RHODOPSIN KINASE - STUDIES ON THE SEQUENCE OF AND THE RECOGNITION MOTIF FOR MULTIPHOSPHORYLATIONS

Peptides of 10-12 amino acids in length, which overlapped with the seq uence of the last 20 amino acids in the C-terminal tail of rhodopsin, were synthesised and used as substrates far rhodopsin kinase. In all c ases the phosphorylation of the peptides was found to be greatly stimu lated (> 20-fold) by the presence of light-activated rhodopsin (Rho). The incorporation of P-32 at seven Ser/Thr residues that are the pote ntial sites of phosphorylation was quantified, and the results were an alyzed in terms of two parameters. First, a global comparison of phosp horylation at each site was made when the propensity for the modificat ion was found to be in the order: Ser 343 > Ser 338 > Thr 336 > Ser 33 4, Thr 342 > Thr 335, Thr 340. Second, the peptides were aligned on a hypothetical template with the residue to be phosphorylated occupying the P-position, and the manner in which the nature of the surrounding resides effected the phosphorylation was assessed. It was found that t he optimal phosphorylation of the P-site Ser/Thr occurs if it has at l east one residue on the amino side and five on the acyl side and also contains a neutral residue, preferably small (A, P, S, T) at the P+4 p osition. The salient features of the two analyses are combined into a model, acid it is speculated that the multiphosphorylation of rhodopsi n involves a sequence in which the first modification occurs at Ser 34 3, second at Ser 338, third at Thr 336, and fourth at Thr 342; the rem aining three residues (Ser 334, Thr 335, and Thr 340) are poorly phosp horylated, and a choice from among these is discussed. These findings extend and in broad terms confirm the previous conclusions drawn from structural studies on the phosphorylated receptor.