Regulation of EGF Receptors by G protein receptor

描述(由申请人提供)：本申请的目的是 探索G蛋白偶联受体(GPCR)之间的串扰 受体酪氨酸激酶(RTK)，尤其是表皮生长因子 受体(EGFR)。我们，像其他人一样，已经观察到 GPCRs导致转磷酸化和激活EGFR。我们还 做出了这一新发现，GPCRs的先前激活导致了一种深刻的 EGFR的脱敏。这项应用的目的是获得 对脱敏过程的机械性见解。 在具体目标1中，我们将确定需要哪条信号通路(S 用于GPCR对EGF受体的脱敏和转磷酸化。我们 将主要使用原代培养的细胞类型具有内源性gpr和 这些研究将通过明智地使用转基因细胞而得到加强。 在这个目标中要回答的主要问题是：什么信号分子联系 GPCR激活对EGFR的磷酸化和反式激活？它们是一样的吗 是那些导致EGFR脱敏的基因吗？我们将主要关注角色 蛋白激酶C、非受体酪氨酸激酶Src和钠质子 I型交换机(NHE-I)。GPCR是否需要EGFR的内吞作用 表皮生长因子受体脱敏？是否需要肝素结合EGF(HB-EGF) EGFR的脱敏或反式激活？为了回答这个问题 问题，我们将使用一些补充方法来中和 原代培养细胞中的天然HB-EGF。 在特定的目标2中，我们将确定EGFR的细胞内命运 刺激GPCRs。EGFR针对的是什么隔室 GPCR的S激活了？内部化了哪些其他信令组件 使用EGFR(GPCR、NRTK、NHE-1、HB-EGF)，它们是否通过 同样的道路？需要什么样的酶途径来下调 EGFR？我们将研究溶酶体、蛋白酶体和锌调节的作用。 金属蛋白酶在这个过程中的作用。这些研究解决了我们在 了解调节RTK功能的GPCR信号。

DESCRIPTION (provided by applicant): The purpose of this application is to explore the cross talk that occurs between G protein-coupled receptors (GPCR's) and receptor tyrosine kinases (RTK's), especially the epidermal growth factor receptor (EGFR). We, like others, have made the observation that activation of GPCR's leads to transphosphorylation and activation of EGFR's. We have also made the novel finding that prior activation of GPCR's leads to a profound desensitization of the EGFR's. The purpose of this application is to gain mechanistic insights into the desensitization process. In Specific Aim 1, we will determine which signaling pathway(s) is/are required for desensitization and transphosphorylation of the EGF receptor by GPCR's. We will mainly use primary cultures of cell types that have endogenous GPCR's and EGFR's. Those studies will be augmented by judicious use of transfected cells. Major questions to be answered in this aim are: What signaling molecules link GPCR activation to EGFR phosphorylation and transactivation? Are they the same as those that induce EGFR desensitization? We will focus primarily on the roles of protein kinase C, the non-receptor tyrosine kinase Src, and sodium proton exchanger type I (NHE-I). Is endocytosis of EGFR required for GPCR-mediated desensitization of EGFR? Is heparin-bound EGF (HB-EGF) required for either desensitization or transactivation of the EGFR? In order to answer this question, we will use a number of complementary methods to neutralize the native HB-EGF in primary cells in culture. In Specific Aim 2, we will determine the intracellular fate of the EGFR's after stimulation of GPCR's. To what compartments are the EGFR targeted after transactivation by GPCR' s? What other signaling components are internalized with the EGFR (GPCR, NRTK, NHE-1, HB-EGF), and are they processed through the same pathways? What enzymatic pathways are required for down-regulation of the EGFR? We will study the roles of lysosomes, proteasome, and zinc-regulated metalloproteinases in this process. These studies address important gaps in our knowledge of GPCR signals that regulate the functions of RTK's.