Abstract
Purpose :
Cytosolic carboxyl-termini of G-protein coupled receptors (GPCRs) play important roles in dimerization, folding, and membrane transport. Characterized by a small hydrophobic helix and a long flexible tail, C-termini interact with cytosolic partner proteins that couple GPCRs to and modulate signaling pathways. Additionally, GPCRs feature a variety of transport signals within their C-termini, including E(X)3LL, F(X)6LL, and VxPx motifs. However, the mechanism(s) by which these sequences regulate GPCR trafficking remain opaque. Here we examine the functions of C-terminal tail domains in transport of Xenopus laevis rhodopsin to ciliary compartments.
Methods :
The role of the rhodopsin C-terminal domains in its localization in the outer segments of photoreceptors was investigated using transgenic X. laevis. Several X. laevis rhodopsin gene mutation and truncation constructs were generated based on multiple sequence alignment and structural information from mammalian rhodopsins and EGFP was appended to the C-termini. Transgenic tadpoles expressing constructs under the Xenopus opsin promoter were generated by REMI and the distribution of gene product was quantified in rods using confocal microscopy.
Results :
Contrary to previous reports, truncation of the rhodopsin C-terminus at residue 310 resulted in significant mislocalization of rhodopsin to the photoreceptor cell body, showing that the C-terminus is indeed essential for proper outer segment localization. Addition of VxPx to this truncated construct did not rescue outer segment localization. A previous study of zebrafish rhodopsin showed that methionine 317 (M317), which is conserved across all vertebrate species examined to date except Xenopus which possesses a leucine at this site (L317), results in partial mislocalization of the protein to the cell body. This was completely mitigated by replacing M317 with L. We show here that the reciprocal experiment, generating L317M mutation in Xenopus rhodopsin, results in its partial mislocalization.
Conclusions :
Our results show that residue 317 and the VxPx motif are essential for efficient rhodopsin trafficking. L317 allows the most efficient rhodopsin localization to rod outer segments. Remarkably, vertebrates possess rhodopsins with the inefficient M317 and, thus, their rods have chronic, low-level rhodopsin mislocalization.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.