Hormone-sensitive adenylate cyclase reflects the interaction of three plasma membrane proteins: the catalytic protein (C), a GTP-binding regulatory protein (G/F), and a hormone receptor. We propose to study the mechanisms of these events with particular interest in the role of membrane lipids and bilayer structure on their regulation. These studies will utilize unilamellar vesicles composed of synthetic phospholipids into which the proteins are incorporated. The stimulation of C by liganded G/F in such vesicles has been achieved in our laboratory. We will study the phospholipid requirement for this interaction with reference to its specificity for lipids, a probable effect of bilayer packing on its efficiency, and the distinction between C-G/F binding and the activation of C. In support of this work, C will be purified by a combination of conventional and affinity techniques. Pure G/F is available now from several sources. We can now reconstitute Beta-adrenergic receptors into vesicles, and our two procedures will be improved to utilize partially purified receptors, increase the number of receptors per vesicle, and improve the coupling with added G/F/ Coupling will be monitored both by catalysis of the activation of G/F by nucleotides and by equilibrium and kinetic measurements of cooperative binding interactions in the system. We will study the effects of bilayer environment on G/F-receptor coupling, possible requirements for specific lipids, and the effect of bilayer packing and fluidity on the rates of formation and dissociation of variously liganded receptor- G/F complexes. Our goal is to develop a kinetic map of the ligand-mediated protein-protein interactions of the three-protein system and understand how discrete steps are regulated by membrane structure.
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