称为脂质筏的膜微域，可以通过其对洗涤剂的耐药性和梯度离心的浮力来隔离。大鼠肝膜中的内源性RGS16和COS细胞中的过表达RGS16，而不是RGS16的非层状半胱氨酸突变体，该突变体位于脂质筏上。然而，用甲基-B-环糊精通过处理脂质筏的破坏不会改变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RGS盒中包含CYS-98的肽片段。这些结果表明，氨基末端棕榈酰化促进了RGS16的脂质筏的靶向，从而允许棕榈酰化不足的半胱氨酸残基。 异三聚体G蛋白G12和G13在其序列，信号伴侣和细胞效应（例如致癌转化和细胞骨架重组）中密切相关。但是，G12和G13可以通过不同的途径作用，结合不同的蛋白质并在某些效应子上显示相反的作用。我们研究了膜上G12和G13的隔室化，因为其他G蛋白位于脂质筏中，富含胆固醇和鞘脂的膜微域。在冷，非离子洗涤剂提取细胞和梯度离心后分离脂质筏。 galpha 12在脂质筏部分中，而galpha 13与脂质筏无关。 C11在Galpha 12上的突变阻止了其棕榈酰化，部分使Galpha 12从脂质筏上移动。特异性抑制HSP90的Geldanamycin处理导致野生型Galpha 12的部分损失，并从脂质筏中完全丧失C11突变体，并且在可溶性部分中出现了较高的分子量Galpha 12。这些结果表明，酰基化和HSP90相互作用将Galpha 12局部到脂质筏上。 HSP90只能充当支架和伴侣，仅在离散的膜结构域中维持功能性Galpha 12，从而解释G12和G13的某些非重叠函数以及对这些有效的细胞调节剂的控制。 大多数异三聚体G蛋白α亚基是通过棕榈酰化后的翻译后修饰的，棕榈酰化是一种动态调节的可逆过程。我们分析了Galpha palmitoylation对其细胞内分布的影响以及与β-肾上腺素能受体（BAR）搭配并刺激腺苷酸环化酶的能力。 Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc- cells, which lack endogenous Galpha s, showed that wild-type Galpha s was predominantly localized at the plasma membrane, but the mutant C3A-Galpha s, which does not incorporate [3H]palmitate, was mostly associated with intracellular membranes.与这种错误定位一致，C3a-galpha s显示异丙肾上腺素或GTPGAMMAS刺激的腺苷酸环化酶激活或GTPGAMMAS敏感的高亲和力激动剂结合，它们都存在于野生型Galpha S表达细胞中。 C3a-Galpha与棒（bar-（c3a）galpha s）的融合，部分挽救了其诱导高亲和力激动剂结合的能力，并在异丙肾上腺素或GTPGAMMAS处理后刺激腺苷酸环化酶活性。与wt-galpha s和bar（bar-galpha s）融合蛋白的结果相比，bar-（c3a）galpha的融合蛋白在与受体和效应子偶联时的效率约为一半。表达棒状 - 甲胺的羟胺化学去甲酰胺化的化学二酰胺降低了高亲和力激动剂结合和腺苷酸环化酶激活的程度与在Bar-（C3a）Galpha S表达膜中观察到的程度相似。总之，这些发现表明，棕榈酰化确保了Galpha s的适当定位，并促进了Galpha S与棒和腺苷酸环化酶的双分子相互作用。