ER-to-Golgi traffic and signal regulation of GPCRs

描述(申请人提供)：G蛋白偶联受体(GPCRs)构成细胞表面受体的超家族，调节多种细胞功能。GPCRs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最充分的例子之一。然而，GPCRs出口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的反应中的功能作用。在这一大目标下，这项建议侧重于两个重要问题：1)GPCRs如何从ER出口？2)GPCRs如何从ER运输到高尔基？我们的初步数据表明，由一个苯丙氨酸(F)和一个双亮氨酸(L)组成的基序被六个残基{F(X)6Ll}隔开，是从内质网输出α2B肾上腺素能(AR)和血管紧张素Ⅱ1型(AT1R)受体所必需的。这一主题在许多GPCR中高度保守，因此可能为它们的出口提供一个共同机制。我们的数据还表明，在神经母细胞瘤-神经胶质瘤NG108和人胚胎肾HEK293细胞系中，不同的GPCR可能使用不同的途径移动到细胞表面。非糖基化的α2B-AR使用一种新的、尚未确定的途径。具体目的是：1)明确F(X)6LL基序在调节内质网gpr输出中的作用机制。我们将确定F(X)6LL基序是否是内质网GPCR输出的常见密码，确定F(X)6LL基序是否作为ER输出信号和/或调节受体折叠，并确定F(X)6LL基序的结构-功能关系。2)明确α2B-AR从内质网到高尔基体转运的新途径。我们将定义参与α2B-AR转运的细胞内隔室和运输小泡，并确定糖基化是否改变了α2B-AR转运途径。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通过靶向GPCR转运来治疗疾病的新治疗策略的可能性。

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.