This proposal involves the development and use of photoactive radioactive compounds which will be used to identify various domains within the beta- adrenergic receptor (betaAR), the GTP-binding regulatory proteins (G- protein), and adenylyl cyclase. We will develop novel antagonist and agonist radioiodinated photoactive compounds which are selective for the catechol portion of the beta2AR ligand binding site. These compounds will be used to photoaffinity label the ligand binding domain of the baculovirus-expressed Sf9 beta2-adrenergic receptor (rbeta2AR). Partial N-terminal or complete sequence will be obtained for [125I]photolabeled peptides using a variety of photoprobes to """"""""map"""""""" the binding domain of the beta2AR. Novel radioiodinated forskolin photolabels with a potential for high insertion yield will be prepared and the forskolin binding site in adenylyl cyclases (types I, IV, and VI) will be identified. Adenylyl cyclase (I, V, VI) will be overexpressed in Sf9 cells and purified as a polyhistidine epitope-tagged enzyme. Intramolecular and intermolecular domains of Gs, Gi, Go, and Gt will be identified by use of radiolabel transfer from """"""""tethered"""""""" molecules. This work will involve the preparation of novel radioactive NAD+ photoactivatable molecules and of sulfhydryl """"""""tethered"""""""" photoactivatable molecules. Specific peptides derived from the sequence of the beta2AR, Gsalpha, Gtalpha will be derivatized to produce photoaffinity peptides (photopeptides) to identify interacting domains between beta2AR-Gs and Gs-beta2AR, between alphat and rhodopsin, and between alphat and cGMP phosphodiesterase. The location and intramolecular domains of the sulfhydryl and disulfides of the rbeta2AR and rhodopsin will be assessed by titration with sulfhydryl- specific reagents. Label transfer experiments will be performed to probe the intramolecular structure of the beta2AR. An assessment of the alpha- helical character of the beta2AR transmembrane 5 (TM 5) segment will be performed by use of the Nuclear Overhauser Effect (NOE) in NMR experiments. The approaches outlined in this proposal will be applicable to the study of intramolecular and intermolecular interactions between polypeptides in a variety of proteins. These experiments will increase our understanding of catecholamine beta-receptors which function to control autonomic functions such as heart rate, blood pressure, neuronal function, and metabolic states of liver, adipose, and muscle.
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