Functionally Discrete Mimics of Light-Activated Rhodopsin Identified Through Expression of Soluble Cytoplasmic Domains

Published

December 15, 2000

Author(s)

N G. Abdulaev, T Ngo, Ruby I. Chen, Z H. Lu, K D. Ridge

Abstract

Seven-helical integral membrane receptors mediate signal transduction between extracellular stimuli and intracellular signaling cascades. Numerous studies on the seven-transmembrane-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal tail in the binding and activation of proteins involved in signal 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 a mutant form of thioredoxin. The purpose of the thioredoxin fusion is to act as a soluble scaffold for restraining the ends of the cytoplasmic loops, much like the transmembrane helices in rhodopsin, thereby allowing the grafted fragments sufficient conformational freedom to fold to a structure that mimic the protein binding sites on rhodopsin. All of the fusion proteins are expressed to high levels in E. 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 cytoplasmic surface of light-activated 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, or phosphopeptide stimulated arrestin binding. These results suggest that specific segments of the cytoplasmic loops of bovine opsin are capable of folding to a functional conformation in the absence of the transmembrane helices.

Citation

Journal of Biological Chemistry

Volume

275

Issue

50

Pub Type

Journals

Keywords

domains, membrane protein, photoreceptor, protein folding, signal transduction

Citation

Abdulaev, N. , Ngo, T. , Chen, R. , Lu, Z. and Ridge, K. (2000), Functionally Discrete Mimics of Light-Activated Rhodopsin Identified Through Expression of Soluble Cytoplasmic Domains, Journal of Biological Chemistry (Accessed December 11, 2023)