Tyrosine kinases in G protein mediated signaling.

描述（由申请人提供）：在过去的十年中，许多G蛋白偶联受体（gpcr）传递影响细胞分化和生长的信号已经变得很明显，包括刺激Ras家族gtpase和激活丝裂原活化蛋白（MAP）激酶途径。许多常见的临床疾病，包括压力过载心脏肥大、心肌纤维化、血管平滑肌内膜增生、合成代谢骨重塑和前列腺肥大，已被证明与这些gpcr介导的信号有关。先前对gpcr用于控制酪氨酸蛋白激酶活性和调节ERK1/2 MAP激酶级联的机制的研究表明，这些反应通常是新的信号事件的结果，例如gpcr、Src家族酪氨酸激酶和β -阻滞蛋白结合的MAP激酶之间形成复合物，以及gpcr与经典受体酪氨酸激酶之间的串导。新出现的数据表明，不同的信号机制导致空间分离和功能不同的MAP激酶池的形成。这个应用程序有两个广泛的目标。首先是详细描述gpcr通过β -抑制蛋白支架和转激活表皮生长因子（EGF）受体介导的ERK激活的分子机制。第二个目标是了解这些新信号如何整合以确定细胞对GPCR激活的反应。该提案的第一个具体目标是，在细胞模型系统中使用野生型和突变型gpcr，确定ERK在β -抑制蛋白支架上激活的机制，包括异源三聚体G蛋白亚基的作用。G蛋白效应器，Src激酶和Ras gtp酶。第二个具体目标是，利用一种由配体供体和配体受体细胞群组成的新型混合细胞检测系统，以及直接检测gpcr刺激的肝素结合EGF释放，确定gpcr控制基质金属蛋白酶依赖性EGF受体释放的机制，并表征金属蛋白酶非依赖性EGF受体转激活的机制。第三个目的是验证gpcr刺激的ERK激酶激活的后果是由激活机制决定的假设。这些实验将在β -抑制素敲除的成纤维细胞中使用内源性表达的GPCR，确定β -抑制素支架和EGF受体反激活在控制特定胞底、膜和核ERK底物磷酸化中的作用，以及对GPCR刺激的转录反应。

DESCRIPTION (provided by applicant): Over the past decade, it has become apparent that many G protein-coupled receptors (GPCRs) transmit signals that influence cellular differentiation and growth, including stimulation of Ras family GTPases and activation of mitogen-activated protein (MAP) kinase pathways. Many common clinical conditions, incIuding pressure overload cardiac hypertrophy, myocardial fibrosis, neointimal hyperplasia of vascular smooth muscle, anabolic bone remodeling, and prostate hypertrophy have been shown to involve these GPCR-mediated signals. Prior study of the mechanisms that GPCRs use to control the activity of tyrosine protein kinases and regulate the ERK1/2 MAP kinase cascade has revealed that these responses are often the result of novel signaling events, such as the formation of complexes between GPCRs, Src family tyrosine kinases, and beta-arrestin-bound MAP kinases, and cross talk between GPCRs and classical receptor tyrosine kinases. Emerging data suggest that different signaling mechanisms lead to the formation of spatially separate and functionally distinct MAP kinase pools. This application has two broad goals. The first is to characterize in detail the molecular mechanisms underlying GPCR-mediated ERK activation via beta-arrestin scaffolds and transactivated epidermal growth factor (EGF) receptors. The second goal is understand how these novel signals are integrated to determine the cellular response to GPCR activation. The first specific aim of the proposal is to determine, using wild type and mutant GPCRs in cellular models systems, the mechanism of ERK activation on beta-arrestin scaffolds, including the role of heterotrimeric G protein subunits. G protein effectors, Src kinases, and Ras GTPases. The second specific aim is to determine, using a novel mixed cell assay system consisting of ligand donor and ligand accepter cell populations, as well as direct assays of GPCR-stimulated heparin-binding EGF release, the mechanisms used by GPCRs to control matrix metalloprotease-dependent release of EGF receptor ligands, and to characterize mechanisms of metalloprotease-independent EGF receptor transactivation. The third aim is to test the hypothesis that the consequences of GPCR-stimulated ERK kinase activation are determined by the mechanism of activation. These experiments will employ endogenously-expressed GPCRs in beta-arrestin knockout fibroblasts, into which beta-arrestin expression has or has not been stably restored, to determine the role of beta-arrestin scaffolds and EGF receptor transactivation in controlling the phosphorylation of specific cytosole, membrane and nuclear ERK substrates, and the transcriptional response to GPCR stimulation.