INTERACTING PARTNERS OF G PROTEIN GAMMA SUBUNITS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 通过异三聚体G蛋白的信号转导对多种细胞反应是必不可少的，包括神经传递、激素反应、嗅觉转导、光转导、细胞迁移和细胞凋亡。作为对控制这些不同途径的大量G蛋白偶联受体(GPCR)的响应，脊椎动物进化了多种亚型的α、β和伽马亚基，这些亚基选择性地结合形成异源三聚体G蛋白。斑马鱼基因组有26个α亚基、9个贝塔亚基和17个伽马亚基，如果所有组合都组合在一起，那么总共有3978个潜在的不同的杂三聚体。虽然已经在体外研究了单个G蛋白亚型的信号伙伴和结合亲和力，但许多异源三聚体的内源性功能及其功能冗余的程度仍未确定。为了解决体内含有特定GGamma亚基的异源三聚体的功能多样性，我们表达了GGamma亚基的显性负向版本，以干扰已知的GPCR介导的事件-原始生殖细胞(PGC)迁移所必需的信号。我们表明，绝大多数预烯基化缺陷的GGamma亚基可以通过改变信号成分的亚细胞定位来扰乱PGC的迁移。这种干扰表现为PGC无法定向迁移。我们确定了野生型GGamma亚基的一个独特的子集，能够逆转这种半显性的负面影响，这表明GGamma亚基在体内具有不同的和重叠的信号能力。为了了解GGamma蛋白结构域在GGamma信号能力差异中所起的作用，我们构建了GGamma嵌合体。对这些嵌合体的分析表明，中心区域和c-末端区域中的多个区域和基序影响GGamma介导PGC定向迁移所需的信号通路的能力。我们的结果还表明，预烯基化缺陷的GGamma亚基可以用来在体内干扰GPCR介导的信号事件。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Signaling through heterotrimeric G proteins is essential for a variety of cellular responses including neurotransmission, hormonal response, olfactory transduction, phototransduction, cell migration and apoptosis. In response to the multitude of G protein coupled receptors (GPCR) that control these diverse pathways, vertebrates have evolved multiple isoforms of the alpha, beta and gamma subunits that selectively associate to form heterotrimeric G proteins. The zebrafish genome has 26alpha, 9beta and 17gamma subunits, which would make a total of 3978 potential different heterotrimers, if all combinations were made. Although the signaling partners and binding affinities of individual G protein isoforms have been investigated in vitro, the endogenous function of many heterotrimers and the extent of their functional redundancy remain uncharacterized. To address the functional diversity of heterotrimers containing specific Ggamma subunits in vivo, we expressed dominant negative versions of the Ggamma subunits to disrupt the signaling necessary for a known GPCR-mediated event, primordial germ cell (PGC) migration. We show that the vast majority of prenylation-deficient Ggamma subunits can disrupt PGC migration by altering the subcellular localization of signaling components. This disruption manifests in an inability of PGCs to migrate directionally. We identified a distinct subset of wild type Ggamma subunits that have the ability to reverse this semi-dominant negative effect, suggesting that Ggamma subunits have distinct and overlapping signaling capacities in vivo. To understand the roles Ggamma protein domains have in contributing to differences in Ggamma signaling capacity, we constructed Ggamma chimeras. Analysis of these chimeras demonstrated that multiple regions and motifs within the central and c-terminal regions influence the ability of Ggamma to mediate the signaling pathways necessary for directional PGC migration. Our results also indicate that prenylation-deficient Ggamma subunits can be used to disrupt GPCR-mediated signaling events in vivo.