Sites of arrestin action during the quench phenomenon in retinal rods.
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.
Zuckerman
R
R
Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia 19104.
Cheasty
J E
JE
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
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