D.A. Schwinn, M.D., A.Oganesian, Ph.D.
The long-term objective of this research is to understand the role of stress (acute and chronic) on perioperative outcome in humans. Within this context, our laboratory has focused on examining mechanisms underlying regulation of one of the stress hormone (catecholamine) binding receptor families, the alpha1-adrenergic receptors (alpha1 ARs). Our previous studies characterized subtype specific regulation of alpha1a AR transcription, desensitization, and internalization. One key finding that emerged was that alpha1a ARs have the unique ability to continuously signal in the presence of agonist in situations where alpha1b and alpha1dARs (and indeed many G protein-coupled receptors [GPCRs]) concurrently dampen (desensitize and/or down regulate) signaling. Indeed, our recent indicate that alpha1a AR trafficking mechanisms are distinct from other alpha1 AR subtypes since alpha1a AR displays a unique ability to constitutively recycle, independent of agonist stimulation. Underlying mechanisms remain unknown, but it is becoming increasingly clear that GPCRs are able to interact directly with an array of cytosolic that directly modulate receptor dimerization, trafficking, and transcription. The focus of this competitive renewal is to elucidate these processes with the overall hypothesis that alpha1a ARs couple to regulatory pathways distinct from other ARs. We propose two specific aims designed to identify novel alpha1a AR coupling (including clathrin, Gq-independent, and caveolin associated pathways). In the first aim we will investigate the ability of alpha1a ARs to interact directly with specific proteins in these pathways (e.g., beta-arrestins) using quantitative immunoprecipitation approaches in classic cellular models of both stably transfected rat-1 fibroblasts and neonatal cardiomyocytes. We will utilize novel soluble competitors that target intracellular loops to determine distinct alpha1a AR intracellular regions involved in alpha1a AR signaling and identify the proteins to which they bind using targeted proteomics. In a parallel second aim, we will examine mechanisms underlying both constitutive and agonistinduced alpha1a AR cycling using highly characterized functional and subcellular localization assays. Perturbation of model systems using engineered mutants for both alpha1a AR, as well as targeted factors within relevant pathways, be used to independently confirm results. Mutation of putative phosphorylation sites will be used to further mechanisms underlying alpha1a AR trafficking. Identification of alpha1a AR-responsive pathways is an important in understanding the role of catecholamines in stress responses with the ultimate goal of identifying novel designed to improve therapeutic strategies for acute and chronic disease.