Mechanisms Regulating GPCR Trafficking

总结 这项提案的长期目标是了解G蛋白偶联受体（GPCR）的调控 通过内吞运输进行信号传导。GPCR包含在细胞中表达的最大的信号传导受体家族。 哺乳动物基因组，介导细胞对各种刺激的反应，并控制大量的生理反应。 GPCR信号转导失调与神经系统疾病、心血管疾病和糖尿病有关。 癌症进展，使这类受体成为近一半临床使用的药物的靶点。除了 在脱敏过程中，GPCR运输对于受体信号传导的时间和空间控制至关重要。这是 最好的例子是蛋白酶激活受体-1（PAR 1），一种凝血蛋白酶凝血酶的GPCR。PAR1 在血管生理、发育和肿瘤进展中具有重要作用，是一种重要的药物 目标与大多数GPCR类似，激活的PAR 1的信号传导迅速脱敏。我们还发现 活化的PAR 1内化和溶酶体分选对于凝血酶信号传导的保真度是关键的， 适当的细胞反应。我们进一步发现，激活的PAR 1运输在细胞中失调， 转移性乳腺癌，因此受体粘附到细胞表面，持续地发出信号， 促进肿瘤进展。PAR 1运输调节异常的机制尚不清楚 重要的是要理解。许多GPCR被泛素修饰，并被分选到溶酶体中， 与ESCRT机制的泛素结合组分的相互作用。然而，并非所有GPCR都需要 包括PAR 1的溶酶体分选的直接泛素化。我们最近发现了一种新的溶酶体分类 途径，绕过受体泛素化和泛素结合ESCRT的要求， 由阿利克斯提供。阿利克斯是一种CHMP 4/ESCRT-III相互作用蛋白，通过其V 结构域和介导的PAR 1溶酶体分选。我们还鉴定了A类GPCR的子集， YPXnL基序，表明该途径可能适用于其他GPCR。我们的初步研究进一步 表明ALIX相互作用蛋白抑制蛋白结构域蛋白-3（ARRDC 3）调节PAR 1， 降解ARRDC 3似乎是一种肿瘤抑制因子，其表达在浸润性乳腺癌中丢失。 显示PAR 1运输失调的癌。此外，ARRDC 3的异位表达促进了 浸润性乳腺癌中PAR 1的溶酶体分选。拟议的研究将促进我们的理解 ARRDC 3和阿利克斯如何在正常人和正常人中调节GPCR细胞内运输和信号传导， 癌细胞该提案的具体目标是：1）确定ARRDC 3是否调节GPCR分选 晚期核内体/多泡体，2）描绘溶酶体中阿利克斯功能的调节机制 GPCR的分类，和3）检查ARRDC 3在癌症中失调的GPCR运输中的作用。

Summary The long-term goal of this proposal is to understand the regulation of G protein-coupled receptor (GPCR) signaling by endocytic trafficking. GPCRs comprise the largest family of signaling receptors expressed in the mammalian genome, mediate cellular responses to diverse stimuli and control vast physiological responses. Dysregulated GPCR signaling has been implicated in neurological disorders, cardiovascular diseases and cancer progression, making this receptor class the target of nearly half the drugs used clinically. In addition to desensitization, GPCR trafficking is crucial for the temporal and spatial control of receptor signaling. This is best exemplified by protease-activated receptor-1 (PAR1), a GPCR for the coagulant protease thrombin. PAR1 has important functions in vascular physiology, development and tumor progression and is an important drug target. Similar to most GPCRs, signaling by activated PAR1 is rapidly desensitized. We also found that activated PAR1 internalization and lysosomal sorting is critical for the fidelity of thrombin signaling and appropriate cellular responses. We further discovered that activated PAR1 trafficking is dysregulated in metastatic breast carcinoma, consequently the receptor recycles to the cell surface, signals persistently and promotes tumor progression. The mechanisms responsible for dysregulation of PAR1 trafficking are not known and important to understand. Many GPCRs are modified with ubiquitin and sorted to lysosomes through interactions with ubiquitin-binding components of the ESCRT machinery. However, not all GPCRs require direct ubiquitination for lysosomal sorting including PAR1. We recently discovered a novel lysosomal sorting pathway that bypasses the requirement for receptor ubiquitination and ubiquitin-binding ESCRTs that is mediated by ALIX. ALIX, a CHMP4/ESCRT-III interacting protein, bound to an YPX3L motif of PAR1 via its V domain and mediated PAR1 lysosomal sorting. We also identified a subset of class A GPCRs containing YPXnL motifs, suggesting that this pathway may be applicable to other GPCRs. Our preliminary studies further indicate that the ALIX-interacting protein arrestin-domain containing protein -3 (ARRDC3) regulates PAR1 degradation. ARRDC3 appears to function as a tumor suppressor and its expression is lost in invasive breast carcinoma that exhibit dysregulated PAR1 trafficking. Moreover, ectopic expression of ARRDC3 promoted lysosomal sorting of PAR1 in invasive breast carcinoma. The proposed studies will advance our understanding of how ARRDC3 and ALIX function to regulate GPCR intracellular trafficking and signaling in normal and cancer cells. The specific aims of the proposal are to: 1) determine whether ARRDC3 regulates GPCR sorting to late endosomes/multivesicular bodies, 2) delineate the regulatory mechanisms of ALIX function in lysosomal sorting of GPCRs, and 3) examine the role of ARRDC3 in dysregulated GPCR trafficking in cancer.