Regulation of CXCR4 Signaling

描述（由申请人提供）：趋化因子受体CXCR4是一种重要的GPCR，涉及许多人类疾病，包括HIV， WHIM综合征和癌症。CXCR4功能调控的详细机制及其在癌症中的作用目前尚不清楚。GPCR的激动剂依赖性信号主要受GPCR激酶（GRKs）和抑制因子的调控，激活GPCR的GRK磷酸化通常是调控过程的第一步。CXCR4在激动剂激活后迅速磷酸化，尽管磷酸化的具体位点、涉及的激酶和功能作用尚未明确。在这个应用中，我们建议使用分子、生化和细胞策略来更好地表征正常细胞和癌细胞中调节CXCR4表达和功能的机制。我们的初步研究表明，在HEK293细胞中，GRKS可能在激动剂特异性磷酸化CXCR4中起主要作用。此外，当GRKS、GRK6、arrestin-2或arrestin-3被敲低时，可以观察到Ca2+动员增强，而ERK1/2的激活因GRK2的缺失而增强，但因GRKS、GRK6、arrestin-2或arrestin-3的缺失而降低。这些初步研究表明，grk和抑制可能对CXCR4信号的调控存在差异。我们将通过解决几个重要问题来验证位点特异性磷酸化CXCR4介导信号传导动力学和特异性的假设。CXCR4中哪些特异性残基在激动剂刺激下被磷酸化？哪些激酶介导位点特异性磷酸化？CXCR4磷酸化的功能作用是什么？位点特异性磷酸化的功能作用背后的机制是什么？这些机制在癌症中有功能失调吗？我们的研究将为了解CXCR4调控的机制提供重要的见解，并为控制CXCR4功能提供潜在的治疗方法。

DESCRIPTION (provided by applicant): The chemokine receptor CXCR4 is an essential GPCR that has been implicated in a number of human diseases including HIV, WHIM syndrome, and cancer. The detailed mechanisms involved in the regulation of CXCR4 function and its role in cancer are currently poorly understood. Agonist- dependent signaling of GPCRs is principally regulated by GPCR kinases (GRKs) and arrestins with GRK phosphorylation of an activated GPCR often being the initial step in the regulatory process. CXCR4 is rapidly phosphorylated following agonist activation, although the specific sites of phosphorylation, the kinases involved, and the functional roles are not well defined. In this application, we propose to use molecular, biochemical, and cellular strategies to better characterize the mechanisms that regulate CXCR4 expression and function in normal and cancer cells. Our initial studies suggest that GRKS may play the major role in agonist-specific phosphorylation of CXCR4 in HEK293 cells. In addition, enhanced Ca2+ mobilization is observed when GRKS, GRK6, arrestin-2, or arrestin-3 are knocked down while ERK1/2 activation is enhanced by the loss of GRK2 but decreased by the loss of GRKS, GRK6, arrestin-2, or arrestin-3. These initial studies suggest that GRKs and arrestins may differentially regulate CXCR4 signaling. We will test the hypothesis that site-specific phosphorylation of CXCR4 mediates the kinetics and specificity of signaling by addressing several important questions. What specific residues in CXCR4 are phosphorylated in response to agonist stimulation and what kinases mediate site-specific phosphorylation? What are the functional roles of CXCR4 phosphorylation and what mechanisms underlie the functional effects of site-specific phosphorylation? Are any of these mechanisms dysfunctional in cancer? Our studies should provide important insight into the mechanisms involved in CXCR4 regulation as well as provide potential therapeutic approaches for controlling CXCR4 function.