Abstract
Purpose :
Rhodopsin delivery to newly forming outer segment discs is crucial for vision, yet the underlying mechanisms remain poorly understood. Cytosolic carboxy-termini of G-protein coupled receptors, including a conserved hydrophobic helix 8 (H8), play important roles in dimerization, folding, and transport to the functional locations within cells. Studies of adrenergic receptors (α- and βAR) and vasopressin receptors (V2R) show that H8 is involved in intramolecular hydrophobic interactions with the first transmembrane helix, TM1, that are critical for folding and transport to the plasma membrane. We examined if similar interactions are important for rhodopsin transport to the photoreceptor outer segments.
Methods :
Xenopus rhodopsin-EGFP (xRho-EGFP) constructs containing mutations in H8 and/or TM1were generated based on multiple sequence alignment and structural information from βAR and mammalian rhodopsins. Transgenic Xenopus tadpoles expressing constructs under the Xenopus opsin promoter were generated and the distribution of gene products was quantified in rods using confocal microscopy.
Results :
Our homology modeling between xRho and βAR suggests that there are two residues in H8 (F313 and L317) and four residues in TM1 (M57, V61, L68, L76) that form a hydrophobic interaction network. Mutagenesis experiments where L317 and M57 are replaced with hydrophilic aspartic acid (D) or arginine (R) resulted in significant mislocalization of xRho to inner segment membranes. However, replacing V61 with R had little impact. Interestingly, except for Xenopus, all vertebrate rhodopsins possess M at position 317 and, except for a few amphibians, L at position 57. Flipping these residues (xRho-L317M; M57L) resulted in small, but significant mislocalization to the inner segment and xRho-L317M, where M remains at position 57 resulted in significantly worse mislocalization.
Conclusions :
Our results show that hydrophobic interactions between H8 and TM1 are essential for efficient rhodopsin trafficking to the outer segment. Moreover, the identity of the hydrophobic amino acid at the specific H8 or TM1 positions is important for efficient outer segment localization. Of all the naturally occurring combinations, the L317 interaction with M57 found only in Xenopus rhodopsin produces the most efficient outer segment rhodopsin transport.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.