ER-to-Golgi traffic and signal regulation of GPCRs

描述（由申请人提供）：G蛋白偶联受体（gpcr）构成细胞表面受体的超家族，调节多种细胞功能。gpcr从内质网（ER）通过高尔基体到细胞表面的出口运输缺陷与多种人类疾病的发病机制有关，肾源性尿崩症是研究得最好的例子之一。然而，gpcr输出的分子机制仍然知之甚少。我们的总体目标是确定GPCR出口贩运的分子机制及其在调节细胞对激素和药物反应中的功能作用。在这一广泛目标下，本提案侧重于两个重要问题：1)gpcr如何从ER输出？gpcr如何从ER转运到高尔基体？我们的初步数据表明，由间隔6个残基{F(x)6LL}的苯丙氨酸(F)和双亮氨酸(L)组成的基序对于从内质网输出alpha2b -肾上腺素能（AR）和血管紧张素1型（AT1R）受体是必需的。这个基序在许多gpcr中高度保守，因此可能为它们的输出提供了一个共同的机制。我们的数据还表明，在神经母细胞瘤-胶质瘤NG108和人胚胎肾HEK293细胞系中，不同的gpcr可能通过不同的途径移动到细胞表面。非糖基化的alpha2B-AR使用一种新的，尚未定义的途径。具体目的是：1)确定F(x)6LL基序介导GPCR从ER输出的机制。我们将确定F(x)6LL基序是否是GPCR从ER输出的共同代码，确定F(x)6LL基序是否作为ER输出信号和/或调节受体折叠，并定义F(x)6LL基序的结构-功能关系。2)确定α 2b - ar从内质网转运到高尔基体的新途径。我们将定义参与alpha2B-AR运输的细胞内腔室和运输囊泡，并确定糖基化是否改变了alpha2B-AR运输途径。这些研究将为GPCR输出的分子机制及其对受体功能的控制提供新的重要信息。这些信息可能有助于开发新的治疗策略，通过靶向GPCR转运来治疗疾病。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) constitute a super-family of cell surface receptors that regulate a variety of cell functions. Defective export trafficking of GPCRs from the endoplasmic reticulum (ER) through the Golgi to the cell surface is associated with the pathogenesis of a variety of human diseases, nephrogenic diabetes insipidus being one of the best-studied examples. However, the molecular mechanism underlying the export of GPCRs remains poorly understood. Our overall objective is to define the molecular mechanism of GPCR export trafficking and its functional role in regulating cellular responses to hormones and drugs. Under this broad objective, this proposal focuses on two important questions: 1) how GPCRs export from the ER? 2) How GPCRs transport from the ER to the Golgi? Our preliminary data have demonstrated that the motif consisting of a phenylalanine (F) and double leucine (L) spaced by six residues {F(x)6LL} is required for export of the alpha2B-adrenergic (AR) and angiotensin ll-type 1 (AT1R) receptors from the ER. This motif is highly conserved in many GPCRs and thus may provide a common mechanism for their export. Our data also indicate that different GPCRs may use different routes to move to the cell surface in neuroblastoma- glioma NG108 and human embryonic kidney HEK293 cell lines. Non-glycosylated alpha2B-AR uses a novel, as yet undefined pathway. The Specific Aims are: 1) To define the mechanism of the F(x)6LL motif in mediating GPCR export from the ER. We will determine if the F(x)6LL motif is a common code for GPCR export from the ER, determine if the F(x)6LL motif functions as an ER export signal and/or regulates receptor folding, and define the structure-function relationship of the F(x)6LL motif. 2) To define the novel pathway for alpha2B-AR transport from the ER to the Golgi. We will define intracellular compartments and transport vesicles involved in alpha2B-AR transport and determine if glycosylation alters alpha2B-AR transport pathway. These studies will provide new and important information regarding the molecular mechanisms underlying GPCR export and its control on receptor function. Such information may help exploit the possibility of developing new therapeutic strategies for treating disease by targeting GPCR transport.