Sites of arrestin action during the quench phenomenon in retinal rods

• Published in: FEBS letters 1988-10, Vol.238 (2), p.379-384
• Main Authors: Zuckerman, Ralph, Cheasty, James E.
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-GMP Phosphodiesterases - metabolism; Adenosine Triphosphate - pharmacology; Animals; Antigens - physiology; Arrestin; arrestin, 48 kDa protein, S-antigen of retinal rods; Autoantigens; Biological and medical sciences; Cattle; Cell Membrane - metabolism; Cell physiology; cGMP, cyclic guanosine 3′:5′-monophosphate; Cross-Linking Reagents; DTT, dithiothreitol; Enzyme Activation; Eye Proteins - physiology; Fundamental and applied biological sciences. Psychology; Immunoblotting; Membrane Proteins; Molecular and cellular biology; Mops, 3-(N-morpholino)-propanesulfonic acid; PDE, cGMP phosphodiesterase; Phosphodiesterase; Phosphodiesterase Inhibitors; Photochemistry; Photoreceptor; Photoreceptor Cells - metabolism; Quench; R, photolyzed rhodopsin; RDMs, retinal rod disk membranes; Rhodopsin - metabolism; Succinimides; sulfo-SADP, sulfosuccinimidyl (4-azidophenyldithio)-propionate; Transducin - metabolism; transducin, G-protein of retinal rods; Vision, photoreception
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(88)80516-7

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, photoactivatable 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 β-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*) catalyses the formation of an activated form of arrestin, which dissociates from R* in the presence of ATP, and binds to PDEs, thereby deactivating them.