CONTROL OF G PROTEIN SIGNALING: ROLE OF THE RGSS

G 蛋白信号传导的控制：RGSS 的作用

• 批准号: 6288950
• 负责人: JOHN H KEHRL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 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/6288950
• 关键词: G protein; biological signal transduction; gene targeting; genetically modified animals; human tissue; laboratory mouse; protein sequence; protein structure function; receptor coupling; recombinant proteins; yeasts

We have discovered a protein family termed RGSs that impair signal transduction through pathways that use seven transmembrane receptors and heterotrimeric G proteins. Such receptors, when activated following the binding of a ligand such as a hormone or chemokine, trigger the G alpha subunit to exchange GTP for GDP; this causes the dissociation of G alpha and G beta-gamma subunits and downstream signaling. RGS proteins bind G alpha subunits and function as GTPase activating proteins (GAPs), thereby deactivating the G alpha subunit and facilitating their re-association with G beta-gamma. We have shown that RGS proteins modulate signaling through chemokine receptors and that they can inhibit chemotaxis. RGS1 expressing B lymphocytes fail to migrate in response to the chemokine SDF-1. Conversely, RGS1 deficient B cells obtained from mice in which the RGS1 gene has been disrupted by gene targeting have an enhanced chemotaxic response to SDF-1. Also, RGS proteins can inhibit the intracellular signaling triggered by a constitutively active G-protein coupled receptor (GPCR) such as the Kaposi?s sarcoma-associated herpes virus (KSHV-GPCR). The KSHV-GPCR has been shown to activate the Gi, Gq, G12 subfamilies of heterotrimeric G- proteins. Certain RGS proteins, in particular RGS2 and RGS3, are very efficient inhibitors of Gq signaling. Their effectiveness depends on the N-terminus of the RGS protein plus the RGS domain, the region required for its GAP activity. Certain key residues in the N-terminus have been identified as important. We have also shown that certain RGS proteins can directly inhibit the activation of adenylyl cyclase, thereby providing a mechanism by which certain RGS proteins can inhibit Gs induced cAMP production. We have demonstrated that RGS14 is highly expressed in lymphocytes and its expression levels are modulated by signaling through the B-cell and T-cell antigen receptors. RGS14 is also unique in that it is an inhibitor of G13 signaling. A yeast two- hybrid screen with RGS14 has been completed and several novel genes have been identified that encode proteins that interact with RGS14. To study the role of RGS proteins in lymphocyte development and function, RGS1 and RGS14 have been transgenically overexpressed. The consequences of their transgenic expression are now being analyzed. Furthermore RGS1 gene targeting has been accomplished and RGS3 and RGS14 are in progress. To study the consequences of persistent heterotrimeric G- protein signaling in lymphocyte development and function, transgenic mice expressing GTPase deficient G12 and G15 are being created. RGS3 and RGS4 have been shown to be phosphorylated and this phosphorylation is likely important in their translocation from the cytosol to membrane. - RGS, G-protein, chemokine, chemotaxis, lymphocytes, cAMP.

我们发现了一个称为 RGS 的蛋白质家族，它通过使用七个跨膜受体和异三聚体 G 蛋白的途径损害信号转导。这些受体在与激素或趋化因子等配体结合后被激活，触发 G α 亚基将 GTP 交换为 GDP；这会导致 G α 和 G β-γ 亚基以及下游信号传导的解离。 RGS 蛋白结合 G α 亚基并充当 GTP 酶激活蛋白 (GAP)，从而使 G α 亚基失活并促进其与 G β-γ 重新结合。我们已经证明，RGS 蛋白通过趋化因子受体调节信号传导，并且可以抑制趋化性。表达 RGS1 的 B 淋巴细胞无法响应趋化因子 SDF-1 进行迁移。相反，从 RGS1 基因已被基因靶向破坏的小鼠中获得的 RGS1 缺陷 B 细胞对 SDF-1 的趋化反应增强。此外，RGS 蛋白还可以抑制由组成型活性 G 蛋白偶联受体 (GPCR)（例如卡波西肉瘤相关疱疹病毒 (KSHV-GPCR)）触发的细胞内信号传导。 KSHV-GPCR 已被证明可以激活异源三聚体 G 蛋白的 Gi、Gq、G12 亚家族。某些 RGS 蛋白，特别是 RGS2 和 RGS3，是非常有效的 Gq 信号传导抑制剂。它们的有效性取决于 RGS 蛋白的 N 末端加上 RGS 结构域（其 GAP 活性所需的区域）。 N 末端的某些关键残基已被确定为重要的。我们还表明，某些RGS蛋白可以直接抑制腺苷酸环化酶的激活，从而提供了某些RGS蛋白可以抑制Gs诱导的cAMP产生的机制。我们已经证明，RGS14 在淋巴细胞中高表达，其表达水平受到 B 细胞和 T 细胞抗原受体信号传导的调节。 RGS14 的独特之处还在于它是 G13 信号传导的抑制剂。酵母与 RGS14 的双杂交筛选已经完成，并且已鉴定出编码与 RGS14 相互作用的蛋白质的几个新基因。为了研究 RGS 蛋白在淋巴细胞发育和功能中的作用，RGS1 和 RGS14 已被转基因过表达。目前正在分析它们转基因表达的后果。此外，RGS1基因靶向已经完成，RGS3和RGS14正在进行中。为了研究持续异源三聚体 G 蛋白信号传导对淋巴细胞发育和功能的影响，我们创建了表达 GTP 酶缺陷的 G12 和 G15 的转基因小鼠。 RGS3 和 RGS4 已被证明被磷酸化，并且这种磷酸化可能对于它们从细胞质到膜的易位很重要。 - RGS、G 蛋白、趋化因子、趋化性、淋巴细胞、cAMP。