The surface of visual arrestin that binds to rhodopsin

[摘要] Purpose: The binding of visual arrestin to phosphorylated, activatedrhodopsin serves as a model for studying the inactivation process of alarge class of G-protein coupled receptor systems. In this study, wecombine the use of insertional mutagenesis, fluorescence labeling, andscanning alanine mutagenesis to identify the surface of interactionbetween arrestin and rhodopsin.Methods: The ten amino acid myc tag (EQKLISEEDL) was inserted ineleven loop structures that connect βstrands and the taggedarrestins were heterologously expressed in yeast. Binding competitionassays were performed with these proteins, using an anti-myc monoclonalantibody. Site specific cysteines were also substituted in selected loopstructures in arrestin. These cysteines were labeled with a fluorescentreporter to assess the proximity of the introduced cysteine withrhodopsin in the bound complex.Results: Competitive inhibition of arrestin binding to lightactivated, phosphorylated rhodopsin with an anti-myc antibody showedthat all competitive sites lay along a single surface encompassing theN- and C-terminal domains. Fluorescence labeling of these loopstructures and subsequent interaction with rhodopsin indicates closeapposition of loops 68-78 and 248-253 to rhodopsin in the receptor boundstate. Scanning mutagenesis of loop 248-253 implicates Ser-251 and/orSer-252 as a potential interaction point with rhodopsin.Conclusions: Our results clearly suggest a surface of arrestin towhich rhodopsin binds upon light activation and phosphorylation. Thissurface encompasses elements from both the N- and C-terminal domains ofarrestin.

[学科分类] 生物化学/生物物理