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

描述（由申请人提供）：众所周知，七种跨膜受体（7 TMR）家族，也称为G蛋白偶联受体（GPCR），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体生理学。目前全球市场上大约50%的药物靶向7 TMR，包括β 2-肾上腺素能受体（β 2AR），一种具有重要心血管意义的受体，使GPCR成为最重要的一类药物靶点。经典的GPCR信号转导伴随着G蛋白的激活，随后是GPCR激酶介导的受体磷酸化和β-抑制蛋白（β-arrestin，β-arrestin）介导的受体脱敏和信号终止。此外，在过去的十年中，β arrs作为网格蛋白和AP 2的内吞衔接子，以及作为包括MAPK和AKT途径的组分在内的多种信号蛋白的分子支架，已经获得了重要性。受体活化后引起的β-受体的这种多效性功能，因此提出了β-受体与GPCR的偶联是高度调节和动态的（即GPCR-β-受体复合物显示出一定的构象可塑性）的前提。我们最近的发现证明了“β arr 1-偶联β 2AR”复合物存在两种不同的构象，这一假设变得更加明显;（一）“尾巴”（或悬挂）构象，其中β arr 1主要与受体的磷酸化C-末端尾部偶联，和（ii）"核心（或紧密）构象“，其中C-末端尾部偶联的β arr 1进一步与受体跨膜核心接合。这些独特的β 2AR-β arr 1构象异构体亚群表明在β 2AR-β arr 1复合物群体中存在功能选择性或“偏向激动作用”，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β arrs的多功能（例如，受体脱敏与信号传导支架），它们的结构基础和功能相关性仍有待确定。因此，本研究的总体目标有三个方面：（1）开发相对高通量的体外形成β 2 AR-β arr 1复合物的方法;（2）通过系统的β arr 1诱变和电子显微镜对复合物的超微结构分析来发现β 2 AR-β arr 1构象的结构决定因素;和（3）通过各种成熟的细胞和药理学研究来描述β 2AR-β arr 1构象状态的功能相关性，包括：（i）共聚焦显微镜;（ii）高亲和力配体结合;（iii）基于荧光素酶的β arr 1募集和内化测定;和（iv）第二信使cAMP测定和ERK 1/2信号传导。该项目的成功完成将描述不同的β 2AR-β arr 1构象的功能相关性（即受体的“核心”与β β arr 1的“尾部”偶联状态），从而为开发基于结构的药物设计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.