Role of beta-arrestins in chemokine receptor signaling

项目总结 异三聚体G蛋白偶联受体C-X-C基序受体4及其同源配体 CXCL12在健康和疾病中发挥着重要作用。大量证据表明，CXCR4信号 与癌症的进展有关。CXCR4在肿瘤中的表达和信号转导与不良预后相关2-5 主要是因为表达CXCR4的癌细胞在CXCL12所在的远处解剖部位定植， 导致转移性疾病，这是大多数癌症相关死亡的原因。CXCR4信号调节几个 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， 这通过几个离散的信号通路发生。然而，人们对这些机制仍然知之甚少。焦点 这一建议的重点是调节CXCR4介导的趋化作用的信号转导机制 CXCL12。我们最近报道，CXCR4介导的趋化作用是通过一种新的机制发生的，涉及一种 内吞接头蛋白b-arrestin1(Barr1)与STAM1(barr1：STAM1)形成复合体。这个 Barr1：STAM1复合体不作用于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CXCL12驱动趋化所必需的。FAK是 通常与整合素信号和焦点黏附动力学有关，但FAK功能的这些方面不是 受barr1：STAM1复合体调控。尽管我们做出了贡献，但barr1：STAM1如何激活FAK下游 CXCR4促进趋化作用的机制目前仍知之甚少。这项建议的总体目标是填补 知识鸿沟。基于我们已发表的和初步的研究，我们假设G蛋白依赖 Barr1：STAM1信号在空间和时间上控制CXCR4依赖所需的FAK活性 趋化性。为了验证这一假设，我们将追求以下具体目标：目标1.阐明 CXCR4位点特异性磷酸化对FAK的激活；目的2.鉴定结构和生物物理 Barr1与STAM1相互作用的性质；目的3.阐明barr1：STAM1的功能作用 趋化性复杂。由于我们提案的机械性重点，我们将使用细胞培养模型和 其他体外方法跨越细胞和分子生物学、遗传学、生物化学和 生物物理学加上先进的活细胞成像策略和质谱学方法。在……结束时 在这个项目中，我们将学习到新的信号转导机制，barr1：STAM1通过这些机制协作 激活FAK促进趋化作用。这一点意义重大，因为它将揭示CXCR4的新方面 暗示这可能是治疗上的靶向。

PROJECT SUMMARY 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.