fine distinct classes of biochemical defects in human rhodopsin and further show that amino acid substitu- tions in class I1 reside within the transmembrane and.
Vol. 268, No. 35, Issue of December 15, pp. 26646-26649,1993 Printed in U S A .
THE JOURNAL OF BIOIDGICAL CHEMI~Y 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Rhodopsin Mutations Responsiblefor Autosomal Dominant Retinitis Pigmentosa CLUSTERING OF FUNCTIONAL CLASSES ALONG THE POLYPEPTIDE CHAIN* (Received for publication, July 7, 1993, and in revised form, August 20, 1993)
Ching-Hwa SungSM, Carol M. DavenportSB, and Jeremy NathansSBn 1I ** From the Departments of Wolecular Biology and Genetics, Iweuroscience, and **Ophthalmology and the $Howard Hughes Medical Institute, Johns Hopkins University Schoolof Medicine, Baltimore, Maryland 21205
Over40 mutations in the rhodopsin gene have been del 68-71 P347S, Vy5M del255 identified in patients with autosomal dominant retinitis pigmentosa. Twenty-one of these mutations have been introduced into a human rhodopsin cDNAby site-directed mutagenesis, and the encoded proteins have been C167R produced bytransfection of a human embryonic kidney P53R cell line (2935). Three of the mutant proteins (GSlV, . P267L V34SM, and P347S) resemble the wild type in yield, reG51V H211P generability with ll-cie-retinal, and accumulation in the H211R plasma membrane (class I). The remaining 18 mutant P171L proteins are produced at lower levels, regenerate variDl 90N ably or not at all with 11-cis-retinal, and accumulate partially or predominantly in the endoplasmic reticuG106R lum (class 11). Together with an earlier analysis of 13 CAI., Schneider, B., Agarwal, mutant rhodopsins (Sung, P23L N., Papermaster,D. S., and Nathans,J. (1991)Proc. Nutl. / G182S Acad. Sci. U.S. A. 88,8840-8&44), these experiments deE181K fine distinct classes of biochemical defects in human FIG.1. Transmembrane model of human rhodopsin showing rhodopsin and further show that amino acid substitulocations and identities of 21 ADRP mutations studied.N and C tions in class I1 reside within the transmembrane and represent amino and carboxyl termini, respectively. The amino termiextracellular domains, whereas classI mutants cluster nus faces the extracellular space. in the first transmembrane domain and at the extreme carboxyl terminus. of all patients withADRP (reviewed by Nathans et al. (1992)). Formost of the rhodopsin mutations,the DNA sequence changes havebeen shown to cosegregate with the diseasepheRhodopsin is the light-absorbing protein that mediates vi- notype in affected families. sion at low light levels. Like other visual pigments,it consists To elucidate the biochemical defect(s) associated with each of a chromophore (ll-cis-retinal) bound to a n integral mem- mutation, our approach has been to produce both wild-type brane protein(opsin). In mammals, rhodopsin accumulates toa and mutant proteins in a tissue culture expression system level of 5 x lo7 moleculedrod outer segment andis synthesized and to characterize the recombinant human opsins with rethroughout life at a rate of 5 x lo6 molecules/rodday (Knowles spectto yield, abilityto recombine with 11-cis-retinal, and and Dartnall, 1977). Interest in rhodopsin structure and func- subcellular localization. In an initial studyof 13 mutant rhotion has been stimulated by the recent finding of rhodopsin dopsins (Sung et al.,1991), two classes were obsemed. Class I gene mutations in a subset of patients with retinitis pigmen- mutants (three examples) resembled the wild type in yield, tosa, a n inherited diseasethat leads toa progressive degenera- low degree of aggregation upon SDS gel electrophoresis, rea concomitant lossof vision (Heckenlively, generability with 11-cis-retinal, and subcellular localization. tion of the retina and 1988). Forty-three mutations, 39 of which lead to amino acid Class I1 mutants (10 examples) accumulated to significantly substitutions, have been reported in the heterozygous state lower levels, appeared predominantly as aggregates upon SDS among patients with autosomal dominant retinitis pigmentosa gel electrophoresis, regenerated with ll-cis-retinal to variable (ADRP),l and together, these mutationsaccount for one-fourth extents or not at all, and were transported inefficiently to the plasma membrane (PM). Members of class I1 differ in the se* This work was supported by the Howard Hughes MedicalInstitute verity of their biochemical defects: some proteins failed to decosts of publi- tectably exit theendoplasmic reticulum (ER)or to reconstitute and the National Retinitis Pigmentosa Foundation. The cation of this article were defrayed in part by the payment of page with 11-cis-retinal (classIIa), while othersaccumulated in charges. This article must thereforebe hereby marked “aduertisement” both the ER and PM (class IIb) and, in two cases, produced in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. TI To whom correspondence should be addressed: 805 PCTB, 725 N. small quantities of photolabile visual pigment.Presumably, Wolfe St., Johns Hopkins University Schoolof Medicine, Baltimore,MD the class I1 mutants are variably defective in protein folding 21205.Tel.: 410-955-4679; Fax: 410-614-0827. a n d o r stability. A recent study inwhich four ADRP mutations The abbreviations used are: ADRP, autosomal dominant retinitis were introduced at the corresponding locations in bovine rhoER, endoplasmic reticulum; pigmentosa;PM,plasmamembrane; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-l-propanesulfonic acid; mAb, monoclonal antibody. Aminoacid substitutions are referred letter amino acid designation,followed by the codon number, followed to by the identity of the wild-type residue, abbreviated using the single- by theintroducedresidue, e.g. arginine 135 + glycine is R135G).
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26646
Rhodopsin Mutations in Retinitis Pigmentosa PWL
P53R
G51V
OM)
005-
Qo5
del 68-71
LlPW
OM)-
"-+
om
-005 P171L
QM)
-0051
FIG.2. Examples of photobleaching difference absorption spectra for 21 mutant rhodopsins.Each spectrum was obtained from a 0.4-ml sample containing solubilized membranes prepared from 15 IO-cm diameterplates of transiently transfected cells. Two independent experiments were conducted for each mutant; yields varied by
