Functional consequences of different conformational states of beta-2-adrenergic receptor-beta-arrestin complexes

描述(申请人提供)：七个跨膜受体家族(7TMR)，也被称为G蛋白偶联受体(GPCRs)，众所周知，通过影响从心血管到神经内分泌到感觉的几乎所有生物系统的功能来调节人类生理。目前全球上市的药物中约有50%以7TMR为靶点，其中包括具有重要心血管意义的受体--2肾上腺素能受体，这使得GPCRs成为最重要的药物靶点之一。经典的GPCR信号转导伴随着G蛋白的激活，继而是GPCRK介导的受体磷酸化，以及B arrestin(？arr)介导的受体脱敏和信号终止。此外，在过去的十年中，ARRs作为胞内连接蛋白和AP2的受体，以及各种信号蛋白的分子支架，包括MAPK和AKT通路的组成部分，都具有重要的意义。？arRs的这种多效性功能是在受体激活后激发的，从而提出了？arr与GPCRs的偶联是高度调控和动态的前提(即gpcr？？arr复合体表现出一定的构象可塑性)。这一假设随着我们最近的发现证明存在两种不同的构象：(I)“尾(或悬挂)构象”，其中？arr1主要偶联到受体的磷酸化的C-末端尾部；(Ii)“核心(或紧密)构象”，其中C-末端尾部偶联的？arr1进一步与受体跨膜核心结合。这些独特的？2AR？？arr1构象亚群表明，在？2AR？？arr1复合体中存在功能选择性或“偏向激动症”，这揭示了开发临床相关偏向疗法的潜在途径。虽然这种构象差异强调了çarRs的多种功能(例如，受体脱敏与信号支架)，但它们的结构基础和功能相关性仍有待确定。因此，这项研究建议的总体目标有三个方面：(1)开发相对高通量的方法，在体外形成？2AR？？arr1复合体；(2)通过系统的？arr1诱变和电子显微镜对复合体的超微结构分析，发现？2ar？？arr1构象的结构决定因素；以及(3)通过各种成熟的细胞和药理学研究，描述？2ar？？arr1构象状态的功能相关性；(Ii)高亲和力配体结合；(Iii)基于报告的？arr1募集和内化分析；ND(Iv)第二信使cAMP测定和ERK1/2信号转导。该项目的成功完成将勾勒出不同的？2AR-？arr1构象的功能相关性(即？arr1的受体‘核心’与‘尾部’耦合状态)，从而为基于结构的药物设计的开发提供强有力的新基础。

DESCRIPTION (provided by applicant): The family of seven transmembrane receptors (7TMRs), alternately referred to as G protein-coupled receptors (GPCRs), are well known to regulate human physiology by impacting the function of virtually every biological system from cardiovascular to neuro-endocrine to sensory. Approximately, 50% of current pharmaceutical drugs marketed worldwide target 7TMRs, including the ß2-adrenergic receptor (ß2AR), a receptor of great cardiovascular significance, making GPCRs one of the most important classes of drug targets. Classical GPCR signaling ensues with the activation of G-proteins, followed by GPCR-kinase mediated receptor phosphorylation and ß-arrestin (ßarr) mediated receptor desensitization and signal termination. Additionally, in the past decade, ßarrs have gained significance as endocytic adaptors for clathrin and AP2, and as molecular scaffolds for a variety of signaling proteins including the components of MAPK and AKT pathways. Such pleiotropic function of ßarrs, elicited following receptor activation, thus puts forward the premise that ßarr coupling to GPCRs is highly regulated and dynamic (i.e. GPCR-ßarr complexes display a certain conformational plasticity). This presumption has become apparent with our recent findings demonstrating the existence of two distinct conformations of the 'ßarr1-coupled ß2AR' complex; identified as (i) the 'tail (or hanging) conformation', where ßarr1 is primarily coupledto the phosphorylated C-terminal tail of the receptor, and (ii) the 'core (or tight) conformation', where the C-terminal tail-coupled ßarr1 is further engaged with the receptor transmembrane core. These unique subsets of ß2AR-ßarr1 conformers suggest the occurrence of functional selectivity or 'biased agonism' within the population of ß2AR-ßarr1 complexes, which unveils potential avenues for developing clinically relevant biased therapeutics. While such conformational distinctions underscore the versatile functions of ßarrs (e.g., receptor desensitization vs. signaling scaffold), their structural bases and functional relevance remain to be determined. Accordingly, the overall goal of this research proposal is three-fold: (1) to develop relatively high-throughput methods for forming ß2AR-ßarr1 complexes in vitro; (2) to discover the structural determinants of ß2AR-ßarr1 conformations by systematic ßarr1 mutagenesis and ultrastructural analyses of complexes by electron microscopy; and (3) to delineate the functional relevance of ß2AR-ßarr1 conformational states through a variety of well-established cellular and pharmacological studies including: (i) confocal microscopy; (ii) high-affinity ligand binding; (iii) reporter-based ßarr1 recruitment and internalization assays; nd (iv) second messenger cAMP assays and ERK1/2 signaling. Successful completion of this project will delineate the functional relevance of distinct ß2AR-ßarr1 conformations (i.e. receptors' 'core' vs. 'tail' coupled states of ßarr1), and thus provide a powerful novel basis fo development of structure-based drug design.