Exposure of tissues or cells in culture to catecholamines results not only in activation of adenylate cyclase, but initiates as well a set of reactions leading to functional down-regulation of the B-adrenergic receptor (BAR). Current evidence is supportive of the hypothesis that the down-regulation process is the result of a sequence of BAR modifications; namely, receptor phosphorylation followed by receptor endocytosis followed by receptor degradation. We propose to test this hypothesis. One series of experiments will test the hypothesis that BAR are internalized via the clathrin-coated pit mechanism. To this end clathrin-coated vesicles (CCV) and endosomes will be isolated and their association with BAR established. CCV will be isolated with the use of antibodies to clathrin and endosomes will be isolated by free-flow electrophoresis and density gradient centrifugation techniques. Another series of experiments will involve determination of the correlation of BAR phosphorylation and BAR functionality. Phosphorylation will be determined by autoradiography of BAR separated on SDS-PAGE after purification by affinity chromatography. BAR functional status will be assessed in membranes by standard adenylate cyclase assay techniques and by reconstitution procedures utilizing purified BAR, phospholipids and guanine nucleotide binding proteins. Finally, the mechanism of agonist-induced BAR degradation will be studied in an attempt to link this process to the process of agonist-induced endocytosis of BAR.
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