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 activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin. All of the fusion proteins are expressed to relatively high levels in Escherichia coli and can be purified using a two- or three-step chromatography procedure. Biochemical studies show that some of the fusion proteins effectively mimic the activated conformation(s) of rhodopsin in stimulating G-protein or competing with the light-activated rhodopsin/G-protein interaction, in supporting phosphorylation of the carboxyl-terminal opsin fragment by rhodopsin kinase, and/or phosphopeptide-stimulated arrestin binding. These results suggest that specific segments of the cytoplasmic surface of rhodopsin can adopt functionally discrete conformations in the absence of the connecting transmembrane helices and retinal chromophore.