Role of beta-arrestins in chemokine receptor signaling

摘要 异源三聚体G蛋白偶联受体（GPCR）C-X-C基序受体4（CXCR 4）及其同源配体 CXCL 12在健康和疾病中发挥重要作用。大量的证据表明，CXCR 4信号传导 与癌症进展有关癌症中CXCR 4的表达和信号传导与肿瘤生长不良相关2 -5， 主要是因为表达CXCR 4的癌细胞定殖于CXCL 12所在的远处解剖部位， 导致转移性疾病，这是大多数癌症相关死亡的原因。CXCR 4信号调节几种 与癌症进展有关的细胞生理学方面。这包括定向细胞迁移和细胞存活， 其通过几个离散的信号传导途径发生。然而，人们对这些机制仍然知之甚少。重点 这一建议的一个重要方面是调节CXCR 4介导的趋化性的信号转导机制， CXCL 12.我们最近报道了CXCR 4介导的趋化性通过一种新的机制发生， 内吞衔接蛋白b-抑制蛋白1（barr 1）和STAM 1（barr 1：STAM 1）之间形成的复合物。的 barr 1：STAM 1复合物不作用于Akt或ERK-1/2信号通路，而是必需的， 激活粘着斑激酶（FAK），这也是CXCL 12驱动的趋化性所必需的。FAK是 通常与整合素信号传导和粘着斑动力学有关，但FAK功能的这些方面不是 由barr 1：STAM 1复合物调控。尽管我们的贡献如何bar 1：STAM 1激活FAK下游 CXCR 4促进趋化性的机制仍然知之甚少。本提案的总体目标是填补 知识差距。根据我们发表的和初步的研究，我们假设G蛋白依赖性 barr 1：STAM 1信号传导在空间和时间上控制CXCR 4依赖性免疫应答所需的FAK活性。 趋化性为了检验这个假设，我们将追求以下具体目标：目标1。阐明的作用 CXCR 4位点特异性磷酸化对FAK活化的影响; Aim 2.为了确定结构和生物物理 barr 1与STAM 1相互作用的性质;目的3。为了阐明barr 1的功能作用：STAM 1 趋化性复杂。由于我们的提案的机械焦点，我们将使用细胞培养模型， 其他体外方法跨越细胞和分子生物学、遗传学、生物化学和生物化学领域的技术， 生物物理学加上先进的活细胞成像策略和质谱分析方法。结束时 在这个项目中，我们将了解到barr 1：STAM 1合作的新的信号转导机制， 激活FAK以促进趋化性。这是重要的，因为它将揭示CXCR 4的新方面 可以作为治疗靶点的信号。

ABSTRACT The heterotrimeric G protein-coupled receptor (GPCR) C-X-C motif receptor 4 (CXCR4) and its cognate ligand CXCL12 play important roles in health and disease. A large body of evidence indicates that CXCR4 signaling is linked to cancer progression. CXCR4 expression and signaling in cancer correlates with poor prognosis2-5, mainly because cancer cells expressing CXCR4 colonize distant anatomical sites where CXCL12 is located, resulting in metastatic disease, the cause of most cancer related deaths. CXCR4 signaling regulates several aspects of cell physiology linked to cancer progression. This includes directed cell migration and cell survival, which occur via several discrete signaling pathways. Yet the mechanisms remain poorly understood. The focus of this proposal is on the signal transduction mechanisms that regulate CXCR4-mediated chemotaxis towards CXCL12. We recently reported that CXCR4-mediated chemotaxis occurs via a novel mechanism involving a complex formed between endocytic adaptor proteins b-arrestin1 (barr1) and STAM1 (barr1:STAM1). The barr1:STAM1 complex does not act on Akt or ERK-1/2 signaling pathways, but instead is necessary for activating focal adhesion kinase (FAK), which is also necessary for CXCL12 driven chemotaxis. FAK is typically linked to integrin signaling and focal adhesion dynamics, but these aspects of FAK function are not regulated by the barr1:STAM1 complex. Despite our contribution how barr1:STAM1 activates FAK downstream of CXCR4 to promote chemotaxis remains poorly understood. The overall objective of this proposal is to fill in knowledge gaps. Based on our published and preliminary studies we hypothesize that G protein-dependent barr1:STAM1 signaling spatially and temporally controls FAK activity required for CXCR4-dependent chemotaxis. To test this hypothesis we will pursue the following specific aims: Aim 1. To elucidate the role of CXCR4 site-specific phosphorylation on FAK activation; Aim 2. To identify the structural and biophysical properties of the barr1 interaction with STAM1; Aim 3. To elucidate the functional role of the barr1:STAM1 complex in chemotaxis. Because of the mechanistic focus of our proposal we will use cell culture models and other in vitro approaches spanning techniques in cell and molecular biology, genetics, biochemistry and biophysics plus advanced live cell imaging strategies and mass spectrometry approaches. At the conclusion of this project we will have learned novel signal transduction mechanisms by which barr1:STAM1 collaborate to activate FAK to promote chemotaxis. This is significant because it will reveal novel aspects of CXCR4 signaling that could be targeted therapeutically.