Functionally discrete mimics of light-activated rhodopsin identified through expression of soluble cytoplasmic domains

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activatio n of proteins involved in visual transduction and desensitization. In our c ontinuing studies on rhodopsin folding, assembly, and structure, we have at tempted to reconstruct the interacting surface(s) for these proteins by ins erting fragments corresponding to the cytoplasmic loops and/or the carboxyl -terminal tail of bovine opsin either singly, or in combination, onto a sur face loop in thioredoxin. The purpose of the thioredoxin fusion is to provi de a soluble scaffold for the cytoplasmic fragments thereby allowing them s ufficient conformational freedom to fold to a structure that mimics the pro tein-binding sites on light-activated rhodopsin. All of the fusion proteins are expressed to relatively high levels in Escherichia coli and can be pur ified using a two- or three-step chromatography procedure. Biochemical stud ies show that some of the fusion proteins effectively mimic the activated c onformation(s) of rhodopsin in stimulating G-protein or competing with the light-activated rhodopsin/G-protein interaction, in supporting phosphorylat ion of the carboxyl-terminal opsin fragment by rhodopsin kinase, and/or pho sphopeptide-stimulated arrestin binding. These results suggest that specifi c segments of the cytoplasmic surface of rhodopsin can adopt functionally d iscrete conformations in the absence of the connecting transmembrane helice s and retinal chromophore.