Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

Abstract

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe45 --Leu, termination (deletion of C-terminal positions 344-348), and Pro-347 --Leul resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants Met, His, Thr-58 --Arg, Val-87 -* Asp, Gly-89 Asp, Gly-106 -Trp, Arg-135 --Leu, Arg-135 Trp, Tyr-178 -+ Cys, and Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.Retinitis pigmentosa (RP) is a group of inherited disorders that cause a progressive loss of retinal function. The hallmarks ofRP are decreased rod sensitivity, progressive loss of visual fields, a diminished electroretinographic response referable to photoreceptors, and characteristic pigmentary deposits in the retina (1).Recently, some patients with autosomal dominant RP (ADRP) were found to carry mutations in the gene encoding rhodopsin, the visual pigment mediating rod vision (2-5). The mutations cosegregate with RP and are absent from control populations with normal vision. In a previous study of 161 unrelated families with ADRP, 39 were found to carry 1 of 13 different point mutations in the rhodopsin coding region (5). The goal of the present study is to define the biochemical differences between wild-type (wt) rhodopsin and the variants responsible for ADRP. For this purpose we have produced in tissue culture cells each of the 13 mutant human rhodopsins described above and determined their yield, regenerability with 11-cis-retinal, and subcellular localization. MATERIALS AND METHODSTissue Culture Expression. A rhodopsin cDNA clone was isolated from a human retina cDNA library (6, 7), and a fragment containing the entire coding region was inserted into the expression plasmid pCIS (8). In vitro mutagenesis and production of opsin after transient or stable transfection of 293S cells were performed as described (9, 10).Absorbance Spectra. Cells from 20 10-cm plates were collected 60 hr after transient transfection, and membranes were prepared as described (9) except that the final membrane pellet was solubilized in 0.3 ml of 0.1 M sodium phosphate, pH 6.5/1 mM EDTA/1% 3-[(3-cholamidopropyl)-dimethylammoniol-1-propanesulfonate (CHAPS; Sigma). The solubilized sample was regenerated with li-cis-retinal, incubated with 50 mM hydroxylamine for 30 min, and the photobleaching difference spectrum was determined (9).Immunoblotting. Membrane samples prepared from cells 60 hr after transfection were mixed with an equal volume of 2x Laemmli sample buffer (lx = 0.125 M TrisHCl, pH 6.8/4% SDS/20o glycerol/10%o 2-mercaptoethanol/0.012% bromophenol blue), resolved on a SDS/12.5%...

Missense mutation of rhodopsin gene codon 15 found in Japanese autosomal dominant retinitis pigmentosa

“…Glycosylation of asparagine (Asn-15) requires the amino acid sequence Asn-Xaa-(Ser or Thr), where Xaa is any amino acid except proline. Sung et al (1991b) supported this assignment. In transfection of cloned c D N A into tissue culture cells, Thr-17-Met mutation produces only the 31-kDa and 35-kDa protein which appeared to be degraded with no higher molecular mass species observed.…”

Point mutations of rhodopsin gene found in Japanese families with autosomal dominant retinitis pigmentosa (ADRP)

“…Now, more than 26 different kinds of single-base mutations and a few cases of deletion of codon (s) within the coding region of rhodopsin gene have been identified in ADRP patients (Inglehearn et al, 1991;Keens et al, 1991 ;Sung et al, 1991a;Dryja et al, 1991, etc.). Moreover, it has been identified that at least two distinct biochemical defects have been associated with different rhodopsin mutants in ADRP (Sung et al, 1991b). Therefore, these allelic heterogeneities suggest a clinical variability.…”

Next-generation sequencing in health-care delivery: lessons from the functional analysis of rhodopsin

“…We also included two positive controls: P23H, the most common pathogenic mutation in rhodopsin, 4,9,10 where there is clear evidence of pathogenicity using in vitro expression systems and immunocytochemistry, [11][12][13][14][15] and ∆68-71, which spans one of our variants and has been demonstrated to be functionally inactive. [16][17][18] spectrophotometry WT and variant constructs were transiently transfected into human embryonic kidney cells (Supplementary Materials and Methods online).…”