Electrostatic Compensation Restores Trafficking of the Autosomal Recessive Retinitis Pigmentosa E150K Opsin Mutant to the Plasma Membrane

Abstract

Rhodopsin is the rod photoreceptor G protein-coupled receptor responsible for capturing light. Mutations in the gene encoding this protein can lead to a blinding disease called retinitis pigmentosa, which is inherited frequently in an autosomal dominant manner. The E150K opsin mutant associated with rarely occurring autosomal recessive retinitis pigmentosa localizes to trans-Golgi network membranes rather than to plasma membranes of rod photoreceptor cells. We investigated the molecular mechanisms underlying opsin retention in the Golgi apparatus. Electrostatic calculations reveal that the E150K mutant features an overall accumulation of positive charges between helices H-IV and H-II. Human E150K and several other closely related opsin mutants were then expressed in HEK-293 cells. Spectral characteristics and functional biochemistry of each mutant were analyzed after reconstitution with the cis-retinoid chromophore. UV-visible spectra and rhodopsin/transducin activation assays revealed only minor differences between the purified wild type control and rhodopsin mutants. However, partial restoration of the surface electrostatic charge in the compensatory R69E/E150K double mutant rescues the plasma membrane localization of opsin. These findings emphasize the fundamental importance of electrostatic interactions for appropriate membrane trafficking of opsin and advance our understanding of the pathophysiology of autosomal recessive retinitis pigmentosa due to the E150K mutation.

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

“…Previous studies in cell culture systems showed that the E150K mutation caused accumulation of positive charges on the cytoplasmic surface of rhodopsin (17,18). Here, we demonstrate that mouse rods expressing E150K rhodopsin undergo severe and rapid retinal degeneration.…”

“…Most of these mutations cause adRP, accounting for 30%-40% of the total RP cases, whereas only a few, including severe truncation of the opsin gene (14), are inherited in an autosomal recessive pattern. The c.448G>A (p.E150K) mutation in patients was the first reported missense mutation in the opsin gene associated with arRP (15,16), and the resulting mutant protein has since been characterized in vitro by biochemical methods (17,18). Another mutation that causes arRP, G284S, is listed in RetNet, but without further biochemical characterization.…”

“…There are several mechanisms potentially leading to disease in E150K patients. The E150K mutation substituting a positively charged Lys for the negatively charged Glu residue could result in disrup-tion of the higher-order organization of rhodopsin (19,(23)(24)(25) or cause aberrant transport of this protein to the ROS (17,18). Even if the protein can properly traffic and oligomerize in rod discs, the crystal structure of rhodopsin (26) indicates that residue 150 interacts with Arg69 in the loop connecting helices I and II (C-I loop), and because almost the entire cytoplasmic surface of rhodopsin is engaged in the interaction with its G protein transducin (27,28), signaling by the mutant protein could also be defective.…”

Computational and Experimental Analysis of the Transmembrane Domain 4/5 Dimerization Interface of the Serotonin 5-HT1A Receptor

“…Modified cellular distribution of GPCR mutants is a well documented phenomenon, which may occur naturally, sometimes causing pathologic conditions (Pulagam and Palczewski, 2010), or in site-directed mutagenesis (Milligan, 2009). We return to this issue under Discussion, whereas the rest of this work focuses on mutants that localize at the plasma membrane: W175A, R176K, Y198F, R151K/R152K, L159A/ L166A, and R176K/Y198F.…”

Functional characterisation of naturally occurring mutations in human melanopsin

“…Both hydrophobic and non-polar TM1 0.0002 (C) T62 H-bond between TM1 and TM2 [ 73 ] Y146C rs200099863 Conserved in all species Both polar and similar hydrophobicity TM3 0.0002 (G) E113 Schiff Base Counterion [ 49 ] R168C b rs143641898 Conserved in all species Positive charge to polar. Increased hydrophobicity TM3 / IL2 0.0005 (T) R135 c Conserved DRY motif [ 47 ] S183P rs151123640 Conserved in most species (T in Amphioxus) Polar to non-polar IL2 0.0002 (C) E150 c Required for normal membrane trafficking [ 74 ] R186H b rs141089672 Conserved in most species (K in Chicken and Zebrafish) Both positive charge, increase in hydrophobicity IL2 0.0005 (A) Not conserved G208S rs549998450 Conserved in all species Non-polar to polar. Introduce large side chain TM4 EL2 0.0004 (A) G174 c Unknown V213M rs202171086 Conserved in Opn4M, l/V in Opn4X Both hydrophobic and non polar EL2 0.0002 (A) I179 Unknown ...…”