This proposal involves the development and use of photoactive radioactive compounds which will be used to identify various domains within the B-adrenergic receptor (BAR), the GTP-binding regulatory proteins (G protein), and adenylate cyclase. We will develop new radioiodinated photoactive compounds which are highly selective beta AR ligands. These compounds will be used to photoaffinity label the catecholamine binding domain of the beta AR in situ within the membrane and in digitonin-solubilized, affinity column-purified beta-receptor from guinea pig lung (82 subtype) and from human placenta (81 subtype). Radiolabeled peptides will be isolated by sulfhydryl affinity chromatography and/or reverse-phase HPLC using C4 or C8 columns and/or antibody affinity columns using polyclonal antibody raised against defined sequences of the BAR. Partial or complete sequence data will be obtained for the purified peptides and their position identified in the known sequence of the BAR. NAD photoaffinity labels (photo NAD) will be developed and used to specifically ADP-ribosylate G proteins. Initial experiments will be carried out with the bovine retinal rod outer segment rhodopsin-transducing system. In the long term, ADP-ribosylation of pure Gs (with cholera toxin) and pure Gi and Go (with pertussis toxin) will be performed. In situ ADP-ribosylation using membranes enriched in BAR and G protein (e.g., lung and placenta), and nearest neighbor analysis will be performed following photolysis by reversal with enzyme or mercuric acetate and analysis by SDS-PAGE. Derivatization of pure beta AR, rhodopsin, transducing, Gi, Go, and Gs will be performed using radioiodinated photocrosslinking compounds which we have recently developed. Reconstitution of """"""""photo G protein"""""""" will be performed with the appropriate receptor system (e.g., rhodopsin and BAR). Identification of interacting domains between receptor and """"""""photo G"""""""" will be performed. Radioiodinated forskolin photoaffinity labels will be developed to identify the binding site on the adenylate cyclase. The approaches outlined in this proposal will be applicable to study intramolecular and intermolecular interactions between polypeptide chains in a variety of proteins. These experiments will increase our understanding of catecholamine beta-receptors which function to control various autonomic functions, such as heart rate, blood pressure, and metabolic states of liver, adipose, and muscle.
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