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

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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.