Mutagenesis Study of Retinal Entry Pathway of Rhodopsin

Abstract

The visual photoreceptor rhodopsin binds a photosensitive ligand 11-cis-retinal to respond to light stimulation. The regeneration of rhodopsin with 11-cis-retinal completes the photocycle of visual signaling. The molecular determinants of retinal entry can be assessed by measuring the retinal uptake kinetics of site-directed rhodopsin mutants. Here we report an assay for this purpose based on the energy transfer between 11-cis-retinal and a fluorescent probe covalently attached to the receptor. Previously, we developed an approach for site-specific labeling of G protein-coupled receptors by combining amber codon suppression technology and strain-promoted alkyne-azide cycloaddition reaction[1]. We have demonstrated robust fluorescent labeling of receptors without generating a cysteine-free background. We utilized this approach to label rhodopsin with Alexa488 as the FRET donor. Compared to tryptophan fluorescence spectroscopy, our assay exhibits a greater sensitivity and enables the characterization of mutant with slow retinal uptake kinetics. We showed that the heterologously expressed rhodopsin labeled at the second intracellular loop retains the wild-type retinal uptake kinetics. We further introduced a series of substitutions for the residues located in proximity of the retinal β-ionone binding pocket. We observed three orders of magnitude changes in retinal uptake kinetics. By varying the residue substitution we further studied the effect of hydrogen bonding and sterical hindrance. The insights into retinal entry not only shed light on a physiologically relevant process, but also facilitate understanding of retinal diseases.[1] H Tian, TP Sakmar, T Huber. Site-specific labeling of genetically encoded azido groups for multicolor, single-molecule fluorescence imaging of GPCRs. Methods in Cell Biology. In press.