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（gpcr）的细胞内运输和信号传导的时空调节是细胞对激素综合反应的一个关键方面。事实上，gpcr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gpcr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反应中的作用。在这一广泛的目标下，本研究的重点是通过使用α 2b -肾上腺素能受体（α 2b - ar）作为模型GPCR，阐明神经母细胞瘤-胶质瘤NG108和人胚胎肾HEK293细胞系中新生GPCR从内质网（ER）输出到细胞表面的机制，以及GPCR介导的丝裂原激活蛋白激酶途径的激活。我们已经证明，alpha2B-AR从内质网输出是由一个高度保守的三精氨酸（3R）基序调制的。3R基序介导受体与选择性Sec24亚型的相互作用，Sec24亚型是copii涂层运输囊泡的组成部分。我们的研究还揭示了GGAs的新功能[单体高尔基定位，3-适应蛋白耳结构域同源性，ADP核糖基化因子（ARF）结合蛋白]。GGAs与α 2b - ar结合，是α 2b - ar从反式高尔基网络（TGN）转运到质膜所必需的。此外，我们已经确定了一种新的信号通路，其中二色氨酸基序介导的，激动剂依赖的alpha2B-AR与小GTPase ARF1的相互作用决定了受体激活传统的Raf1-MEK-ERK1/2级联。具体目的是：1)阐明COPII囊泡介导的α 2b - ar从内质网输出的机制；2)确定GGAs在α 2b - ar从TGN转运到细胞表面中的功能；3)明确α 2b - ar -和arf1介导的激活Raf1-MEK-ERK1/2通路的功能和机制。总的来说，这些研究将揭示gpcr出口贩运和信号调节的新分子机制。从这些研究中产生的信息可能为设计药物治疗涉及gpcr异常运输和功能的疾病开辟新的方向。

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.