Beta2-adrenergic receptor mutagenesis for studies of biased agonism

描述（由申请人提供）：7 -跨膜受体（7TMRs），也被称为G蛋白偶联受体（gpcr），在心血管功能调节中起主要作用，是许多最流行的心血管疾病治疗的靶点（例如，ß-阻滞剂靶向的ß2-肾上腺素能受体（ß2AR））。这些受体的激活长期以来一直被认为是通过G蛋白的作用启动信号级联反应。最近的发现表明，7TMRs也启动了由ß- arrs介导的非g蛋白依赖性信号事件，ß- arrs除了在7TMR脱敏中最初被描述的作用外，还可以作为多种信号蛋白的支架。更明显的是，某些配体可以优先激活7tmr的多种下游效应的亚群，这种现象被称为“偏倚激动作用”，可以用来开发具有较少脱靶效应的“偏倚药物”。然而，偏倚信号的分子决定因素的表征和对这种现象的治疗潜力的研究一直受到缺乏探测偏倚的强大试剂的阻碍。因此，该提案的目标是识别一系列ß2AR的“偏倚突变体”

DESCRIPTION (provided by applicant): Seven-transmembrane receptors (7TMRs), also known as G protein-coupled receptors (GPCRs), serve cardinal roles in the regulation of cardiovascular function and are the targets of many of the most prevalent therapies for cardiovascular disease (e.g., the ß2-adrenergic receptor (ß2AR) targeted by ß-blockers). Activation of these receptors has long been known to initiate signaling cascades through the action of G proteins. More recent discoveries have demonstrated that 7TMRs also initiate non-G protein-dependent signaling events mediated by ß-arrestins (ßarrs), which, in addition to their originally characterized role in 7TMR desensitization, serve as scaffolds for a variety of signalin proteins. It has further become apparent that certain ligands can preferentially activate subsets of 7TMRs' multiple downstream effects, a phenomenon known as "biased agonism" that could be exploited to develop "biased drugs" with fewer off-target effects. However, characterization of the molecular determinants of biased signaling and investigation of this phenomenon's therapeutic potential have been hindered by the dearth of robust reagents for probing bias. Accordingly, the objective of this proposal is to identify a series of "biased mutants" of the ß2AR for use in diverse studies. The ß2AR has been characterized in vivo and in vitro far more extensively than any other 7TMR, making it a logical target for studies of biased signaling, but no strongly biased ß2AR ligands are known. Since receptor mutagenesis is a validated alternative to ligands for biasing 7TMR signaling, we aim first to systematically screen libraries of ß2AR mutants for those exhibiting biased signaling. We will construct focused, semi-rationally designed libraries of mutants and blindly screen constructs for G protein activation, ßarr recruitment, and expression in a multiplexed, medium-throughput assay format. Second, we will subject candidate mutants from this screen to a panel of assays to confirm their bias more rigorously and evaluate their potential for use in downstream applications. Successful completion of these objectives will provide a valuable repository of tools for the study of biased signaling, which could ultimately guide the design of next- generation medications targeting the ß2AR and other 7TMRs that possess novel therapeutic profiles and improved specificities compared to existing drugs.