BETA-ADRENERGIC RECEPTOR STRUCTURE AND DESENSITIZATION

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The overall goal of this proposal is the elucidation of the complex mechanisms which cause the rapid desensitization and sensitization of beta2-adrenergic receptor (beta2AR) stimulation of adenylyl cyclase (AC). The knowledge gained will help in the understanding of diseases such as asthma and hypertension in which these mechanisms may be altered, and in the rationale for pharmacological intervention. This proposal has three specific aims which are focused on determining the role of phosphorylation/dephosphorylation in the regulation of the responsivity of AC to beta2AR stimulation. The first aim is to determine the precise domains of the beta2AR involved in the cAMP-dependent protein kinase, the protein kinase C (PKC) and the beta-adrenergic receptor kinase pathways of desensitization. beta2AR mutants will be constructed, expressed in L cells and their regulation of AC characterized in response to various treatments which elicit desensitization. To measure the stoichiometry of phosphorylation, mutant beta2AR's will be constructed containing foreign epitopes with the intent of developing a rapid high recovery procedure for purification based on epitope binding to affinity columns. The second aim is to assess the hypothesis that PKC-mediated phosphorylation and inactivation of either the Gi/alpha subunits of the GTP-binding proteins, or the catalytic subunit of AC is the mechanism of the 4Beta-phorbol 12-myristate 13-acetate (PMA) and purinergic receptor- induced sensitizations of AC. This will be accomplished through the use of immunoprecipitation to measure phosphorylation of Gi/alpha subunits and AC catalytic subunits, construction of mutants of these proteins to aid in purification and analysis of domains involved, and reconstitution studies to determine if Gi function can be restored. PKC isozyme involvement in the PMA and purinergic sensitizations will be identified by measurement of translocation by immunoprecipitations and phorbol dibutyrate binding. The third aim is to identify phosphatases involved in the desensitization/sensitization in intact cells through the use of phosphatase inhibitors.