The goal of this proposal is to understand how phosphorylation of membrane receptors regulates their activation of GTP-binding regulatory proteins (N proteins). Receptor phosphorylation is correlated with a decreased ability of receptor to activate N proteins. This has been demonstrated with the Beta-adrenergic receptor and rhodopsin, both of which activate specific N proteins resulting in the snythesis of cyclic AMP or the hydrolysis of cyclic GMP, respectively. The kinases involved in receptor phosphorylation are poorly characterized, as is the structural alteration of the receptor that occurs after phosphorylation that decreases its ability to activate N proteins. To address this problem the binding constants for the interaction of the N protein, transducin, with phosphorylated and non-phosphorylated rhodopsin will be determined. Rhodopsin activation of transducin measured by GTP binding and GTPase activity will be assessed with the different forms of the receptor. Two specific kinases will be used to phosphorylate rhodopsin. One kinase is rhodopsin kinase which phosphorylates only bleached rhodopsin. The second kinase is the Ca2+/phospholipid-dependent protein kinase which efficiently phosphorylates both bleached and unbleached rhodopsin. Synthetic peptides will be used to prepare antibodies against specific cytoplasmic sites of rhodopsin. The antibodies and peptides will be used to identify the transducin binding site on rhodopsin. The Beta-adrenergic receptor also has been isolated and will be structurally characterized. Initial experiments will involve fragmentation and microsequencing, with particular emphasis on hydrophilic peptides and peptides containing the receptor phosphorylation sites. The limited sequence information will be used to begin defining the structure of the receptor, and to prepare synthetic peptides for antibody production against the receptor sequences.
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