In vitro assay for trans-phosphorylation of rhodopsin by rhodopsin kinase.
Trans-phosphorylation of rhodopsin refers to a reaction in which a rhodopsin kinase molecule that has been activated by a light-activated rhodopsin molecule collides with and phosphorylates a second molecule of rhodopsin that has not been activated by light. It has been invoked as a mechanism for high-gain phosphorylation, a phenomenon that is observed at low bleaching levels where up to several hundred moles of phosphate are added to the rhodopsin pool per mole of photolyzed rhodopsin. Trans-phosphorylation is an appealing mechanism to propose for high-gain phosphorylation, but it has not been tested directly because of the difficulty inherent in unambiguous identification of light-activated and dark forms of rhodopsin present in the same reaction mixture. We report here a direct assay for trans-phosphorylation of rhodopsin. The assay is based on the use of a split receptor mutant of rhodopsin, SR(1-4/5-7), in which the fully functional protein is assembled from two separately expressed fragments. Because of different electrophoretic mobilities, SR(1-4/5-7) and wild-type rhodopsin can be monitored independently for phosphorylation while in the same reaction mixture. Thus, if wild-type rhodopsin is exposed to light and then incubated in the dark with SR(1-4/5-7), ATP, and rhodopsin kinase, phosphorylation of SR(1-4/5-7) would be a clear demonstration that trans-phosphorylation has occurred. Despite numerous attempts using several different experimental configurations, we have been unable to detect trans-phosphorylation of dark rhodopsin with this system.
Rim
J
J
The Graduate Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254, USA.
Faurobert
E
E
Hurley
J B
JB
Oprian
D D
DD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D005136:Eye Proteins / Q000378:metabolism*; D051606:G-Protein-Coupled Receptor Kinase 1; D066298:In Vitro Techniques; D008027:Light; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*
D005136:Eye Proteins; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1