FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2143409306> ?p ?o ?g. }

• W2143409306 endingPage "22298" @default.
• W2143409306 startingPage "22289" @default.
• W2143409306 abstract "Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus. Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus. Retinitis pigmentosa (RP) 2The abbreviations used are: RP, retinitis pigmentosa; arRP, autosomal recessive RP; adRP, autosomal dominant RP; BTP, 1,3-bis(tris(hydroxymethyl)-methylamino)propane; DM, n-dodecyl-β-d-maltoside; DSP, dithiobis(succinimidyl propionate); Endo H, endoglycosidase H; Gt, transducin, rod photoreceptor G protein; GPCR, G protein-coupled receptor; Meta II (or Rho*), photoactivated Rho; PNGase F, peptide:N-glycosidase F; Rho, rhodopsin; WT, wild type; ER, endoplasmic reticulum; GTPγS, guanosine 5′-O-(thio)triphosphate; DTT, dithiothreitol; PBS, phosphate-buffered saline.2The abbreviations used are: RP, retinitis pigmentosa; arRP, autosomal recessive RP; adRP, autosomal dominant RP; BTP, 1,3-bis(tris(hydroxymethyl)-methylamino)propane; DM, n-dodecyl-β-d-maltoside; DSP, dithiobis(succinimidyl propionate); Endo H, endoglycosidase H; Gt, transducin, rod photoreceptor G protein; GPCR, G protein-coupled receptor; Meta II (or Rho*), photoactivated Rho; PNGase F, peptide:N-glycosidase F; Rho, rhodopsin; WT, wild type; ER, endoplasmic reticulum; GTPγS, guanosine 5′-O-(thio)triphosphate; DTT, dithiothreitol; PBS, phosphate-buffered saline. refers to a group of inherited degenerative retinal diseases that display heterogeneous genetic backgrounds and clinical phenotypes (1Dryja T.P. Li T. Hum. Mol. Genet. 1995; 4: 1739-1743Crossref PubMed Scopus (248) Google Scholar). Many genetic loci have been reported to cause this retinopathy, including mutations in the opsin gene (RetNet, www.sph.uth.tmc.edu/RetNet/). Rod visual pigment rhodopsin (Rho) consists of the apoprotein, opsin, and the covalently bound chromophore, 11-cis-retinal (2Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2003; 65: 851-879Crossref PubMed Scopus (196) Google Scholar, 3Palczewski K. Annu. Rev. Biochem. 2005; 75: 743-767Crossref Scopus (551) Google Scholar). Although more than 100 opsin mutations are associated with RP (the Human Gene Mutation Data base, archive.uwcm.ac.uk/uwcm/mg/ns/1/120347.html), only a few have been reported to be associated with autosomal recessive retinitis pigmentosa (arRP). The first reported case of arRP associated with a mutation in the opsin gene is a nonsense mutation at codon 249 (4Rosenfeld P.J. Cowley G.S. McGee T.L. Sandberg M.A. Berson E.L. Dryja T.P. Nat. Genet. 1992; 1: 209-213Crossref PubMed Scopus (328) Google Scholar) that eliminates helices VI and VII (containing the retinal binding site Lys296), cytoplasmic helix 8, and the C terminus of opsin (Fig. 1A). Even heterozygous carriers of this mutation had affected electroretinograms, indicating an abnormality in rod photoreceptor function (4Rosenfeld P.J. Cowley G.S. McGee T.L. Sandberg M.A. Berson E.L. Dryja T.P. Nat. Genet. 1992; 1: 209-213Crossref PubMed Scopus (328) Google Scholar). This mutant was partially characterized (5Ridge K.D. Lee S.S. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). E150K is missense opsin mutation associated with arRP (6Kumaramanickavel G. Maw M. Denton M.J. John S. Srikumari C.R. Orth U. Oehlmann R. Gal A. Nat. Genet. 1994; 8: 10-11Crossref PubMed Scopus (75) Google Scholar); however, prior to this work the effects of the mutation had not been biochemically characterized. Glu150 is located in the proximity of the C-terminal region of the second cytoplasmic loop, embedded at the edge of the phospholipid bilayer (Fig. 1, A and B). Based on the crystal structure of Rho (7Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5003) Google Scholar), this residue may have electrostatic interaction with Arg69 on C-I. In general, residues on the cytoplasmic side, especially in C-II, C-III, and helix 8, play important roles in the activation of a cognate G protein, transducin (Gt) (8Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4819-4821Crossref PubMed Scopus (179) Google Scholar, 9Filipek S. Krzysko K.A. Fotiadis D. Liang Y. Saperstein D.A. Engel A. Palczewski K. Photochem. Photobiol. Sci. 2004; 3: 628-638Crossref PubMed Scopus (153) Google Scholar, 10Franke R.R. Konig B. Sakmar T.P. Khorana H.G. Hofmann K.P. Science. 1990; 250: 123-125Crossref PubMed Scopus (305) Google Scholar, 11Natochin M. Gasimov K.G. Moussaif M. Artemyev N.O. J. Biol. Chem. 2003; 278: 37574-37581Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 12Abdulaev N.G. Ridge K.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12854-12859Crossref PubMed Scopus (77) Google Scholar, 24Ernst O.P. Hofmann K.P. Sakmar T.P. J. Biol. Chem. 1995; 270: 10580-10586Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Another arRP mutation, G284S, is listed in www.sph.uth.tmc.edu/RetNet/ without any further characterization. We focused our research on the E150K mutant of opsin to understand the biochemical mechanisms underlying arRP and further characterize opsin biosynthesis by studying this recombinant opsin mutant in vitro. We explored whether the mutation interferes with phototransduction by affecting the binding and activation of Gt as suggested previously (6Kumaramanickavel G. Maw M. Denton M.J. John S. Srikumari C.R. Orth U. Oehlmann R. Gal A. Nat. Genet. 1994; 8: 10-11Crossref PubMed Scopus (75) Google Scholar) or causes disruption of the cellular trafficking. Materials—11-cis-Retinal was a gift from Dr. R. Crouch (University of South Carolina) through a contract with the National Institutes of Health. Human eyes were obtained from Lions Eye Bank (Seattle, WA and Portland, OR). The mouse anti-Rho (C-terminal) monoclonal antibody 1D4 was purchased from the University of British Columbia (Dr. R. Molday). The mouse anti-Rho (N-terminal) monoclonal antibody B6-30N was a generous gift from Dr. P. Hargrave (University of Florida). The rabbit anti-Rho IgG was a generous gift from Dr. E. L. Kean (Case Western Reserve University). The anti-Gtα subunit (Gtα) monoclonal antibody was a generous gift from Dr. H. E. Hamm (Vanderbilt University Medical Center). The anti-Gtβ subunit (Gtβ) polyclonal antibody was a generous gift from Dr. O. G. Kisselev (St. Louis University School of Medicine). Rabbit anti-calreticulin IgG, Cy3-conjugated anti-FLAG, and fluorescein isothiocyanate-conjugated anti-c-Myc mouse monoclonal antibodies were purchased from Sigma-Aldrich. Mouse monoclonal anti-GM130, anti-P230, and anti-Vti1b monoclonal antibodies were purchased from BD Biosciences (Franklin Lakes, NJ). Cy3-conjugated goat anti-mouse IgG or goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA). Hoechst33342 dye and Alexa 488-conjugated goat anti-rabbit or mouse IgG were purchased from Invitrogen. N-Dodecyl-β-d-maltoside (DM) was purchased from Anatrace Inc. (Maumee, OH). GTPγS was purchased from Sigma-Aldrich. Endoglycosidase H (Endo H) and peptide: N-glycosidase F (PNGase F) were purchased from New England Biolabs. Dithiobis(succinimidyl propionate) (DSP) was purchased from Pierce. Brefeldin A was obtained from EMD Biosciences (San Diego, CA). The synthetic 1D4 peptide was ordered from United Biochemical Research, Inc. (Seattle, WA). Other resins and columns for chromatography were purchased from Amersham Biosciences. Cell Lines and Growth Conditions—Tetracycline-inducible HEK293 cells stably transfected with constructs encoding human WT and E150K opsins were generated according to the manufacturer's procedure (T-REx-293™; Invitrogen), grown in the Dulbecco's modified Eagle's medium containing high glucose (Invitrogen) at 37 °C in the presence of 5% CO2. Unless otherwise mentioned, the cells were harvested after 48 h of tetracycline induction (1 μg/ml). Brefeldin A was added to the stable cell lines 1 h after tetracycline induction, at a final concentration of 2.5 μg/ml, and the cells were fixed 1 h after the brefeldin A treatment or left to recover for another 10 h before fixation. To add FLAG or c-Myc to the C terminus of the protein, WT or E150K opsin constructs were cloned in frame into the pCMV-Tag4 or pCMV-Tag5 vectors, respectively (Stratagene). After selection of the cloned constructs and confirmation of the sequence, HEK293 cells were cotransfected with 50% of each vector using Lipofectamine (Invitrogen). Two days post-transfection (48 h), the cells were then fixed with 2% of paraformaldehyde for 5 min. Electrophoresis and Immunoblotting—All of the protein separations were performed on 12% SDS-PAGE gels. Silver staining and immunoblotting (Immobilon-P polyvinylidene difluoride; Millipore) were carried out according to standard protocols. Sample buffer without dithiothreitol (DTT) was used with DSP cross-linked samples for the nonreducing condition. Anti-Rho 1D4 and anti-Rho B6-30N antibodies were used to detect the corresponding C-terminal and N-terminal epitopes. Alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Promega) were used as secondary antibodies. Protein bands were visualized with the nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt (BCIP) color development substrate (Promega). Pigment Reconstitution—Harvested cells expressing opsins were homogenized with a glass-glass homogenizer on ice and then centrifuged at 2,000 × g in a bench top centrifuge for 2 min to remove nuclei and cellular debris. The cell membranes were then pelleted by centrifugation at 14,000 × g for 5 min. All of the procedures employing reconstituted pigment or retinoids were performed under dim red light unless mentioned otherwise. The membranes were washed three times with 37 mm NaCl, 5.4 mm Na2HPO4, 2.7 mm KCl, and 1.8 mm KH2PO4, pH 7.4, and incubated with a final concentration of 50 μm 11-cis-retinal in the presence of protease inhibitors (protease inhibitor mixture; Sigma-Aldrich) at room temperature overnight to generate Rho. Purification of Opsin—Opsin or Rho was purified using anti-Rho C-terminal 1D4 antibody (13Molday R.S. MacKenzie D. Biochemistry. 1983; 22: 653-660Crossref PubMed Scopus (351) Google Scholar) immobilized on CNBr-activated Sepharose™ 4B (14Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (385) Google Scholar, 15Zhu L. Jang G.F. Jastrzebska B. Filipek S. Pearce-Kelling S.E. Aguirre G.D. Stenkamp R.E. Acland G.M. Palczewski K. J. Biol. Chem. 2004; 279: 53828-53839Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, a 4.6 × 12-mm column was packed with 300 μl of 2 mg 1D4/ml Sepharose beads. The incubated cell membranes were pelleted and homogenized with PBS, using a glass-to-glass homogenizer. Soluble proteins in the supernatant were removed by centrifugation at 14,000 × g for 5 min, and the pellet was then solubilized in buffer A (20 mm DM, 10 mm 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP), pH 7.5, containing 500 mm NaCl). The supernatant was loaded onto the 1D4 immunoaffinity column after a 20-min centrifugation at 125,000 × g at 4 °C, followed by a thorough washing with buffer B (2 mm DM, 10 mm BTP, pH 7.5, and containing 500 mm NaCl) at a flow rate of 0.5 ml/min. The purified protein was eluted with buffer C (500 μm TETSQVAPA in buffer B, pH 6.0) at room temperature. The protein concentration was determined from absorption at 280 and 500 nm using a Hewlett-Packard 8452A UV-visible spectrophotometer. Chemical Cross-linking with DSP—The cross-linking experiments were performed as described previously (16Jastrzebska B. Maeda T. Zhu L. Fotiadis D. Filipek S. Engel A. Stenkamp R.E. Palczewski K. J. Biol. Chem. 2004; 279: 54663-54675Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Suda K. Filipek S. Palczewski K. Engel A. Fotiadis D. Mol. Membr. Biol. 2004; 21: 435-446Crossref PubMed Scopus (62) Google Scholar). Briefly, the cell membranes were suspended in 100 mm sodium phosphate buffer, pH 8.0. DSP in Me2SO was added to reach a final concentration of 50 μm, and the reaction was allowed to proceed on ice for 30 min. The concentration of Me2SO did not exceed 2% of the reaction volume. To quench the reaction, 1 m Tris-HCl, pH 7.5, was added to a final concentration of 100 mm. The cell membranes were then washed with phosphate buffer and pelleted for opsin purification. Deglycosylation of Opsin—Approximately 3 μg of immunoaffinity-purified opsin was incubated with 100 units of Endo H or PNGase F at room temperature for 1 h (deglycosylation of opsin purified from cells) or at 4 °C for 24 h (for purified DSP cross-linked opsin), and the products were analyzed by SDS-PAGE. Photosensitivity of the Visual Pigment—UV-visible absorption spectra were measured with freshly purified Rho at 20 °C (15Zhu L. Jang G.F. Jastrzebska B. Filipek S. Pearce-Kelling S.E. Aguirre G.D. Stenkamp R.E. Acland G.M. Palczewski K. J. Biol. Chem. 2004; 279: 53828-53839Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In brief, photoactivation spectra were taken at intervals of 5, 10, 20, 30, and 180 s of exposure to light (applied with a long pass wavelength filter, >490 nm, at a distance of 20 cm). To trap the protonated retinylidene Lys296, concentrated H2SO4 was added to adjust the final pH to 1.9-2.0 after bleaching the sample for 3 min. The rate of photoactivation was determined using a plot of A496 nm against bleaching time. Rates of Meta II Decay—Rho (10 nm)in2mm DM, 10 mm BTP, pH 6.0, containing 100 mm NaCl was used to measure Meta II decay. The increase in intrinsic Trp fluorescence results from hydrolysis of the protonated Schiff base and release of all-trans-retinal from Rho (18Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Immunoaffinity-purified Rho was bleached by a Fiber-Lite illuminator (>490-nm filter applied) for 30 s from a distance of 15 cm, immediately followed by fluorescence measurements. The fluorometer slit was set to 2.5 μm at 295 nm for excitation and 8 μm at 330 nm for emission on a PerkinElmer Life Sciences LS 50B luminescence spectrophotometer. A thermostat was applied to stabilize the temperature of the cuvette at 20 °C during the measurement. Both fitting curves had R2 values of over 0.99. Gt Binding and Activation Assay—Gt was purified as described previously (16Jastrzebska B. Maeda T. Zhu L. Fotiadis D. Filipek S. Engel A. Stenkamp R.E. Palczewski K. J. Biol. Chem. 2004; 279: 54663-54675Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In the binding assay, affinity-purified Rho (WT or E150K) was mixed with a molar equivalent amount of Gt in 20 mm BTP, pH 7.5, containing 120 mm NaCl, 1 mm MgCl2, 1 mm DTT, and 2 mm DM. After bleaching Rho, the mixture was incubated for 15 min on ice to allow the complex to form. Gt dissociation was induced by the addition of 100 μm GTPγS (Sigma-Aldrich) on ice for 30 min before loading onto a size exclusion column (15Zhu L. Jang G.F. Jastrzebska B. Filipek S. Pearce-Kelling S.E. Aguirre G.D. Stenkamp R.E. Acland G.M. Palczewski K. J. Biol. Chem. 2004; 279: 53828-53839Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), using 20 mm BTP, pH 7.5, containing 120 mm NaCl, 1 mm MgCl2, and 1 mm DTT. In the fluorescence assay, the ratio of Gt to Rho was adjusted to 10:1 with Rho at ∼2.5 nm. The bleached sample was incubated for 10 min at 20 °C with continuous low speed stirring. The fluorescence was measured as described by Farrens et al. and others (19Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1104) Google Scholar, 20Heck M. Schadel S.A. Maretzki D. Bartl F.J. Ritter E. Palczewski K. Hofmann K.P. J. Biol. Chem. 2003; 278: 3162-3169Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), employing excitation and emission wavelengths at 300 and 345 nm, respectively. Immunocytochemistry—HEK293 cells were cultured on 35-mm glass-bottomed dishes (MatTek Corporation), and opsin expression was induced by 1 μg/ml tetracycline for 48 h after overnight attachment. The cells were then fixed with 2% paraformaldehyde in PBS (11.4 mm sodium phosphate, pH 7.4, containing 136 mm NaCl) for 5 min and washed with PBS. To block nonspecific labeling, the cells were incubated with 1.5% normal goat serum in PBST (PBS with 0.1% Triton X-100) for 15 min at room temperature. The cells were then incubated overnight at 4 °C with purified mouse anti-Rho 1D4 or rabbit anti-Rho antibody with PBST, along with one of the rabbit anticalreticulin IgG, mouse anti-GM130, anti-Golgin84, anti-P230, or anti-Vti1b monoclonal antibodies for double labeling. The cells were then rinsed in PBST and incubated with Cy3-conjugated goat anti-mouse IgG or goat anti-rabbit IgG for detection of opsin immunofluorescence and with Hoechst 33342 dye for DNA staining. For detection of other antigens, the cells were also incubated with Alexa 488-conjugated goat anti-rabbit or mouse IgG. For transient transfected cells, FLAG-tagged WT opsin was labeled with Cy3-conjugated anti-FLAG antibody, whereas E150K opsin was labeled with fluorescein isothiocyanate-conjugated anti-c-Myc antibody along with the DNA stain. After labeling with secondary antibodies, the cells were rinsed in PBST and mounted with 50 μl 2% 1,4-diazabicyclo (2,2,2)-octane in 90% glycerol to retard bleaching. For confocal imaging, the cells were analyzed on a Zeiss LSM510 laser scanning microscope (Carl Zeiss, Inc.). Image contrast and brightness were adjusted by Adobe Photoshop CS. Modeling—A phospholipid enhanced molecular model of Rho in the oligomeric state was constructed using the Rho network structure 1N3M from the Protein Data Bank. A three component lipid bilayer mimicking the rod disc membranes was composed of phospholipids with phosphatidylcholine head groups on the intradiscal side and phosphatidylethanolamine together with phosphatidylserine head groups on the cytoplasmic side. All three types of phospholipids contained the saturated stearoyl chain (18:0) in the sn1 position and the polyunsaturated docosahexaenoyl chain (22:6) in the sn2 position. The detailed procedure of the membrane embedding, solvation, and equilibration of the protein-membrane-water system was described previously (21Liang Y. Fotiadis D. Maeda T. Maeda A. Modzelewska A. Filipek S. Saperstein D.A. Engel A. Palczewski K. J. Biol. Chem. 2004; 279: 48189-48196Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The equilibration phase was performed at a constant temperature of 300 K and a constant pressure of 1013 hPa for 1 ns. Opsin mutants were subjected to additional molecular dynamics simulation for a subsequent 500 ps. E150K Opsin Exhibits a Different Glycosylation Pattern than WT Opsin—The arRP-related E150K Rho and WT human and bovine Rho as controls were immunoaffinity-purified from HEK293 cells and analyzed by UV-visible spectroscopy, SDS-PAGE, and immunoblotting. After normalization for the number of cells, the expression level of the mutant was ⅛ that of WT Rho as determined by immunoblotting (supplemental Fig. S1) and based on the amount of isolated mutant and WT Rho. The spectra of the human mutant and WT Rho were indistinguishable from each other (λmax = 496 nm) but shifted from bovine Rho (λmax = 498 nm) (Fig. 2A). Both proteins were purified to similar levels as judged by A280 nm/A500 nm ratio, which was 1.6-1.8 in most preparations (Fig. 2A, inset). In SDS-PAGE analysis, the monomeric form of WT Rho migrated as a broad band (∼40-55 kDa), because of heterogenous N-linked glycosylation in HEK293 cells. However, the majority of monomeric E150K opsin migrated with an apparent molecular mass of ∼50-58 kDa (Fig. 2B), which is slightly higher than that of WT opsin. These differences in the apparent molecular mass could result from altered post-translational glycosylation. Hence, Endo H and PNGase F were used to assess the status of glycosylation of the mutant and WT opsins. Endo H cleaves only the ER products containing high mannose structures and some hybrid oligosaccharides at the GlcNAc-GlcNAc bond of its chitobiose core from N-linked glycoproteins (22Maley F. Trimble R.B. Tarentino A.L. Plummer Jr., T.H. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (635) Google Scholar). Mature proteins are resistant to Endo H once they exit the ER and enter the Golgi. The amidase PNGase, cleaves the innermost GlcNAc-Asn bond from N-linked glycoproteins (22Maley F. Trimble R.B. Tarentino A.L. Plummer Jr., T.H. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (635) Google Scholar). Both silver-stained gels (Fig. 2C) and immunoblots (Fig. 2D) showed that E150K and WT opsins were mostly resistant to deglycosylation by Endo H. These data suggest that the majority of E150K opsin passed the ER processing and underwent early trimming such as the cleavage of the high mannose form of the N-linked glycans in the Golgi apparatus. These properties distinguish E150K mutants from the most extensively studied adRP opsin mutant, P23H, which is sensitive to Endo H cleavage (23Illing M.E. Rajan R.S. Bence N.F. Kopito R.R. J. Biol. Chem. 2002; 277: 34150-34160Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The fact that after PNGase F treatment the two E150K opsin bands merged into one with the same apparent molecular mass as deglycosylated monomeric WT opsin (∼35 kDa) (Fig. 2, C and D) excluded the possibility that the lower molecular mass resulted from partial degradation. E150K opsin formed more SDS-resistant oligomers than WT opsin, suggesting a higher propensity of the mutant to aggregate (Fig. 2B). The significance of this observation is unclear. The difference in apparent molecular masses between WT and E150K opsins was also observable for the SDS-induced dimer forms. E150K Mutant Rho and Gt Activation—The ability of the mutant to couple to Gt was investigated by size exclusion chromatography and fluorescence assays (Fig. 3, A and B). Upon light activation, Rho* bound Gtαβγ, and the Rho*·Gtαβγ complex was detected by gel filtration, where Rho and Gt were eluted in the same fractions (Fig. 3A, left panels). Upon the addition of nonhydrolyzable GTP analog, GTPγS, Rho, and Gt were eluted in different fractions (Fig. 3A, right panels) indicating that Gtα·GTP (Gtα·GTPγS) had dissociated to free Gtβγ and Rho*. In the direct intrinsic fluorescence assay (18Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), E150K Rho* activated Gtα with an initial activation rate of k0 ∼0.112 min−1, a rate that was faster than the activation by human WT Rho, k0 ∼0.071 min−1, but similar to activation by bovine WT Rho k0 ∼0.114 min−1. No changes in fluorescence were observed in the absence of Gt, and no constitutive Gt activation was detected with purified E150K opsin in the absence of light or chromophore (Fig. 3C) in the presence or absence of phospholipids to stabilize opsins (26Fasick J.I. Lee N. Oprian D.D. Biochemistry. 1999; 38: 11593-11596Crossref PubMed Scopus (80) Google Scholar). No Gt activity was observed using the whole membrane preparations (data not shown). These results suggest that mutated E150K did not affect G protein binding and activation. The increase in the Gt activation for the E150K mutant could be a result of modified photoactivation properties of the mutant. The active form of Rho, Meta II (or Rho*), had a faster decay rate (τE150K = 11.8 min) than that of WT Rho (τWT = 13.6 min), indicating that the release rate of all-trans-retinal from the binding pocket was faster (Fig. 3D). Both Rho proteins were photoactivated in DM, but again the decrease of A498 nm was faster for the mutant (supplemental Fig. S2), consistent with the rate of decay of Meta II. Additionally, DM-solubilized E150K opsin regenerated with 11-cis-retinal ∼50% more slowly than its WT counterpart. This observation could be a result of lower stability of the mutant in the detergent. Collectively, these data suggest that E150K Rho is correctly folded at the protein core around the binding pocket and undergoes similar, but not identical, photoactivation processes. Chemical Cross-linking of Opsin with DSP—WT opsin forms covalently linked dimers when expressed in HEK293 cells and treated with DSP (16Jastrzebska B. Maeda T. Zhu L. Fotiadis D. Filipek S. Engel A. Stenkamp R.E. Palczewski K. J. Biol. Chem. 2004; 279: 54663-54675Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Suda K. Filipek S. Palczewski K. Engel A. Fotiadis D. Mol. Membr. Biol. 2004; 21: 435-446Crossref PubMed Scopus (62) Google Scholar). Based on the crystal structure of bovine Rho (7Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5003) Google Scholar, 27Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (626) Google Scholar), there are several Lys residues from each Rho molecule of the dimer that lie within the accessible range of the DSP spacer (1.2 nm). When the membranes from cells expressing E150K opsin or WT opsin were treated with DSP, the formation of dimers was observed (Fig. 4). We used DTT in control experiments to reduce the disulfide bond within the cross-linker to exclude the possibility of nonspecific oligomerization. Similar results were obtained from both WT and E150K opsins, suggesting that the mutation did not induce dramatic changes in either the global folding of individual mutant opsin or the intermolecular interactions between opsins within dimers. E150K Opsin Partially Colocalizes with cis/Medial Golgi Marker—Confocal laser scanning microscopy was used to study the localization of WT and mutant opsins in HEK293 cells. WT opsin was properly folded and transported to the plasma membrane as previously reported (Fig. 5; see also supplemental Fig. S3) (14Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (385) Google Scholar). The adRP-related opsin mutation, P23H, is known to be ubiquitinylated and targeted for degradation by the ubiquitin-proteasome system while expressed in HEK293 or COS-7 cells (23Illing M.E. Rajan R.S. Bence N.F. Kopito R.R. J. Biol. Chem. 2002; 277: 34150-34160Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 28Chapple J.P. Grayson C. Hardcastle A.J. Saliba R." @default.
• W2143409306 created "2016-06-24" @default.
• W2143409306 creator A5001609547 @default.
• W2143409306 creator A5042148986 @default.
• W2143409306 creator A5045657649 @default.
• W2143409306 creator A5062898522 @default.
• W2143409306 creator A5069500879 @default.
• W2143409306 creator A5071314651 @default.
• W2143409306 creator A5073856478 @default.
• W2143409306 creator A5081013058 @default.
• W2143409306 date "2006-08-01" @default.
• W2143409306 modified "2023-10-13" @default.
• W2143409306 title "Autosomal Recessive Retinitis Pigmentosa and E150K Mutation in the Opsin Gene" @default.
• W2143409306 cites W1513724643 @default.
• W2143409306 cites W1559365105 @default.
• W2143409306 cites W1562615854 @default.
• W2143409306 cites W1571589296 @default.
• W2143409306 cites W1590595627 @default.
• W2143409306 cites W1843009580 @default.
• W2143409306 cites W1866273988 @default.
• W2143409306 cites W1923091024 @default.
• W2143409306 cites W1970512473 @default.
• W2143409306 cites W1979030108 @default.
• W2143409306 cites W1980823970 @default.
• W2143409306 cites W1982675033 @default.
• W2143409306 cites W1982846458 @default.
• W2143409306 cites W1993343824 @default.
• W2143409306 cites W1993697025 @default.
• W2143409306 cites W1994019984 @default.
• W2143409306 cites W1994108158 @default.
• W2143409306 cites W2002232559 @default.
• W2143409306 cites W2005277840 @default.
• W2143409306 cites W2007149985 @default.
• W2143409306 cites W2008975313 @default.
• W2143409306 cites W2012582259 @default.
• W2143409306 cites W2019742616 @default.
• W2143409306 cites W2024747540 @default.
• W2143409306 cites W2028291036 @default.
• W2143409306 cites W2030697532 @default.
• W2143409306 cites W2039170982 @default.
• W2143409306 cites W203942036 @default.
• W2143409306 cites W2043565870 @default.
• W2143409306 cites W2052717453 @default.
• W2143409306 cites W2054308102 @default.
• W2143409306 cites W2059441263 @default.
• W2143409306 cites W2068534497 @default.
• W2143409306 cites W2072073925 @default.
• W2143409306 cites W2072144082 @default.
• W2143409306 cites W2073420203 @default.
• W2143409306 cites W2079178498 @default.
• W2143409306 cites W2079302947 @default.
• W2143409306 cites W2089021583 @default.
• W2143409306 cites W2097948220 @default.
• W2143409306 cites W2102463040 @default.
• W2143409306 cites W2107312415 @default.
• W2143409306 cites W2111297610 @default.
• W2143409306 cites W2113736979 @default.
• W2143409306 cites W2115710616 @default.
• W2143409306 cites W2120504835 @default.
• W2143409306 cites W2129369040 @default.
• W2143409306 cites W2130035243 @default.
• W2143409306 cites W2133524076 @default.
• W2143409306 cites W2140128594 @default.
• W2143409306 cites W2147631462 @default.
• W2143409306 cites W2149963584 @default.
• W2143409306 cites W2156070415 @default.
• W2143409306 cites W2160119905 @default.
• W2143409306 cites W2168486543 @default.
• W2143409306 cites W2169889031 @default.
• W2143409306 cites W2169961890 @default.
• W2143409306 cites W2414461651 @default.
• W2143409306 cites W4238224698 @default.
• W2143409306 doi "https://doi.org/10.1074/jbc.m602664200" @default.
• W2143409306 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1618956" @default.
• W2143409306 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16737970" @default.
• W2143409306 hasPublicationYear "2006" @default.
• W2143409306 type Work @default.
• W2143409306 sameAs 2143409306 @default.
• W2143409306 citedByCount "23" @default.
• W2143409306 countsByYear W21434093062012 @default.
• W2143409306 countsByYear W21434093062013 @default.
• W2143409306 countsByYear W21434093062014 @default.
• W2143409306 countsByYear W21434093062015 @default.
• W2143409306 countsByYear W21434093062017 @default.
• W2143409306 countsByYear W21434093062020 @default.
• W2143409306 countsByYear W21434093062022 @default.
• W2143409306 crossrefType "journal-article" @default.
• W2143409306 hasAuthorship W2143409306A5001609547 @default.
• W2143409306 hasAuthorship W2143409306A5042148986 @default.
• W2143409306 hasAuthorship W2143409306A5045657649 @default.
• W2143409306 hasAuthorship W2143409306A5062898522 @default.
• W2143409306 hasAuthorship W2143409306A5069500879 @default.
• W2143409306 hasAuthorship W2143409306A5071314651 @default.
• W2143409306 hasAuthorship W2143409306A5073856478 @default.
• W2143409306 hasAuthorship W2143409306A5081013058 @default.
• W2143409306 hasBestOaLocation W21434093061 @default.
• W2143409306 hasConcept C104317684 @default.
• W2143409306 hasConcept C202033177 @default.

• next