STRUCTURE AND FUNCTION IN RHODOPSIN - PEPTIDE SEQUENCES IN THE CYTOPLASMIC LOOPS OF RHODOPSIN ARE INTIMATELY INVOLVED IN INTERACTION WITH RHODOPSIN KINASE

Phosphorylation of light-activated rhodopsin by the retina-specific en zyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic fac e of rhodopsin, and the binding results in activation of the enzyme wh ich then phosphorylates rhodopsin at several serine and threonine resi dues near the carboxyl terminus, To map the RK binding sites, we prepa red two sets of rhodopsin mutants in the cytoplasmic CD and EF loops, In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences, In the second set of mutants, th e charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E 249) were replaced by neutral amino acids in groups of 1-3 per mutant, The deletion and replacement mutants in the CD loop showed essentiall y no phosphorylation, and they appeared to be defective in binding of RK, Of the mutants in the EF loop, that with a deletion of 13 amino ac ids, was also defective in binding to RK while the second mutant conta ining a replacement sequence bound RK but showed a reduction of about 70% in V-max for phosphorylation, The mutants containing charged to ne utral amino acid replacements in the CD and EF loops were all phosphor ylated but to different levels, The charge reversal mutant E134R/R135E showed a 50% reduction in V-max relative to wild-type rhodopsin, Repl acements of charged residues in the EF loop decreased the K-m by 5-fol d for E239Q and E247Q/K248L/E239Q, In summary, both the CD and EF cyto plasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.