PHARMACOLOGY OF CARDIAC A1 ADENOSINE RECEPTORS

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The major goals of the proposed research are the elucidation of the molecular structure of the A1 adenosine receptor and the biochemical mechanism(s) involved in the transduction of information from the interaction of adenosine with the receptor to the production of intracellular signals. Adenosine is released from cells under normal and pathophysiological conditions, such as ischemia and hypoxia and can interact with A1 adenosine receptors to directly inhibit adenylate cyclase, modulate K+ channels, blunt the effect of catecholamines, induce bradycardia and AV nodal block, inhibit lipolysis and induce sedation. The mechanisms and mode of coupling of the A1 adenosine receptor (A1AR) to the various effector systems remain largely unknown. Which guanine nucleotide regulatory proteins (G proteins) the A1AR couple to and how specificity and selectivity are maintained for these diverse pathways remains to be determined. These studies will (1) complete the purification of the A1AR to homogeneity, (2) study the purified A1AR in reconstitution systems with G proteins derived from both purification of Gi and Go from bovine brain and with recombinant G protein (alpha subunits), (3) determine if the A1AR is a substrate for protein kinases and the functional consequences of phosphorylation, (4) begin to ascertain the mechanisms involved in the "tight" coupling of A1AR with G proteins and which G proteins the A1AR couples to and (5) to obtain protein sequence data from the purified A1AR to clone the A1AR using an oligonucleotide hybridization approach. These biochemical and molecular biologic approaches are complimentary to in vivo animal and cell culture receptor regulation and cell biology studies currently ongoing in the Principal Investigator's laboratory. The ultimate goal of the proposed research is to understand the structure and function of the components of the A1AR transmembrane signalling apparatus both in health and disease so that we can manipulate its components for therapeutic benefit.