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

β2-肾上腺素能受体-β-抑制蛋白复合物不同构象状态的功能后果

• 批准号: 8983644
• 负责人: Thomas J Cahill
• 金额: $ 4.81万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8983644
• 关键词: Adrenergic Antagonists; Adrenergic Receptor; Adverse effects; Affinity; American; Applications Grants; Arrestins; Biochemical; Biological Assay; Biological Models; Biology; C-terminal; Cardiovascular Diseases; Cardiovascular system; Cause of Death; Cells; Clathrin Adaptors; Clinical; Complex; Confocal Microscopy; Congestive Heart Failure; Coronary Arteriosclerosis; Coupled; Coupling; Cyclic AMP; Development; Disease; Drug Design; Drug Targeting; Electron Microscopy; Endocrine; Family; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Health; Human; Hypertension; In Vitro; Knowledge; Lead; Ligand Binding; Ligands; MAP Kinase Gene; MAPK3 gene; Mediating; Membrane Proteins; Methods; Molecular; Molecular Conformation; Mutagenesis; Mutate; Outcome; Output; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phosphorylation; Physiological; Physiology; Population; Property; Proto-Oncogene Proteins c-akt; Receptor Activation; Receptor Signaling; Regulation; Reporter; Research Proposals; Resolution; Second Messenger Systems; Sensory; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Tail; Testing; Therapeutic; Transcription Factor AP-2 Alpha; Transmission Electron Microscopy; Treatment Protocols; United States; Woman; Work; base; beta-2 Adrenergic Receptors; beta-arrestin; biological systems; biophysical properties; clinically relevant; conformer; desensitization; drug market; functional outcomes; insight; men; mutant; next generation; novel; novel therapeutics; particle; receptor; reconstitution; reconstruction; scaffold; second messenger; structural biology

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.

描述（由申请人提供）：7个跨膜受体（7TMRs）家族，也称为G蛋白偶联受体（gpcr），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体生理。目前全球市场上约有50%的药物靶向7tmr，包括ß2-肾上腺素能受体（ß2AR），这是一种对心血管有重要意义的受体，这使得gpcr成为最重要的药物靶点之一。经典的GPCR信号传导伴随着g蛋白的激活，随后是GPCR激酶介导的受体磷酸化和ß-阻滞蛋白（ßarr）介导的受体脱敏和信号终止。此外，在过去的十年中，ßarrs作为网格蛋白和AP2的内吞接头，以及包括MAPK和AKT通路成分在内的多种信号蛋白的分子支架具有重要意义。ßarrs的这种多效性，引发了受体的后续激活，从而提出了ßarr与GPCR偶联是高度调控和动态的前提（即GPCR-ßarr复合物表现出一定的构象可塑性）。我们最近的发现证明了‘ßarr1偶联ß2AR’复合体的两种不同构象的存在，这一假设变得明显；确定为(i)“尾部（或悬挂）构象”，其中ßarr1主要偶联到受体磷酸化的c末端尾部，以及（ii）“核心（或紧密）构象”，其中c末端尾部偶联的ßarr1进一步与受体跨膜核心结合。这些独特的ß2AR-ßarr1构象亚群表明，在ß2AR-ßarr1复合体群体中存在功能选择性或“偏倚激动作用”，这揭示了开发临床相关偏倚疗法的潜在途径。虽然这种构象上的区别强调了ßarrs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，本研究计划的总体目标有三个方面：(1)开发相对高通量的体外形成ß2AR-ßarr1复合物的方法；(2)通过系统的ßarr1突变和电子显微镜下复合物的超微结构分析，发现ß2AR-ßarr1构象的结构决定因素；(3)通过各种成熟的细胞和药理学研究来描述ß2AR-ßarr1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）基于记者的ßarr1招募和内部化分析；（iv）第二信使cAMP测定和ERK1/2信号传导。该项目的成功完成将描述不同ß2AR-ßarr1构象的功能相关性（即受体的“核心”与“核心”）。“尾”耦合状态ßarr1)，从而为基于结构的药物设计的发展提供了强大的新基础。