Rhodopsin's Carboxyl-Terminal Threonines Are Required for Wild-Type Arrestin-Mediated Quench of Transducin Activation in Vitro
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- 作者：Michael T. Brannock ; Ke Weng ; and Phyllis R. Robinson
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：March 23, 1999
- 年：1999
- 卷：38
- 期：12
- 页码：3770 - 3777
- 全文大小：85K
- 年卷期：v.38,no.12(March 23, 1999)
- ISSN：1520-4995
文摘
Many recent reports have demonstrated that rhodopsin's carboxyl-terminal serine residues arethe main targets for phosphorylation by rhodopsin kinase. Phosphorylation at the serines would thereforebe expected to promote high-affinity arrestin binding. We have examined the roles of the carboxyl serineand threonine residues during arrestin-mediated deactivation of rhodopsin using an in vitro transducinactivation assay. Mutations were introduced into a synthetic bovine rhodopsin gene and expressed inCOS-7 cells. Individual serine and threonine residues were substituted with neutral amino acids. Theability of the mutants to act as substrates for rhodopsin kinase was analyzed. The effect of arrestin on theactivities of the phosphorylated mutant rhodopsins was measured in a GTP
S binding assay involvingpurified bovine arrestin, rhodopsin kinase, and transducin. A rhodopsin mutant lacking the carboxyl serineand threonine residues was not phosphorylated by rhodopsin kinase, demonstrating that phosphorylationis restricted to the seven putative phosphorylation sites. A rhodopsin mutant possessing a singlephosphorylatable serine at 338 demonstrated no phosphorylation-dependent quench by arrestin. Theseresults suggest that singly phosphorylated rhodopsin is deactivated through a mechanism that does notinvolve arrestin. Analysis of additional mutants revealed that the presence of threonine in the carboxyltail of rhodopsin provides for greater arrestin-mediated quench than does serine. These results suggestthat phosphorylation site selection could serve as a mechanism to modulate the ability of arrestin to quenchrhodopsin.
S binding assay involvingpurified bovine arrestin, rhodopsin kinase, and transducin. A rhodopsin mutant lacking the carboxyl serineand threonine residues was not phosphorylated by rhodopsin kinase, demonstrating that phosphorylationis restricted to the seven putative phosphorylation sites. A rhodopsin mutant possessing a singlephosphorylatable serine at 338 demonstrated no phosphorylation-dependent quench by arrestin. Theseresults suggest that singly phosphorylated rhodopsin is deactivated through a mechanism that does notinvolve arrestin. Analysis of additional mutants revealed that the presence of threonine in the carboxyltail of rhodopsin provides for greater arrestin-mediated quench than does serine. These results suggestthat phosphorylation site selection could serve as a mechanism to modulate the ability of arrestin to quenchrhodopsin.