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 se rine 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 seri ne and threonine residues during arrestin-mediated deactivation of rhodopsi n using an in vitro transducin activation assay. Mutations were introduced into a synthetic bovine rhodopsin gene and expressed in COS-7 cells. Indivi dual serine and threonine residues were substituted with neutral amino acid s. The ability of the mutants to act as substrates for rhodopsin kinase was analyzed. The effect of arrestin on the activities of the phosphorylated m utant rhodopsins was measured in a GTP gamma S binding assay involving puri fied bovine arrestin, rhodopsin kinase, and transducin. A rhodopsin mutant lacking the carboxyl serine and threonine residues was not phosphorylated b y rhodopsin kinase, demonstrating that phosphorylation is restricted to the seven putative phosphorylation sites. A rhodopsin mutant possessing a sing le phosphorylatable serine at 338 demonstrated no phosphorylation-dependent quench by arrestin. These results suggest that singly phosphorylated rhodo psin is deactivated through a mechanism that does not involve arrestin. Ana lysis of additional mutants revealed that the presence of threonine in the carboxyl tail of rhodopsin provides for greater arrestin-mediated quench th an does serine. These results suggest that phosphorylation site selection c ould serve as a mechanism to modulate the ability of arrestin to quench rho dopsin.