Rhodopsin's carboxyl-terminal threonines are required for wild-type arrestin-mediated quench of transducin activation in vitro.
Many recent reports have demonstrated that rhodopsin's carboxyl-terminal serine residues are the main targets for phosphorylation by rhodopsin kinase. Phosphorylation at the serines would therefore be expected to promote high-affinity arrestin binding. We have examined the roles of the carboxyl serine and threonine residues during arrestin-mediated deactivation of rhodopsin using an in vitro transducin activation assay. Mutations were introduced into a synthetic bovine rhodopsin gene and expressed in COS-7 cells. Individual serine and threonine residues were substituted with neutral amino acids. The ability of the mutants to act as substrates for rhodopsin kinase was analyzed. The effect of arrestin on the activities of the phosphorylated mutant rhodopsins was measured in a GTPgammaS binding assay involving purified bovine arrestin, rhodopsin kinase, and transducin. A rhodopsin mutant lacking the carboxyl serine and threonine residues was not phosphorylated by rhodopsin kinase, demonstrating that phosphorylation is restricted to the seven putative phosphorylation sites. A rhodopsin mutant possessing a single phosphorylatable serine at 338 demonstrated no phosphorylation-dependent quench by arrestin. These results suggest that singly phosphorylated rhodopsin is deactivated through a mechanism that does not involve arrestin. Analysis of additional mutants revealed that the presence of threonine in the carboxyl tail of rhodopsin provides for greater arrestin-mediated quench than does serine. These results suggest that phosphorylation site selection could serve as a mechanism to modulate the ability of arrestin to quench rhodopsin.
Brannock
M T
MT
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Weng
K
K
Robinson
P R
PR
D000818:Animals; D019393:Arrestin / Q000494:pharmacology; D019556:COS Cells; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D012243:Rhodopsin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism*
D019393:Arrestin; D013912:Threonine; D012694:Serine; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D015293:Transducin