Tyrosine kinases in G protein mediated signaling.

描述(申请人提供)：在过去的十年中，很明显，许多G蛋白偶联受体(GPCRs)传递影响细胞分化和生长的信号，包括刺激RAS家族GTP酶和激活丝裂原激活蛋白(MAP)激酶通路。许多常见的临床情况，包括压力超负荷性心肌肥大、心肌纤维化、血管平滑肌新生内膜增生、同化性骨重塑和前列腺肥大，都被证明与这些GPCR介导的信号有关。先前对GPCRs用于控制酪氨酸蛋白激酶活性和调节ERK1/2 MAP激酶级联反应的机制的研究表明，这些反应通常是新的信号事件的结果，例如GPCRs、Src家族酪氨酸激酶和β-arrestin结合的MAP激酶之间形成复合体，以及GPCRs和经典受体酪氨酸激酶之间的串扰。新出现的数据表明，不同的信号机制导致形成空间上分离的和功能上不同的MAP激酶池。这个应用程序有两个主要目标。第一个是详细研究GPCR通过β-arrestin支架和反式激活的表皮生长因子(EGF)受体激活ERK的分子机制。第二个目标是了解这些新的信号是如何整合的，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GPCRs来确定ERK在β-arrestin支架上的激活机制，包括异源三聚体G蛋白亚基的作用。G蛋白效应物、Src激酶和Ras GTP酶。第二个具体目的是利用一种新的由配体供体和配体受体细胞群体组成的混合细胞检测系统，以及直接测定GPCRs刺激的肝素结合的EGF释放，来确定GPCRs控制基质金属蛋白酶依赖的EGF受体配体释放的机制，并表征金属蛋白酶非依赖的EGF受体反式激活的机制。第三个目的是验证这样一种假设，即GPCR刺激的ERK激酶激活的后果由激活机制决定。这些实验将利用在β-arrestin基因敲除的成纤维细胞中内源性表达的GPCRs，其中β-arrestin表达已经或尚未稳定恢复，以确定β-arrestin支架和EGF受体反式激活在控制特定的胞浆、膜和核ERK底物的磷酸化以及对GPCR刺激的转录反应中的作用。

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.