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 V max 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 E134R͞ R135E showed a 50% reduction in V max relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the K m by 5-fold for E239Q and E247Q͞K248L͞ E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.Light activation of rhodopsin initiates two biochemical cascades, one leading to visual sensitization and the other to desensitization. The primary event in the first cascade is the binding of transducin to light-activated rhodopsin (metarhodopsin II; Meta II), and that in the second is the binding of rhodopsin kinase (RK) to the same photointermediate resulting in its phosphorylation (2-4). While the structural requirements for interaction between Meta II and transducin have been investigated extensively, the study of RK-rhodopsin interaction has begun only recently. Akhtar and coworkers (5) dissected the action of RK into two distinct steps. The first involved binding to Meta II, which resulted in activation of the enzyme. Catalysis of phosphorylation then followed as a second step. Palczweski et al. (6) compared the activation of RK by a number of rhodopsin derivatives and concluded that the cytoplasmic EF loop was involved in binding to RK. More recently, Weiss and coworkers (7) studied the effects of certain amino acid replacements in cytoplasmic loops AB, CD, and EF and suggested the involvement of all the three loops in phosphorylation by RK.We now report on further characterization of the requirements for the binding of RK to Meta II and catalysis of the phosphorylation reaction. Two sets of mutants in the cytoplasmic loops CD and EF ( Fig. 1) (8) have been studied. In one set, pepti...

Conformational equilibria of light-activated rhodopsin in nanodiscs

“…The cytoplasmic ends of helices TM6 and TM7 are apparently involved in the recognition of the G protein, arrestin, and kinase (30)(31)(32), and the conformational heterogeneity in these regions of the activated receptor in nanodiscs may be involved in selective recognition of these or other binding partners. Indeed, the binding of a G protein is shown to select a particular receptor structure that itself has multiple conformations, suggesting flexibility in the complex.…”

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

“…Addition of the peptide corresponding to the third cytoplasmic loop (amino acids 234-245) or a longer peptide (amino acids 231-252), known to contain sites of interaction with transducin (28), arrestin (29), and rhodopsin kinase (30), had no effect on post-Golgi trafficking (30.6 Ϯ 0.9% of total radiolabeled rhodopsin in post-Golgi membrane fraction in control vs. 30.6 Ϯ 2.1% in the presence of the peptide 234-245, four separate experiments). Several other peptides, including the second cytoplasmic loop (amino acids 141-153) and a control N-terminal (intradiscal) domain (amino acids 3-14) also had no effect (data not shown).…”

Rhodopsin kinase: Expression in mammalian cells and a two-step purification

“…Six histidines were inserted at the N terminus between aspartic residue at position two and phenylalanine at position three. The RK(His) 6 gene was also constructed by PCR.…”

“…The HindIII-BamHI DNA fragment containing the RK(His) 6 gene was excised from pCMV5-RK(His) 6 (Fig. 1 AII).…”

“…The nature of this structural change is unknown. Nor is there any detailed information on the interactions between the light-activated rhodopsin and RK, although recent studies indicate that the binding of the enzyme involves the cytoplasmic loops in rhodopsin (6)(7)(8). Study of these questions by the mutagenesis approach requires a suitable system for expression and purification of RK mutants.…”