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 2-adrenergic receptor (2AR), 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 -arrestin (arr) mediated receptor desensitization and signal termination. Additionally, in the past decade, arrs 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 ?arrs, elicited following receptor activation, thus puts forward the premise that arr coupling to GPCRs is highly regulated and dynamic (i.e. GPCR-arr complexes display a certain conformational plasticity). This presumption has become apparent with our recent findings demonstrating the existence of two distinct conformations of the 'arr1-coupled 2AR' complex; identified as (i) the 'tail (or hanging) conformation', where arr1 is primarily coupledto the phosphorylated C-terminal tail of the receptor, and (ii) the 'core (or tight) conformation', where the C-terminal tail-coupled arr1 is further engaged with the receptor transmembrane core. These unique subsets of 2AR-arr1 conformers suggest the occurrence of functional selectivity or 'biased agonism' within the population of 2AR-arr1 complexes, which unveils potential avenues for developing clinically relevant biased therapeutics. While such conformational distinctions underscore the versatile functions of arrs (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 2AR-arr1 complexes in vitro; (2) to discover the structural determinants of 2AR-arr1 conformations by systematic arr1 mutagenesis and ultrastructural analyses of complexes by electron microscopy; and (3) to delineate the functional relevance of 2AR-arr1 conformational states through a variety of well-established cellular and pharmacological studies including: (i) confocal microscopy; (ii) high-affinity ligand binding; (iii) reporter-based arr1 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 2AR-arr1 conformations (i.e. receptors' 'core' vs. 'tail' coupled states of ?arr1), and thus provide a powerful novel basis fo development of structure-based drug design.
In the United States alone, almost 600,000 Americans die every year from cardiovascular disease, making it the leading cause of death for both men and women. Some of the most important drugs, such as ' blockers' (-adrenergic receptor antagonists) used to treat cardiovascular disease, target specific receptors on the outside of cells. In this study, we seek to understand, at a molecular level, the mechanism(s) by which these receptors signal to the inside of cells, laying the basis for next-generation therapeutics fo cardiovascular and other diseases.
| Cahill 3rd, Thomas J; Thomsen, Alex R B; Tarrasch, Jeffrey T et al. (2017) Distinct conformations of GPCR-?-arrestin complexes mediate desensitization, signaling, and endocytosis. Proc Natl Acad Sci U S A 114:2562-2567 |
| Kahsai, Alem W; Wisler, James W; Lee, Jungmin et al. (2016) Conformationally selective RNA aptamers allosterically modulate the ?2-adrenoceptor. Nat Chem Biol 12:709-16 |
| Thomsen, Alex R B; Plouffe, Bianca; Cahill 3rd, Thomas J et al. (2016) GPCR-G Protein-?-Arrestin Super-Complex Mediates Sustained G Protein Signaling. Cell 166:907-919 |
