Probing the Dimerization Affinity of Visual Opsins

Abstract

Visual opsins are the light sensing proteins at the center of human vision. Rhodopsin is the pigment for low-light vision, and three cone opsins serve as the pigments for daytime image-forming vision. Opsin proteins are part of the G protein-coupled receptor (GPCR) family, and rhodopsin has served as something like a prototype for structural studies of GPCRs. Rhodopsin has also been observed to form dimeric complexes in vitro, in tissue, and in cell cultures, including recent work from our lab using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Here we report on our recent investigation of cone opsin dimerization in a heterologous expression system. PIE-FCCS is used to resolve the relative population of dimeric complexes over a range of concentrations in live cell membranes. Surprisingly, the homodimerization affinities of the cone opsins are not identical to rhodopsin and there are significant differences in the dimerization affinity of the three cone opsins. This raises important questions about the physiological role of opsin dimerization, which has wide-ranging implications for GPCR dimerization in general. To investigate these questions we explored the differential dimerization affinity with mutational studies, in which we identify key residues responsible for dimerization. To connect the dimerization affinity to its functional consequences we also measured the spectral shifts in the absorption profiles of the wild-type and mutant cone opsins regenerated with the retinal chromophore. The results of our work provide critical insight into the molecular basis of color vision. They also will inform the ongoing study of GPCR dimerization in cell signaling.