title: Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase.
abstract: Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared 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, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially 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 acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in Vmax for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in Vmax relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the Km by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Thurmond
authors.forename: C
authors.initials: C
authors.lastname: Creuzenet
authors.forename: P J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism; D051606:G-Protein-Coupled Receptor Kinase 1; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012160:Retina / Q000737:chemistry / Q000201:enzymology*; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D015293:Transducin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D003902:Detergents; D005136:Eye Proteins; D005960:Glucosides; D008565:Membrane Proteins; C013110:metarhodopsins; C040358:dodecyl maltoside; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D015293:Transducin