Regulation of the Chemokine Receptor CXCR4 by Ubiquitin

描述(由申请人提供)：本提案的主要目标是阐明趋化因子受体CXCR4受调控的细胞和分子机制。CXCR4基因异常与乳腺癌、HIV、突发奇想综合征以及慢性缺血性心脏病、心绞痛和人类终末期心力衰竭等心血管疾病状态有关。了解CXCR4的调控机制可能为预防和治疗与CXCR4失调相关的病理提供新的策略。CXCR4在E3泛素连接酶AIP4的介导下，在质膜上经历激动剂依赖的泛素化，AIP4作为内体分选信号，靶向于溶酶体中的受体降解。然而，对AIP4识别和泛素化激活的CXCR4及其在CXCR4信号中的作用的细胞和分子机制缺乏深入的了解。我们提供的初步证据表明，AIP4与非视觉拦阻蛋白相互作用，并且磷酸化可能在靶向CXCR4至溶酶体方面发挥作用。我们假设，AIP4介导的CXCR4泛素化之前是磷酸化导致arrestin招募，这是一个适配器，反过来招募AIP4到CXCR4。我们还提供了证据表明，CXCR4上的泛素部分不仅可以作为内体分选信号，还可以作为质膜信号的快速和即时终止子。我们提出了一系列全面的研究，旨在通过GST下拉试验、免疫共沉淀研究、siRNA分析和免疫荧光显微镜实验来研究拦阻蛋白与AIP4在细胞和体外的相互作用。我们的目标是确定决定AIP4如何识别和泛素化激活的CXCR4以调节其信号转导的细胞和分子机制。具体目标如下：1.确定泛素连接酶AIP4如何识别和泛素化激活的CXCR4。2.确定磷酸化在介导AIP4依赖的泛素化和CXCR4降解中的作用。3.确定AIP4依赖的CXCR4泛素化在CXCR4介导的信号转导中的作用。

DESCRIPTION (provided by applicant): The primary goal of this proposal is to elucidate the cellular and molecular mechanisms by which the chemokine receptor CXCR4 is regulated. CXCR4 dysregulation has been associated with several pathologies including breast cancer, HIV, WHIM syndrome and cardiovascular disease sates such as chronic ischemic heart disease, angina and human end-stage heart failure. Understanding the mechanisms regulating CXCR4 may provide new strategies for the prevention and treatment of the pathologies associated with CXCR4 dysregulation. CXCR4 undergoes agonist-dependent ubiquitination at the plasma membrane mediated by the E3 ubiquitin ligase AIP4, which targets the receptor for degradation in lysosomes by serving as an endosomal sorting signal. However, insight into the cellular and molecular mechanisms by which AIP4 recognizes and ubiquitinates activated CXCR4 and its role on CXCR4 signaling is lacking. We provide initial evidence demonstrating that AIP4 interacts with non-visual arrestins and that phosphorylation may play a role in targeting CXCR4 to lysososmes. We hypothesize that AIP4-mediated ubiquitination of CXCR4 is preceded by phosphorylation leading to arrestin recruitment, which serves as an adaptor to in turn recruit AIP4 to CXCR4. We also provide evidence that suggests that the ubiquitin moiety on CXCR4 not only serves as an endosomal sorting signal, but may also serve as a rapid and immediate terminator of signaling at the plasma membrane. We propose a comprehensive series of studies aimed at examining the interaction between arrestins and AIP4 in cells and in vitro using GST pull-down assays, co- immunoprecipitation studies, siRNA analysis and immunofluorescence microscopy experiments. Our objective is to define the cellular and molecular mechanisms that determine how AIP4 recognizes and ubiquitinates activated CXCR4 to regulate its signaling. The following Specific Aims are proposed: 1. To define how the ubiquitin ligase AIP4 recognizes and ubiquitinates activated CXCR4. 2. To determine the role of phosphorylation in mediating AIP4-dependent ubiquitination and degradation of CXCR4. 3. To determine the role of AIP4-dependent CXCR4 ubiquitination on regulation of CXCR4 mediated signaling.