Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase* (rhodopsin phosphorylationyphototransductionydesensitizationysite-directed mutagenesisyG protein-coupled receptors)

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 E134Ry 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 E247QyK248Ly 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, peptide sequences in both loops are either deleted or replaced by indifferent peptide sequences. In the second set, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) are replaced by neutral amino acids in groups of 1–3 per mutant (Fig. 1, Table 1). Studies of the mutants as substrates for phosphorylation by RK in a homogeneous dodecylmaltoside (DM)-solubilized system show that both the CD and EF loops are intimately involved in the binding and interaction of RK with light-activated rhodopsin. A brief report of a part of these results has been made previously (9). MATERIALS AND METHODS Materials. DM was from Anatrace (Maumee, OH). 11-cisRetinal was a gift from R. Crouch (Medical University of South Carolina and National Eye Institute, National Institutes of Health). Cyanogen bromide-activated Sepharose 4B was from Sigma. The enhanced chemiluminescence detection system and horseradish peroxidase-conjugated goat anti-mouse IgG were from Amersham. Anti-rhodopsin mAb, rho-1D4 (10), was purified from a myeloma cell line provided by R. S. Molday (University of British Columbia). It was coupled to cyanogen bromide-activated Sepharose 4B as described (11). Frozen bovine retinae were from J. A. Lawson (Lincoln, NE), and rod outer segments (ROS) were prepared by the method of Papermaster (12). The nitrocellulose membranes were obtained from Schleicher & Schuell, and the PNGase F was from New England Biolabs. Endoproteinase Asp-N was obtained from Boehringer Mannheim, concanavalin ASepharose was from Pharmacia Biotech, and [g-32P]ATP (6000 Ciymmol; 1 Ci 5 37 GBq) was from DuPontyNEN. RhodopsinMutants. Themutant opsin genes corresponding to the mutant proteins in Table 1 are all among those previously described by Franke et al. (8). For convenience, the numbering of the mutants has been changed from that in the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y941715-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: RK, rhodopsin kinase;Meta II, metarhodopsin II; DM, dodecylmaltoside; ROS, rod outer segments; Rho-CT, rhodopsin truncated at C terminus; WT, wild type. *This is paper 23 in the series ‘‘Structure and Function in Rhodopsin.’’ Paper 22 is ref. 1. †R.L.T. and C.C. contributed equally to this work. ‡Present address: R. W. Johnson Pharmaceutical Research Institute, Rt. 202, Raritan, NJ 08869. §Present address: Institut National de la Recherche Agronomique, LEIMA, BP 1627, 44316 Nantes Cedex 03, France. ¶To whom reprint requests should be addressed.