Regulation of CXCR4 Signaling

描述（由申请人提供）：趋化因子受体CXCR 4是一种必需的GPCR，与许多人类疾病（包括HIV、WHIM综合征和癌症）有关。目前对CXCR 4功能调节的详细机制及其在癌症中的作用知之甚少。GPCR的激动剂依赖性信号传导主要由GPCR激酶（GRKs）和抑制蛋白调节，活化的GPCR的GRK磷酸化通常是调节过程的初始步骤。CXCR 4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还没有很好地定义。在本申请中，我们建议使用分子，生物化学和细胞策略，以更好地表征在正常和癌细胞中调节CXCR 4表达和功能的机制。我们的初步研究表明，GRKS可能在激动剂特异性磷酸化HEK 293细胞CXCR 4中发挥主要作用。此外，当GRKS、GRK 6、arrestin-2或arrestin-3被敲低时，观察到增强的Ca 2+动员，而ERK 1/2激活通过GRK 2的损失而增强，但通过GRKS、GRK 6、arrestin-2或arrestin-3的损失而降低。这些初步研究表明，GRKs和抑制蛋白可能差异调节CXCR 4信号。我们将通过解决几个重要问题来检验CXCR 4位点特异性磷酸化介导信号传导动力学和特异性的假设。CXCR 4中哪些特定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？CXCR 4磷酸化的功能作用是什么？位点特异性磷酸化的功能作用机制是什么？这些机制在癌症中是否有功能障碍？我们的研究将为深入了解CXCR 4调控机制提供重要线索，并为控制CXCR 4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.