The mechanism of the photophosphorylation of rhodopsin was studied using several synthetic peptides corresponding to the sequence of the phosphorylation domain. It was found that the dccapeptide (residues 339-348) was effectively phosphorylated by rhodopsin kinase only when incubation was performed in the presence of both rhodopsin and light. These results are interpreted to suggest that in the dark-adapted state rhodopsin kinase exists in an inactive conformation and that this is converted into a catalytically competent form only after interaction with metarhodopsin II (Rho*)
The phosphorylation of a synthetic peptide, corresponding to the C-terminal 11 amino acids of bovine rhodopsin (MI, residues 338 -348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C-terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C-terminal9 -12 amino acids as opposed to other parts of the C-terminus. It was noted that the absence of the two-terminal residues Pro347 and Ala348 impaired peptide phosphorylation.The effect of the decay of metarhodopsin I1 on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively.The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11-residue peptide (MI, residues 338-348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro.The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a 'good' peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the 'kinase-activation hypothesis'. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin. [18, 191 and rhodopsin kinase [20-221. This kinase, in vitro, acts specifically on several serine and threonine residues in the C-terminal domain of bleached rhodopsin [23-261. This dual role of Rho* in the transduction and termination processes is illustrated in Scheme 1.With respect to the mechanism of light-dependent phosphorylation, it was originally assumed that the C-terminal domain of rhodopsin is inaccessible to the kinase but becomes Correspondence to M. Akhtar,
Two types of protein phosphatases were identified in carefully prepared bovine rod outer segments (ROS). Extraction of the ROS with a medium-salt buffer solubilized protein phosphatase activity that was mainly type 2A, since it was active toward phosphorylase a in the absence of divalent cations, was not retained by heparin-Sepharose, dephosphorylated the alpha-subunit of phosphorylase kinase faster that the beta-subunit, and was unaffected by inhibitor 2. Further extraction of the resulting membranes with a high-salt buffer solubilized additional phosphatase activity which was predominantly type 1, since it was retained by heparin-Sepharose and was blocked by inhibitor 2. The molecular mass of the type 2A phosphatase estimated by gel permeation chromatography on Superose 12 was 100 kDa, suggesting it may be the 2A2 form. Only the ROS type 2A phosphatase dephosphorylated opsin and rhodopsin efficiently. Concordant with this finding, the purified catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle dephosphorylated opsin efficiently, while the type 1 catalytic subunit isolated from this tissue was inactive. Together, the results suggest that the ROS type 2A protein phosphatase plays an important role in regenerating rhodopsin from the various phosphorylated species in vivo. The activity of the enzyme per retina (approximately 85 pmol of Pi released/min) is comparable to that of rhodopsin kinase (100 pmol of phosphate transferred/min).
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