Studies on the phosphorylation and dephosphorylation of rhodopsin

Fowles, Charles (1988) Studies on the phosphorylation and dephosphorylation of rhodopsin. University of Southampton, Doctoral Thesis.

Abstract

Photoexcitation of rhodopsin leads to the formation of an active intermediate, presumably metarhodopsin II (symbolised as Rho') that has been proposed to be involved in signal transmission via interaction with transducin (Stryer, L.A. (1986) An.. Rev. Neurosci., 2, 87-119). Rho' also acts as a substrate for rhodopsin kinase and this process culminates in the phosphorylation of several serine and threonine residues at the C-terminal of the protein. The regeneration of rhodopsin from phosphoRho' must involve three stages (i) dissociation of the chromophore (ii) reaction of the resultant protein with 11-cis-retinal and (iii) the removal of protein bound phosphate by a phosphatase. The precise order in whcih these processes occur is as yet undetermined. In the work presented here, the presence of 2 types of protein phosphatases in bovine rod outer segments (ROS) was demonstrated. These phosphatases were fractionated by the selective extraction of ROS with buffers of different ionic strength, followwed by separation on a heparin sepharose column. Only one of these two types of phosphatases was shown to be effective in the dephosphorylation of a sample of `highly' phosphorylated opsin (3-4.5 phosphates/opsin). A systematic study of the properties of this phosphatase suggested that this enzyme belongs to the type 2A class of protein phosphatases. The activity of this phosphatase was greatly stimulated by the presence of protamine sulphate in incubations. Interestingly `partially' phosphorylated opsin (0.15 phosphates/opsin) was less efficientlydephosphorylated than `highly'phosphorylated opsinspecies (3-4.5 phosphates/opsin). The phosphatase was shown to dephosphorylate phosphoopsin and phosphorhodopsin at identical rates and did not exhibit a preferential dephosphorylation of the serine or threonine residues in the protein. These results suggest that the phosphatase may also act on phosphoRho' and thus that no fixed reaction order exists in the process of regeneration of rhodopsin from this species. The cumulative results are discussed with regards to the involvement of the phosphorylation/dephosphorylation processes in the shutdown of the visual transduction system and possible desensitization of this system. The mechanism of photophosphorylation of rhodopsin by rhodopsin kinase was also studied using synthetic peptides corresponding to the sequence of the phosphorylation domain of bovine rhodopsin (The peptides corresponded to residues 318-348, 329-348, 329-339 and 339-348 of rhodopsin). All peptides investigated were shown to inhibit the photophosphorylation of rhodopsin, however the 10mer (residues 339-348) was by far the most efficient at inhibiting this process. This peptide was efficiently phosphorylated by the kinase only when incubation was performed in the presence of both rhodopsin and light. This phosphorylation was shown to be dependent on the presence of metarhodopsin II in decay experiments performed in these studies. This most interesting result is interpreted to suggest that in the dark-adapted state, rhodopsin kinase exists in an inactive conformation and that it is converted into a catalytically competent form only after interaction with metarhodopsin II [see also Fowles, C., Sharma, R. and Akhtar, M. (1988), FEBS Lett. 238, 56-60]. (DX85652)

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