Functional Characterization of Human Rhodopsin Mutations by Fluorescence Imaging

Abstract

Rhodopsin is the membrane receptor responsible for photoreception in the vertebrate retina. Over 120 point mutations in rhodopsin are found to be related with autosomal dominant retinitis pigmentosa (ADRP) and the congenital stationary night blindness (CNSB). Despite of several mutations with intense studies, like P23H, a majority of rhodopsin mutations still need further investigations. In order to have extensive and quick functional characterization of these mutations, here we utilize fluorescence imaging to monitor rhodopsin cellular distribution, which reveals to us much useful information, like if rhodopsin has normal transportation to the cell membrane, interrrupted glycosylation or protein aggregates formation. The experiments are carried out through the following process: First, a series of human rhodopsin mutations were constructed, which include mutations responsible for both ADRP and CNSB, like G89D and G90D. Second, wild-type rhodopsin was expressed in 293S GnTi- cells with homogeneous N-glycosylation for protein detection, T-REx293 cells for glycosylation analysis, and Hela cells for immunofluorescence imaging seperately. Third, we also engineered a fusion protein rho-EGFP for in vivo study, with a green fluorescent protein insterted into human rhodopsin. We found that mutations that cause ADRP are usually misfolded and retained in endoplasmic reticulum and thus have low efficiency of 11-cis-retinal binding. And G90D mutant (causing CNSB) that was considered to have correct protein folding, however, showed an astonishing tendency to form inclusion bodies in our study, which may be related with its interrupted glycosylation in Golgi apparatus. In a word, we have established an effective and convenient system to investigate rhodopsin synthesis and transportation both in vitro and in vivo.