The Retinitis Pigmentosa-Linked Mutations in Transmembrane Helix 5 of Rhodopsin Disrupt Cellular Trafficking Regardless of Oligomerization State

Abstract

G protein-coupled receptors can exist as dimers and higher-order oligomers in biological membranes. The specific oligomeric assembly of these receptors is believed to play a major role in their function, and the disruption of native oligomers has been implicated in specific human pathologies. Computational predictions and biochemical analyses suggest that two molecules of rhodopsin (Rho) associate through the interactions involving its fifth transmembrane helix (TM5). Interestingly, there are several pathogenic loss-of-function mutations within TM5 that face the lipid bilayer in a manner that could potentially influence the dimerization of Rho. Though several of these mutations are known to induce misfolding, the pathogenic defects associated with V209M and F220C Rho remain unclear. In this work, we utilized a variety of biochemical and biophysical approaches to elucidate the effects of these mutations on the dimerization, folding, trafficking, and function of Rho in relation to other pathogenic TM5 variants. Chemical cross-linking, bioluminescence energy transfer, and pulsed-interleaved excitation fluorescence cross-correlation spectroscopy experiments revealed that each of these mutants exhibits a wild type-like propensity to self-associate within the plasma membrane. However, V209M and F220C each exhibit subtle defects in cellular trafficking. Together, our results suggest that the RP pathology associated with the expression of the V209M and F220C mutants could arise from defects in folding and cellular trafficking rather than the disruption of dimerization, as has been previously proposed.

The F220C and F45L rhodopsin mutations identified in retinitis pigmentosa patients do not cause pathology in mice

“…Based on the measured reduction of the outer segment diameter and the corresponding reduction in the disc surface area in F220C mice, we estimate that the amount of rhodopsin transported to the outer segment is approximately 92% of WT. This explanation is consistent with a recent finding that the F220C rhodopsin mutant has a minor trafficking defect in cultured mammalian cells, with only 86% of mutant protein trafficked to the plasma membrane 9 .…”

“…Indeed, the only study to address the effect of disrupted rhodopsin dimerization in intact photoreceptors reported that inhibitory peptides 46 , shown to mildly disrupt rhodopsin dimerization in vitro 47 , caused rhodopsin mislocalization 46 . Yet, another recent study in mammalian cells suggested that F220C mutant rhodopsin does not affect its dimerization 9 . While rhodopsin dimerization within disc membranes has been shown through atomic-force microscopy 48 and cryo-electron microscopy 49 , there is currently no assay for analyzing rhodopsin dimerization during folding and trafficking, precluding us for identifying whether the F220C mutation in rhodopsin does indeed affect its dimerization in vivo, and, subsequently, what the role of rhodopsin dimerization in intact photoreceptors is.…”

“…However, a different study 7 found that a similar F220L rhodopsin mutation did not co-segregate with adRP. Further, both F220C and F220L mutant rhodopsins have been predicted to have normal protein stability and folding in a computational study 8 , although more recent work found that the F220C mutant has a minor trafficking defect in mammalian cell culture 9 .…”

To see or not to see: molecular evolution of the rhodopsin visual pigment in neotropical electric fishes

“…Substitutions that affect rhodopsin kinetics are known to have evolved convergently in distantly related species inhabiting dim-light environments [46,47], including other fishes inhabiting the same rivers of the Amazon basin [37]. Interestingly, mutagenesis studies in bovine rhodopsin have also shown that the F220C substitution experimentally decreases the rate of retinal release [35]. Although the link between these shifts in rhodopsin kinetics and effects on visual physiology remains to be investigated, our results suggest that shifts in important aspects of rhodopsin function are coincident with the evolution of electrosensory systems in gymnotiforms.…”

“…In the meta-II active-state structure of rhodopsin [33], the side chain of site 258 is closer to the phenylalanine at site 220 than any other residue in rhodopsin (figure 2a). The F220C mutation has been shown to impair both rhodopsin dimerization [34] and its kinetic properties [35]. These structural differences in helices 5 and 6, critical domains for rhodopsin dimerization and activation [20], might act to mitigate, or even mask the pathogenic effect of the substitutions at site 220.…”