Rhodopsin: the functional significance of asn-linked glycosylation and other post-translational modifications

Abstract

Rhodopsin, the G-protein coupled receptor in retinal rod photoreceptors, is a highly conserved protein that undergoes several types of post-translational modifications. These modifications are essential to maintain the protein's structure as well as its proper function in the visual transduction cycle. Rhodopsin is N-glycosylated at Asn-2 and Asn-15 in its extracellular N-terminal domain. Mutations within the glycosylation consensus sequences of rhodopsin cause autosomal dominant retinitis pigmentosa, a disease that leads to blindness. Several groups have studied the role of rhodopsin's N-linked glycan chains in protein structure and function using a variety of approaches. These include the generation of a transgenic mouse model, study of a naturally occurring mutant animal model, in vivo pharmacological inhibition of glycosylation, and in vitro analyses using transfected COS-1 cells. These studies have provided insights into the possible role of rhodopsin glycosylation, but have yielded conflicting results.

Figure 1. Rhodopsin and its sites of post-translational modifications

RHO is a 7-pass G-protein coupled receptor (GPCR) that resides both within the disks of the rod outer segments and within the plasma membrane surrounding those disks. The protein undergoes several post-translational modifications that are indicated here in their order of occurrence during the synthesis and intracellular transport of RHO: (1) Schiff base formation upon binding of the chromophoric ligand, 11-cis retinaldehyde, to Lys-296 [white square]; (2) disulfide bond formation between two cysteine resides [grey hexagons]; (3) palmitoylation at two cytoplasmic cysteines [grey spotted circles]; (4) acetylation at its N-terminal methionine [black circle]; (5) phosphorylation at three C-terminal residues [grey diamonds]; (6) ubiquitinylation of an unknown number of cytoplasmic lysines [white spotted circles]; and (7) N-linked glycosylation at its extracellular N-terminus [grey and black diagonal circles]. Cytoplasmic loops I, II, and III are also depicted.

Figure 2. Schematic of the rod photoreceptor cell

It is a polarized neuronal cell type that contains a synaptic region, cell body, inner segment, connecting cilium and outer segment. RHO is synthesized in the rER in the inner segment and is translocated through the Golgi to the base of the outer segments where the formation of new disks and plasma membrane occurs. The RHO protein is found both within the disk and the plasma membrane of the cell.

Figure 3. Conservation of the asparagines (N) involved in N-linked glycosylation

Sequence alignment of the first 50 amino acid residues of RHO from several vertebrate species is shown. The alignment was generated using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/). The glycosylated residues at N-2 and N-15 are in grey and are designated by an arrow at the top of the alignment. (*), indicates that the residue is identical; (:), indicates that the residue is conserved; (.) indicates that the residue is semi-conserved.

Figure 4

Histological and ultrastructural appearance of retinas from non-transgenic mice (A,C) and NOG transgenic mice on a rho^+/− genetic background (B,D). Magnification: A and B, 40×; C and D, 10,00040×. Asterisks (panel D) indicate OS structural abnormalities. Abbreviations: RPE, retinal pigment epithelium; OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.