ER-to-cell surface transport and signal regulation of GPCRs

描述(由申请人提供)：G蛋白偶联受体(GPCRs)细胞内运输和信号的时空调节是细胞对激素综合反应的关键方面。事实上，GPCRs的运输缺陷和功能障碍与许多人类疾病的发病机制有关。我们的总体目标是确定GPCRs成熟和信号传播的分子机制，以及它们在调节细胞对激素和药物的反应中的作用。在这一大的目标下，本研究的重点是以α2B-肾上腺素能受体(α2B-AR)为模型，研究神经母细胞瘤-神经胶质瘤NG108和人胚胎肾HEK293细胞系中新生GPCRs从内质网(ER)输出到细胞表面的机制，以及GPCRs介导的丝裂原活化蛋白激酶通路的激活。我们已经证明了内质网的α2B-AR输出受到一个高度保守的三重精氨酸(3R)基序的调节。3R基序介导受体与选择性SEC24亚型的相互作用，SEC24亚型是COPII包被的运输囊泡的组成部分。我们的研究还揭示了GGAS(高尔基体定位，3-Adaptin EAR结构域同源，ADP核糖化因子(ARF)结合蛋白)的新功能。GAs与α2B-AR结合，是α2B-AR从反式高尔基体网络(TGN)向质膜转运所必需的。此外，我们还发现了一条新的信号通路，在该通路中，α2B-AR与小GTPase ARF1之间的双色氨酸基序介导的激动剂依赖的相互作用决定了受体激活传统的Raf1-MEK-ERK1/2级联反应。其具体目的是：1)阐明COPII囊泡介导的α2B-AR从内质网输出的机制；2)确定GAs在α2B-AR从TGN到细胞表面转运中的作用；3)确定α2B-AR和ARF1介导的激活Raf1-MEK-ERK1/2通路的功能和机制。总体而言，这些研究将揭示出口贩运和GPCRs信号调控的新分子机制。这些研究产生的信息可能为设计治疗涉及GPCRs异常贩运和功能的疾病的药物开辟新的方向。 公共卫生相关性：该提案将研究G蛋白偶联受体的细胞内转运和功能调节。这些受体在生理和病理条件下调节各种细胞功能，是治疗许多疾病的药物的靶点。这些研究的成功完成将为设计治疗涉及G蛋白偶联受体异常运输和功能的人类疾病的药物开辟新的方向。

DESCRIPTION (provided by applicant): The spatiotemporal regulation of intracellular trafficking and signaling of G protein-coupled receptors (GPCRs) is a critical aspect of integrated responses of the cell to hormones. Indeed, defective transport and dysfunction of GPCRs are associated with the pathogenesis of many human diseases. Our overall objective is to define the molecular mechanisms underlying the maturation and signal propagation of GPCRs and their roles in modulating cellular responses to hormones and drugs. Under this broad objective, the focus of the current proposal is to elucidate the mechanisms of nascent GPCR export from the endoplasmic reticulum (ER) to the cell surface and GPCR-mediated activation of the mitogen-activated protein kinase pathway in neuroblastoma-glioma NG108 and human embryonic kidney HEK293 cell lines by using alpha2B-adrenergic receptor (alpha2B-AR) as a model GPCR. We have demonstrated that alpha2B-AR export from the ER is modulated by a highly conserved triple arginine (3R) motif. The 3R motif mediates receptor interaction with selective Sec24 isoforms, components of COPII-coated transport vesicles. Our studies have also revealed a novel function for GGAs [monomeric Golgi-localizing, 3-adaptin ear domain homology, ADP ribosylation factor (ARF)-binding proteins]. GGAs associate with alpha2B-AR and are required for alpha2B-AR transport from the trans-Golgi network (TGN) to the plasma membrane. Furthermore, we have identified a novel signaling pathway in which the di-tryptophan motif-mediated, agonist-dependent interaction of alpha2B-AR with the small GTPase ARF1 dictates the activation of the conventional Raf1-MEK-ERK1/2 cascade by the receptor. The Specific Aims are: 1) to elucidate the mechanism of COPII vesicle-mediated alpha2B-AR export from the ER, 2) to determine the function of GGAs in alpha2B-AR transport from the TGN to the cell surface, and 3) to define the function and mechanism of alpha2B-AR- and ARF1-mediated activation of the Raf1-MEK-ERK1/2 pathway. Overall, these studies will reveal novel molecular mechanisms underlying export trafficking and signal regulation of GPCRs. The information generated from these studies may open new directions for designing drugs to treat diseases involving abnormal trafficking and functioning of GPCRs. PUBLIC HEALTH RELEVANCE: This proposal will study the intracellular trafficking and functional regulation of G protein-coupled receptors. These receptors regulate a variety of cell functions under physiological and pathological conditions and are the targets for drugs to treat many diseases. The successful completion of these studies will open a new direction for designing drugs to treat human diseases involving abnormal trafficking and functioning of G protein-coupled receptors.