title: Sites of arrestin action during the quench phenomenon in retinal rods.
abstract: The target proteins for arrestin (48 kDa protein) action during the quench of cGMP phosphodiesterase (PDE) activation in retinal rod disk membranes were identified by the use of a cross-linking reagent. A heterobifunctional, cleavable, photo-activatable cross-linker (sulfo-SADP) was coupled to purified arrestin. Under precise weak visible light bleach and nucleotide conditions of quench, the cross-linker was UV flash-activated at a time when quench was well established. The target proteins covalently linked to arrestin by cross-linker activation were identified by immunoblotting. In the presence of ATP arrestin cross-linked to both PDE and rhodopsin during the quench phenomenon. Removal of ATP from the reaction mixture essentially abolished the cross-link with PDE, just as ATP omission abolishes quench, but significantly increased the cross-link to rhodopsin. The absence of a cross-link to the plentiful beta-subunit of transductin, as well as the results of competition studies employing arrestin without attached cross-linker, suggest that the observed cross-links are specific and reflect true binding interactions of arrestin during quench. The data are consistent with a model of quench in which photolyzed rhodopsin (R*) catalyzes the formation of an activated form of arrestin, which dissociates from R* in the presence of ATP, and binds to PDEs, thereby deactivating them.
authors.affiliation: Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia 19104.
authors.forename: R
authors.initials: R
authors.lastname: Zuckerman
authors.forename: J E
authors.initials: JE
authors.lastname: Cheasty
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000941:Antigens / Q000502:physiology*; D019393:Arrestin; D001324:Autoantigens; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D005136:Eye Proteins / Q000502:physiology*; D015151:Immunoblotting; D008565:Membrane Proteins; D010726:Phosphodiesterase Inhibitors; D010777:Photochemistry; D010786:Photoreceptor Cells / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D013388:Succinimides; D015293:Transducin / Q000378:metabolism
chemical_list: D000941:Antigens; D019393:Arrestin; D001324:Autoantigens; D003432:Cross-Linking Reagents; D005136:Eye Proteins; D008565:Membrane Proteins; D010726:Phosphodiesterase Inhibitors; D013388:Succinimides; C057345:sulfosuccinimidyl (4-azidophenyldithio)propionate; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin