The long term objective of this project is to relate adrenergic receptor (AR) molecular structure to hormone/drug interaction and evoked physiologic responses. Within this context, the overall focus of this project is molecular characterization of alpha1-adrenergic (alpha1AR) subtypes. alpha1ARs play key roles in the regulation of physiologic processes such as myocardial blood flow, smooth muscle contraction, liver metabolism and the control of systemic arterial blood pressure in man. The availability of clones for two distinct alpha1AR subtypes in our laboratory places us in a unique position to evaluate the structural basis of AR distinct alpha1AR subtypes in our laboratory places us in a unique position to evaluate the structural basis of AR ligand binding and also signal transduction mechanisms utilized by AR subtypes. Hence, our first aim is the evaluation of ligand binding properties and signal transduction systems utilized by alpha1A and alpha1B-ARs. After permanently-transfected cell lines expressing alpha1AR subtypes are established, detailed ligand binding experiments will be conducted in tandem in order to fully characterize alpha1AR subtypes pharmacologically. Potential coupling systems including inositol phospholipid turnover, adenylyl cyclase stimulation or inhibition, arachidonic acid metabolism, and calcium channel coupling will be explored in order to understand the functional responses to stimulation of alpha1AR subtypes. G protein-coupling using recombinant G proteins and purified cloned receptor protein will complete this characterization. once pharmacologic and functional studies are complete, our second specific aim is to localize alpha1A-AR and alpha1B-AR distribution throughout the body. This will initially be done using NOrthern blot analysis. Following cloning of the rat homolog of these receptors, in-situ hybridization studies in rat tissue will be used to further refine the location of alpha1AR subtypes within specific tissues. Finally, our last specific aim is molecular structure-function analysis of alpha1ARs. Key residues responsible for subtype specific binding and differential coupling will be elucidated using chimeric alpha1ARs and specific mutagenesis techniques. By defining key residues responsible for AR subtype specific ligand binding, the development of more selective pharmacologic agents is enhanced. This should ultimately allow the production of more specific drugs that can be used in the managements of cardiovascular diseases with minimal clinical side-effects.
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