A2-ADRENERGIC RECEPTOR: RECONSTITUTION AND PURIFICATION

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Strategies are proposed for the isolation, reconstitution, and purification of Alpha2-adrenergic receptors. These receptors mediate inhibition of adenylate cyclase activity by Alpha2-adrenergic agonists. Initial experiments will focus on solubilization and characterization of the receptors in detergents that will enhance procedures to be used for reconstitution and purification. Incorporation of the protein into phospholipid vesicles will facilitate measurement of the binding of ligands to the receptor. Partial purification should resolve the receptor from the catalytic and regulatory components of adenylate cyclase. Attempts at reconstitution will emphasize the reestablishment of the interaction of the receptor with a regulatory protein that is believed to mediate the inhibition of adenylate cyclase observed in the presence of Alpha-adrenergic agonists. This guanine nucleotide-binding regulatory protein (GI) is distinct from the regulatory protein that mediates stimulatory responses and is available in a purified form. Criteria for successful reconstitution of the receptor will be: 1) association of ligands with the receptor in a fashion similar to that observed in native membranes, 2) observation of effects of Alpha2-adrenergic agonists on the association of guanine nucleotides with GI and effects of guanine nucleotide on the association of agonists with receptors, and 3) restoration of inhibition of adenylate cyclase activity by Alpha-adrenergic agonists. Attempts at purification of these receptors will include the development of techniques that use the GI-protein as a specific probe. Antibodies to the G-protein or other molecules (e.g., avidin) with high affinity for chemical groups attached to the G-protein (e.g., biotin) can be used to isolate complexes of GI and the receptor specifically. Experiments designed to resolve and reconstitute the Alpha2-adrenergic receptor will yield information on the mechanism by which hormones inhibit adenylate cyclase. The techniques developed for reconstitution and purification of this receptor will hopefully be applicable to other receptors that either inhibit or activate this important regulatory enzyme.