Glycosylation on serines and threonines has been described for a variety of proteins, from the ones responsible for the ABO blood types (Bundle, 1995), to structural proteins like glycophorin and collagen, and secreted proteins like mucines. Signaling proteins such as chorionic gonadotropin, erythropoietin, and some interleukins also contain O-linked sugars and it has been suggested that this posttranslational modification prolongs the life of cell surface components and of secreted proteins by protecting them from circulating proteases. The LDL and transferrin receptors, two cell surface proteins required for the utilization of cholesterol and iron from the extracellular space are O-glycosylated (Cummings et al., 1983; Hayes et al., 1992). It has been demonstrated for the transferrin receptor that the presence of the O-linked sugar protects the protein from tryptic-like proteases encountered by the receptor in its normal cycle of internalization and externalization (Rutledge and Enns, 1996). Other membrane proteins such as the Na+/H+ exchangers NHE-1 and NHE-2 have also been reported to be O-glycosylated (Counillon et al., 1994; Tse et al., 1994), but until now this modification had not been identified in receptors involved in signal transduction.
The V2 vasopressin receptor is a heptahelical G protein coupled receptor that mediates the antidiuretic action of arginine vasopressin in the kidney collecting duct (Birnbaumer et al., 1992). As shown for other polytopic receptors of this superfamily, the amino terminus of the protein is extracellular, while the carboxy terminus is intracellular. More specifically, it has been established that the first seventy amino acids of the V2R are sufficient to direct the insertion of the protein into the endoplasmic reticulum with the appropriate orientation (Schuelein et al., 1996). Among the 40 amino acids that constitute the extracellular amino terminus, there is one predicted site for N-linked glycosylation at asparagine 22. Substitution of this asparagine for glutamine (N22Q-V2R) destroys the high mannose acceptor site and results in the synthesis of a protein that migrates faster in polyacrylamide gels than the N-glycosylated receptor protein, and retains unaltered vasopressin binding and coupling to the G protein Gs. The migration of the mutant N22Q-V2R protein in polyacrylamide gels was not altered by treatment with N-peptide glycosidase F (PNGase F), an enzyme that cleaves all sugars attached to asparagine regardless of their composition, corroborating that asparagine 22 is the only site of the V2R that can be used for N-linked glycosylation (Innamorati et al., 1996; Sadeghi et al., 1997a).
Polyacrylamide gel electrophoresis analysis of wild type V2R expressed in HEK 293, COS M6 or MDCK cells had revealed the presence of two protein forms as seen by immunoblots of cell membranes or immunoprecipitation of metabolically labeled receptor. The slower migrating protein was detected as a diffuse band at 45-50 kDa in HEK 293 and COS cells and as a better focused band between 43 and 48 kDa in the MDCK cells. For the three cell types a faster migrating protein of about 37 kDa was also detected. Expression of the mutant N22Q-V2R in HEK 293 and COS cells also produced two receptor bands: one at 40 kDa that was the only one present on the cell surface as revealed by biotinylation of intact cells followed by immunoprecipitation with anti-receptor antibodies (Sadeghi et al., 1998), and a 33 kDa protein (Innamorati et al., 1996; Sadeghi et al., 1997a). The appearance of two bands when the protein lacked N-linked glycosylation suggested the presence of O-glycosylation in this receptor, and led us to investigate whether O-linked sugars were responsible for the difference in migration between the two proteins. Indeed, we found the receptor to be glycosylated in serines and threonines present in the amino terminal segment of the V2R.
Figure 1. PNGase F treatment of wild type and N22Q V2R. HEK 293 cells transfected with cDNAs encoding the wild type and the N22Q V2R were metabolically labeled 48 h after transfection, and the receptor immunoprecipitated after a 2 h chase. Lanes 1-3 show a prominent band of precursor V2R protein that is Endoglycosidase H and PNGase F sensitive (40 kDa, left arrow). The mature glycosylated V2R (vertical line) Endoglycosidase H resistant and PNGase F sensitive migrates as a sharp band of 40 kDa after cleavage of the N-linked sugars. Lanes 4 and 5 show the sharp bands of precursor and mature proteins of N22Q-V2R that did not change their mobility after PNGase F treatment (right arrowhead).
As illustrated in Figure
Figure 2. Glycosidase treatment of membranes containing N22Q-V2R. Cell membranes containing the N22Q-V2R receptor were isolated and aliquoted as described under Materials and methods. After the enzymatic treatments, the samples were analyzed by gel electrophoresis and immunoblot. (A) membranes were treated with 2 µg/20 µl sialidase for 2 h at 22°C in 50 mM Na acetate, pH = 5.0, 9 mM CaCl2, 150 mM NaCl. Prior to O-glycosidase treatments, the membranes were washed twice with 50 mM Tris phosphate, pH 7.0, recovered each time by centrifugation, and incubated in 20 µl of the same buffer containing 1 mU O-glycosidase for 1 h at 22°C. N-Acetylglucosaminidase was incubated with the membrane samples at 5 mg/20 ml for 2 h at 22°C in the same buffer used for sialidase. Membranes subjected to two enzymatic treatments were pelleted by centrifugation and resuspended in the corresponding buffer prior to the addition of each enzyme. (B) illustrates the effect of 1% NP40 on sialidase activity. #, Immature N22Q-V2R protein; arrowheads, PN-Gase F resistant N22Q-V2R protein after cleavage of sialic acid.
As mentioned above, the size of the fully glycosylated receptor synthesized in MDCK cells was different from the one detected in COS and HEK cells; therefore, it was determined whether the protein was O-glycosylated in these cells. The wild type receptor expressed in the MDCK cells was metabolically labeled, immunoprecipitated as described, and while still bound to the protein A Sepharose beads it was treated with glycosidases to identify the type of sugar links it contained. As illustrated in Figure
Figure 3. Sialidase sensitivity of the wild type human V2R expressed in MDCK cells. Stably transfected MDCK cells expressing the wild type V2R were metabolically labeled and the protein was immunoprecipitated as described under Materials and methods. The purified receptor attached to protein A-Sepharose was subjected to treatments with PNGase F as described in the caption to Figure 1, and with sialidase and O-glycosidase as described in the caption to Figure 2. After the enzymatic treatments the proteins were extracted with Laemmli sample buffer and analyzed by SDS-PAGE and fluorography.
An alternative method to explore the presence of O-linked sugars is the use of inhibitors of glycosylation in cells expressing the N22Q V2R. Analogues of N-acetyl-galactosamine containing bulky hydrophobic side chains such as phenyl or benzyl groups have been shown to interfere successfully with the formation of fully glycosylated mucins (Kuan et al., 1989). Later experiments analyzing the effect of the phenyl containing inhibitors on the glycosylation of a cell surface glycoprotein revealed that they interfere with the incorporation of N-acetyl-neuraminic acid, but not with the initial step of O-linked glycosylation (Rettig et al., 1992). Because in transiently transfected cells most of the receptor protein was synthesized between 24 and 48 h after transfection (Sadeghi et al., 1997a), the effect of the inhibitor was tested under those conditions. Transfected cells were exposed overnight to 1 mM phenyl N-acetyl-[alpha]-d-galactosaminide as described in Materials and methods, and the following day a metabolic labeling experiment was carried out in the presence of the inhibitor. After the chase period, cells were lysed, membrane proteins extracted, and the receptor immunoprecipitated from treated and control cells as described under Materials and methods. Aliquots of the receptor were treated with sialidase, sialidase/O-glycosidase, or O-glycosidase alone while still attached to the protein A-Sepharose beads. After treatments performed as described in the figure legends, the proteins were eluted from the beads with Laemmli sample buffer and analyzed by acrylamide gel electrophoresis and fluorography. As shown in the left portion of Figure
Figure 4. Inhibition of O-glycosidation of the N22Q-V2R. Metabolic labeling of the N22Q-V2R was carried out in the absence (left lanes), or presence (right lanes), of 1.0 mM phenyl-N-acetyl-[alpha]-d-galactosaminide. The cells were lysed and the receptor protein extracted and immunoprecipitated as described in Materials and methods. The receptor protein attached to the protein A-Sepharose beads was treated with sialidase, sialidase plus O-glycosidase, and O-glycosidase as described in the Figure 2 caption.
The right portion of Figure
With the help of a data base specializing in proteins containing O-linked sugars, the amino acid sequence of the V2R was analyzed with a program that predicts the most likely sites for O-linked glycosylation based on the compilation of the composition of the glycosylated sites of mucins. The serines and threonines present in the extracellular amino terminal segment of the V2R were considered the most probable site for O-glycosylation by this program. More specifically, the cluster between amino acids 5 and 8, and serines 15 and 18 were identified as the ones with the highest probability for O-glycosylation. Figure
Figure 5. Sites for O-glycosylation in the V2R predicted by the Oglyc-Base database. Composition of the amino terminal segment of the V2R. Possible glycosylation sites are identified by an asterisk.
Mutagenesis of the amino terminus of the V2 receptor was approached with hesitation because this protein, lacking an identifiable signal peptide, could have a cryptic signal encoded in the amino terminus that when altered could reduce receptor expression by interfering with targeting of the protein to the endoplasmic reticulum. The experiments demonstrated that this concern was not justified for the V2 receptor. Expression of the N22Q-V2R was only slightly reduced by the presence of alanines instead of serines and threonines in the amino terminal segment. The cDNAs were transfected into HEK 293 cells, and the size of the receptors produced by the different cDNAs was determined by analyzing the membranes of the transfected cells by immunoblots. As it is shown in Figure
Figure 6. Alanine scanning of N22Q-V2R O-glycosylation. HEK 293 cells were transfected with cDNAs encoding the wild type or mutant V2Rs identified below the photograph. Two days after transfection membranes were prepared from the transfected cells and 10 mg aliquots were analyzed by SDS-PAGE and immunoblots with antibody 3 as described under Materials and methods. (A) and (B) illustrate two different experiments. Arrowheads identify the migration of mature and precursor N22Q-V2R, and the text below the picture identifies the receptor mutations shown.
As illustrated in Figure
The k mutant V2R was expressed transiently in HEK 293 cells to assess by saturation binding assays the impact of the extensive mutagenesis on its ligand binding affinity and level of expression. Figure
Figure 7. Binding affinity and level of expression of the nonglycosylated V2R. Saturation binding assays were performed with intact HEK 293 cells expressing the N22Q mutant V2R (open triangles) or the N22Q mutant V2R containing 9 alanines in the amino terminal segment, k mutant (solid squares). The assay was carried out at 4°C in D-PBS by addition of increasing concentrations of [3H]AVP in the absence and presence of 10 mM unlabeled AVP to determine nonspecific binding. The specific binding obtained in one of the experiments is shown in (A), (B) illustrates the Scatchard analysis of the data. The values obtained were 4.5 ± 0.5 and 4.8 ± 0.4 nM for KD, and 8.1 ± 0.5 and 6.1 ± 0.5 × 106 sites/cell for Bmax for the N22Q and the N22Q mutant k V2R, respectively in four experiments.
Figure 8. Signaling properties of the nonglycosylated V2R. Vasopressin stimulated adenylyl cyclase activity was measured in homogenates of transiently transfected HEK 293 cells expressing the N22Q mutant V2R (open triangles) or the N22Q mutant V2R containing nine alanines in the amino terminal segment, k mutant (solid squares). The results were normalized to the maximal adenylyl cyclase activity obtained in the presence of 100 nM VIP. A representative experiment is shown. Basal adenylyl cyclase activity was 5.63 and 5.18, and maximally stimulated VIP was 26.61 and 25.28 pmol/mg/min for the N22Q V2R and the N22Q k mutant V2R, respectively. After four experiments the EC50 measured were 120 ± 31 and 132 ± 24 pM for the N22Q V2R and the N22Q k mutant V2R, respectively.
These experiments established that the V2R is N-linked glycosylated in the endoplasmic reticulum and O-glycosylated in the Golgi network, and to our knowledge this is the first time both posttranslational modifications have been identified in a G protein coupled receptor. Our inability to predict whether a protein will undergo this derivatization stems from the absence of an identifiable consensus site, and in that aspect these experiments have not clarified the matter but have suggested that the structure of the peptide backbone plays a role in regulating O-linked glycosylation. Since the 40 kDa band was first observed by treating the fully glycosylated receptor with PNGase F, it is clear that the 5-8 cluster and serine 15 undergo O-glycosylation in the presence of N-linked glycosylation at asparagine 22 in the cell types we have analyzed.
Mutagenesis revealed that O-glycosylation can take place at the serines and threonines clusters present at codons 5 to 8, 15, and 21 to 24. Substitution of serines at positions 21, 23, and 24 had a profound effect on the migration of the receptor protein. A weak band normally detected migrating faster than the immature receptor became much stronger at the expense of the 40 kDa band as shown in Figure
Substitution at the 5 to 8 amino acids cluster (Figure
Substitution of as many as 9 of the 40 amino acids that constitute the extracellular amino terminus of the V2R failed to reveal the identity of a segment required for targeting the nascent protein to the endoplasmic reticulum. These data and the ability of the first seventy amino acids of the V2R to mediate localization to the endoplasmic reticulum (Schuelein et al., 1996), suggest that the first transmembrane region itself may be the translocation signal. Neither the level of expression of the receptor nor its function were altered by eliminating the glycosylation sites; thus, one must conclude that contrary to what has been observed with other proteins, sugars are not required for proper folding of the V2R likely because the seven segments traversing the plasma membrane determine the folding and topology of this protein.
Our findings indicate that G protein coupled receptors can be substrates for O-linked glycosylation, and explain the significant alteration in protein migration detected in the V2R once the polypeptide chain acquired the minimum length that allowed exit from the endoplasmic reticulum, and passage to the Golgi network (Sadeghi et al., 1997b), where O-glycosylation takes place (Roettger et al., 1998). Mutagenesis of the amino terminal segment revealed that there is no specific signal for O-glycosylation since the disappearance of some sites promoted the glycosylation of other sites, although at a diminished level, and demonstrated the feasibility of the O-Glyco data base to predict O-glycosylation sites in G protein coupled receptors. Materials
Dulbecco-modified Eagle medium (DMEM) and methionine/cysteine free DMEM were from ICN, Costa Mesa, CA; Hanks buffered salt solution (HBSS), Dulbecco's PBS (D-PBS), penicillin/streptomycin, 0.5% trypsin/5 mM EDTA, Geneticin (G-418), and fetal bovine serum (FBS) were from GIBCO, Grand Island, NY; cell culture plasticware was from COSTAR, Cambridge, MA; arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), isobutylmethylxanthine (IBMX), and phenyl-N-acetyl-[alpha]-d-galactosaminide were from Sigma, St. Louis, MO. Forskolin was from Calbiochem, San Diego, CA. [3H]Arginine vasopressin, specific activity 60-80 Ci/mmol, 35S-Express Protein Labeling Mix, specific activity >1000 Ci/mmol, and [[alpha]-32P]ATP, specific activity 3000 Ci/mmol, were purchased from New England Nuclear, Boston, Mass; Amplify® was purchased from Amersham, Arlington Heights, IL; [3H]cyclic 3[prime],5[prime]-AMP was from ICN Biochemicals, Irvine, CA. PN-Glycosidase F, O-glycosidase, N-acetylgalactosaminidase, and sialidase were from Boehringer Mannheim, Indianapolis, IN. Construction of mutant V2 vasopressin receptors
The mutations at the amino terminus were introduced into the V2R by synthesizing oligonucleotides encoding the desired mutagenized segments and ligating them into the NheI site at codon 36 (Birnbaumer et al., 1992). The resulting constructs were sequenced fully by the dideoxy chain termination method of Sanger et al. (1977). For expression in eucaryotic cells the cDNAs bearing the mutations were cloned into the expression vector pcDNA3 (Invitrogen, Boston, MA). Cell culture
MDCK cells were grown in MEM with Earle's salts containing 10% heat-inactivated FBS, penicillin (50 units/ml) and streptomycin (50 mg/ml). HEK 293 cells were grown in DMEM-high glucose, supplemented with 10% heat-inactivated FBS, penicillin (50 U/ml), and streptomycin (50 µg/µl). Stable expression in HEK 293 and MDCK cells
The cells, kept subconfluent, were transfected by the calcium phosphate precipitation technique of Graham and van der Eb (1973). Cell plating, transfection, and selection were performed as described previously (Innamorati et al., 1997). Cells were maintained with the selection medium containing 400 µg/µl G-418. Transient expression in cells
HEK 293 cells, kept below 75% confluence, were plated at a density of 2.8 × 106 cells per 100 mm dish, respectively, and transfected the following day by a modification of the method of Luthman and Magnusson (1983). Briefly, cells were transfected by replacing the growth medium with 6.7 ml of a mixture of 100 mM chloroquine and 0.25 mg/ml diethylamine-ethyl-dextran in 10% FBS-DMEM containing 2 µg of plasmid DNA. After 2 h at 37°C, the solution was removed and the cells were treated for 1 min with 10% dimethyl sulfoxide in PBS. After rinsing twice with PBS and adding growth medium, the cells were returned to the 37°C. Inhibition of glycosylation
Thirty-six hours after transfection the cells were treated overnight with 1.0 mM phenyl-N-acetyl-[alpha]-d-galactosaminide, an inhibitor of glycosylation. The control plate was treated with vehicle alone (DMSO) (Kuan et al., 1989). The following day the cells were subjected to metabolic labeling as described below. The inhibitor was maintained at the same concentration throughout the experiment. Metabolic labeling with 35S-methionine/35S-cysteine and immunoprecipitation of V2R
Proteins were labeled in 100 mm dishes by a modification of the method published by Keefer and Limbird (1993), 48 h following transfection. After 1 h in methionine/cysteine-free DMEM HG, cells were labeled for 2 h with 2 ml of the same medium containing 5% FBS and 100 µCi of 35S-Express Protein Labeling Mix per plate. After returning to DMEM plus 10% FCS for an additional 2 h (chase), cells were rinsed, and harvested in D-PBS. The cell pellet from each plate was homogenized in 500 µl RIPA buffer (150 mM NaCl, 50 mM Tris·HCl pH 8.0, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS containing protease inhibitors: 0.1 mM PMSF, 1 µg/µl soybean trypsin inhibitor, 0.5 µg/µl leupeptine) by drawing the cells through needles of decreasing gauge (20-25 G) fitted to a 1 ml plastic syringe. Cell extracts were clarified by mixing with 50 µl of a 50% slurry of prewashed Protein A-Sepharose. Prewashed Protein A-Sepharose was prepared by incubating the resin with 25 mg/ml bovine serum albumin (BSA) in RIPA buffer for 1 h, followed by two washes with RIPA buffer alone. The clarified extracts were incubated overnight at 4°C with 9 µg/µl of a peptide purified rabbit polyclonal antibodies raised against a peptide corresponding to the carboxyl terminus (Antibody #3) of the human V2R (Innamorati et al., 1997). The antigen/antibody complexes were then separated by incubating the mixture with prewashed Protein A-Sepharose for 2 h at the same temperature. The beads were centrifuged, incubated three times for 4 min at room temperature with RIPA buffer, and recovered each time by centrifugation. Glycosidase treatments were performed on receptor immobilized on Protein A-Sepharose, proteins were then eluted as usual for 20 min at room temperature with 80 µl of 2× Laemmli buffer containing 10% [beta]-mercaptoethanol. The samples were electrophoresed in 10% polyacrylamide gels and visualized by treating the gel with Amplify®, and exposing the dried gels to Kodak X-Omat film at -70°C. Immunoblot of cell membranes
Transfected cells were washed twice with Dulbecco-PBS containing Ca2+ and Mg2+, harvested with a rubber policeman in the same solution, and collected by centrifugation. The cells were lysed in 20 mM HEPES/1 mM EDTA with 20 strokes of a tight-fitting pestle in a Dounce homogenizer. Unbroken cells and nuclei were separated by centrifugation at 2000 × g at 4°C. The supernatant was centrifuged at 12,000 × g and the pellet collected. This crude membrane preparation was resuspended by pipetting the sample several times in 20 µl of the appropriate buffer. Protein concentration was determined by the method of Bradford and 10 µg aliquots of the membranes were subjected to the treatments indicated in the text. After the treatments the buffers were removed, the membrane samples dissolved in Laemmli buffer and subjected to SDS-PAGE, and the proteins transferred onto a nitrocellulose membrane. After 1 h treatment with 4% fat-free powdered milk in TBS, 10 µg/µl of Antibody 3 in the same solution was added and incubated with the membrane for 3 h at room temperature. After three washes with TBS containing 0.2% Tween 20, the membrane was incubated for 2 h with anti-rabbit IgG coupled to horseradish peroxidase diluted 1:2000 in 5% milk/TBS followed by three washes with TBS/0.2% Tween 20. The position of the receptor band was identified by the ECL reaction. Hormone binding to intact cells
Cells were plated in 12-well plates at a density of 1.0-2.0 × 105 cells /well. Binding assays were performed the following day. Cells were washed twice with ice-cold D-PBS after which each well received 0.5 ml of ice cold D-PBS with 2% BSA and the appropriate dilution of [3H]AVP. Plates were incubated for 2 h on top of crushed ice in the cold room before removal of the binding mixture by aspiration. After quickly rinsing twice with ice-cold D-PBS, 0.5 ml of 0.1 N NaOH was added to each well to extract bound radioactivity. After 30 min at 37°C, the fluid from the wells was transferred to scintillation vials containing 3.5 ml of ULTIMA-FLO M (Packard, Meriden, CT) scintillation fluid for radioassay. Nonspecific binding was determined under the same conditions in the presence of 10 µM unlabeled AVP. Replicate plated wells were trypsinized and their cell content determined to normalize the results as binding sites per cell. Binding experiments were performed at least three times, the data are reported as mean ± the standard error of the mean (SEM). Adenylyl cyclase activity in cell homogenates
Adenylyl cyclase activity was assayed as described previously (Innamorati et al., 1997). The medium contained in a final volume of 50 µl 0.1 mM [[alpha]-32P]ATP (1-5 × 106 c.p.m.), 1.6 mM MgCl2, 10 µM GTP, 1 mM EDTA, 1 mM [3H]cAMP (~10,000 c.p.m.), 2 mM isobutylmethylxanthine (IBMX), a nucleoside triphosphate regenerating system composed of 20 mM creatine phosphate, 0.2 mg/ml (2000 U/mg) creatine phosphokinase, 0.02 mg/ml myokinase (448 U/mg), and 25 mM Tris-HCl, pH 7.4. Incubations were at 32°C for 20 min. Hormones (diluted in 1% BSA) were present at the concentrations indicated on the figures. Reactions were stopped by the addition of 100 µl of a solution containing 40 mM ATP, 10 mM cAMP, and 1% sodium dodecylsulfate. The cAMP formed was isolated by a modification of the standard double chromatography over Dowex-50 and alumina columns (Salomon et al., 1974; Bockaert et al., 1976).
Under these assay conditions, cAMP accumulations were linear with time of incubation for up to 40 min and proportional to the amount of homogenate. The activities were expressed as percent maximal VIP stimulation. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. The experiments were performed at least three times. Prediction of O-glycosylated sites
The predicted amino acid sequence of the human V2R was submitted to the database of O-glycosylated proteins at the Department of Biotechnology, The Technical University of Denmark, Building 206, DK-2800 Lyngby, Denmark (Oglyc-Base, Hansen et al., 1997), to obtain a prediction of the most likely sites for O-glycosylation present in this protein.
This work was supported in part by NIH Grant DK 41-244 to M.B.
Discussion
Materials and methods
Acknowledgments
References
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