An X-Linked NDI Mutation Reveals a Requirement for Cell Surface V2R Expression

Hamid M. Sadeghi, Giulio Innamorati and Mariel Birnbaumer

Department of Anesthesiology and Molecular Biology Institute, University of California Los Angeles School of Medicine, Los Angeles, California 90095


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Function and biochemical properties of the V2 vasopressin receptor (V2R) mutant R337ter, identified in patients suffering from X-linked recessive nephrogenic diabetes insipidus, were investigated by expression in COS.M6 or HEK293 cells. Binding assays and measurements of adenylyl cyclase activity failed to detect function for the truncated receptor, although metabolic labeling demonstrated normal levels of protein synthesis. ELISA assays performed on cells expressing the receptors tagged at the amino terminus with the HA epitope failed to detect V2R R337ter on the plasma membrane. Treatment with endoglycosidase H revealed that the receptor was present only as a precursor form because the mature R337ter V2R, resistant to endoglycosidase H treatment, was not detected. The precursor of V2R-R337ter had a longer half-life than that of the wild type V2R, suggesting that arrested maturation may slow the degradation of the precursor. Unrelated experiments had demonstrated that V2R-G345ter, containing eight additional amino acids, was expressed on the plasma membrane and functioned normally. Receptor truncations longer than 337ter revealed that four of the eight amino acids identified initially provided the minimum length required for the protein to acquire cell surface expression. This was shown by the production of mature receptor (V2R-341ter) detectable in SDS-PAGE, which mediated arginine vasopressin stimulation of adenylyl cyclase activity and bound ligand. In addition, the identity of amino acid 340 was found to play a role in this phenomenon. In conclusion, these data demonstrate that the V2R R337ter is nonfunctional because it does not reach the plasma membrane and that the minimal protein length required for translocation of the V2R to the cell surface is sufficient to confer function to the receptor protein. They also suggest the existence of a protein quality control in the endoplasmic reticulum independent of glycosylation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Patients suffering from X-linked recessive nephrogenic diabetes insipidus experience excessive water loss through the kidney due to improper V2 vasopressin receptor (V2R) function. Many mutations of the V2R have been reported in these patients, including one that encodes a receptor truncated at position 337 (R337ter) (1). This mutant receptor is missing the last 35 amino acids of the carboxyl terminus, truncated shortly after the seventh transmembrane region. This segment of G protein-coupled receptors has been shown to play an important role on cell surface expression, G protein coupling, phosphorylation, and desensitization. Truncation of the carboxyl terminus close to the seven-transmembrane region results in loss of plasma membrane expression of the human CG (hCG) and glucagon receptors (2, 3), but there is no rule about the length of the carboxyl terminus required for cell surface expression of receptors of this superfamily. Part of the uncertainty stems from the inability to identify which amino acids define the boundaries of transmembrane region VII. It has been suggested that folding of the receptor may be affected by a truncation in this location (4). For other receptors of this superfamily, expression is not affected, but truncation of the carboxyl terminus diminishes agonist-dependent phosphorylation and desensitization, as shown by Lattion et al. (5) for the {alpha}1B-adrenoceptors. The impact of receptor truncations in signaling through G proteins is variable. Truncation of the carboxyl terminus of the AT1A receptor results in loss of coupling to Gq (6), while, interestingly, truncation of the carboxyl terminus of the THR receptor produces a constitutively active receptor (7).

In the present study, the function and biochemical properties of the truncated V2R R337ter were investigated. Our results suggested that this truncation yields a receptor protein that remains as a precursor form and does not reach the plasma membrane. We proved that the addition of four amino acids downstream of arginine 337 was sufficient to produce maturation of the V2R and appearance of a functional receptor on the cell surface. These results suggest the existence of chaperones that retain improperly folded proteins in the endoplasmic reticulum independent of glycosylation (8).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations identified in patients suffering from X-linked nephrogenic diabetes insipidus explain the existence of nonfunctional forms of the V2R. In the present study, the properties that rendered the V2R-R337ter nonfunctional were investigated by expressing the protein in transiently transfected cells. The location of this truncation is illustrated in Fig. 1Go. As shown in Fig. 2Go and Table 1Go, neither vasopressin-induced stimulation of adenylyl cyclase activity nor [3H]arginine vasopressin (AVP) binding could be detected in HEK293T or COS.M6 cells transfected transiently with the V2R-R337ter cDNA. This was the first mutant V2R completely negative in terms of ligand-binding activity. The amino acid sequence of the V2R is interrupted after the predicted seven-transmembrane segment; therefore we had hypothesized that it was possible for this truncated receptor to bind ligand and were somewhat surprised by this result. It was possible that this segment was crucial to definition of the hormone-binding pocket, or alternatively, the lack of binding might have indicated absence of the receptor from the cell surface. The stability of the mRNA encoding the R337ter was evaluated because single base mutations may shorten RNA stability. The RNA containing the mutation had an abundance similar to that of the wild type mRNA (data not shown). To examine whether this truncation was missing some amino acids crucial for expression, we used two approaches. One was to prepare a longer truncated receptor, the V2R-G345ter, and the other was to add the amino-terminal portion of the V1a vasopressin receptor from amino acid 328 of the V2R. As illustrated in Fig. 1Go, the V2R-G345ter construct encodes an additional eight amino acids, including the palmitoylated cysteines at codons 341/342. The subsequent experiments were performed in parallel with the three cDNAs: full length, R337ter, and G345ter. All constructs were assayed for expression, binding affinity to AVP in whole cells, and the ability to mediate AVP stimulation of adenylyl cyclase activity.



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Figure 1. Carboxy Terminus of the Human V2R

Schematic presentation of the carboxy terminus of the human V2R. The receptor contains 371 amino acids and the predicted carboxy terminus starts with amino acid 328. Palmitoylation site cysteines at codons 341/342 are shown (CC); the G345ter and R337ter V2Rs shown have termination codons in place of Gly 345 and Arg 337, respectively.

 


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Figure 2. Stimulation of Adenylyl Cyclase Activity by the Truncated V2Rs

AVP-induced stimulation of adenylyl cyclase activity was assayed in homogenates of transiently transfected HEK293T cells expressing the wild type and truncated V2Rs. The results are expressed as percent maximal stimulation obtained with 100 nM vasoactive intestinal peptide (VIP). Adenylyl cyclase activities were: basal, 11.0, 9.0, and 4.0 pmol/mg/min; VIP-stimulated, 50.0, 66.0, and 48.0 pmol/mg/min for the wild type, G345ter, and R337ter receptors, respectively.

 

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Table 1. Representative ELISA Assays Performed in COS.M6 Cells Transiently Transfected with the Wild Type (WT) or Truncated V2 Vasopressin Receptors Tagged with the HA Epitope at the Amino Terminus [HA (N)] as described in Materials and Methods

 
To assay for the presence of receptor proteins on the cell surface, cDNAs encoding the wild type and both truncated V2Rs, tagged with the HA epitope at their amino terminus, were transfected into COS.M6 cells, and the presence of the protein on the cell surface was determined by whole-cell ELISA assay utilizing the 12CA5 monoclonal antibody that interacts specifically with the HA epitope. As shown by the data in Table 1Go, there was no detectable HA epitope attached to the V2R-R337ter on the cell surface, whereas the epitope attached to the full-length and G345ter V2R was measurable and provided values proportional to their ability to bind AVP. Ligand-binding affinities for the wild type and G345ter were similar (data not shown), and as seen in Fig. 2Go, both were able to mediate full stimulation of adenylyl cyclase activity by the hormone.

Synthesis of the epitope-tagged wild type and truncated proteins in transiently transfected COS.M6 cells was examined by metabolic labeling with [35S]methionine/cysteine and immunoprecipitation with the 12CA5 monoclonal antibody. To facilitate visualization of the radioactive bands, the cDNAs were also mutagenized at codon 22 to eliminate the single glycosylation site of the receptor (9). We have previously described that metabolic labeling of transiently transfected cells expressing the human V2R yields a predominant precursor receptor form and a less abundant mature form, both detectable in SDS-PAGE (9). As illustrated in Fig. 3AGo, the three cDNA constructs produced comparable quantities of immature receptor that, as expected, migrated faster than predicted by its molecular mass. The R337ter and G345ter V2R cDNAs produced immature receptors that migrated approximately at 27 kDa, while the wild type receptor produced the expected 33 kDa corresponding to the nonglycosylated immature receptor. In agreement with the binding data, mature receptor forms were detected only for the wild type and G345ter V2R. The bands of mature receptor protein are identified by the arrows at 40 and 35 kDa for the nonglycosylated full-length and the G345ter V2R, respectively. Longer exposure of the gel failed to produce any significant signal corresponding to the mature receptor for the V2R-R337ter (data not shown). These data indicated that the lack of function of this truncation was due to its absence from the plasma membrane, rather than a consequence of reduced mRNA levels or of decreased translation of the mRNA encoding the receptor. The absence of the receptor from the cell surface can be ascribed to posttranslational events, such as the refolding of the protein and its translocation from the endoplasmic reticulum (ER) to the plasma membrane. The incomplete transit of the mutant receptor through the ER and the Golgi apparatus was demonstrated by the sensitivity to endoglycosidase H of the glycosylated V2R-R337ter shown in Fig. 3BGo. This is in contrast with the results obtained with the mature wild type V2R, which migrates as a broad band at 45–55 kDa. This migration is caused by the presence of a sugar moiety that is insensitive to treatment with endoglycosidase H and is evidence of having completed passage across the Golgi network (9). As expected, after treatment with peptide-N-glycosidase F the wild type V2R migrates as a smaller sharp band of 40 kDa, similar to that seen with the nonglycosylated receptor shown in Fig. 3AGo. Thus, the data suggested that although the receptor protein was synthesized in sufficient quantities, truncation at codon 337 inhibited maturation of the V2R. This refolding or maturation process is likely to require the presence of more amino acids after transmembrane VII than those found in this truncated protein. Similar to what has been described for the calnexin/calreticulin system, one could postulate the existence of a protein quality control system in the ER that inhibits the exit of proteins that contain bulky hydrophobic groups on their surface. These groups become buried when these proteins achieve their final mature conformation.



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Figure 3. Metabolic Labeling and Immunoprecipitation of V2R

Panel A, Metabolic labeling with [35S]methionine/cysteine and immunoprecipitation of the full-length and truncated V2Rs expressed in COS.M6 cells were performed as described in Materials and Methods. To facilitate visualization of the bands corresponding to the mature receptor, cDNA encoding the nonglycosylated form of the receptor was used for each construct. Proteins were eluted in 2x Laemmli sample buffer and analyzed by SDS-PAGE followed by fluorography and exposure to Kodak film for 48 h. The arrows point to the mature receptors detected in cells expressing the full length and the G345ter V2R. Panel B, Sensitivity of the glycosylated truncated V2R-R337ter to endoglycosidase H. After metabolic labeling and immunoprecipitation, the receptor protein was eluted with 80 µl of 100 µM peptide in RIPA buffer, and treated with 1 mU endoglycosidase H for 1 h at room temperature. After addition of an equal volume of 2x Laemmli sample buffer, the proteins were analyzed by SDS-PAGE. Fluorography of the samples was performed as described in Materials and Methods, and the dried gel was exposed to Kodak X-Omat 5 film for 48 h at -70 C. C identifies the extract from untransfected COS.M6 cells subjected to the same procedure.

 
Replacement of the carboxyl terminus of the V2R after transmembrane VII by the similar segment of the V1a vasopressin receptor restored expression on the cell surface as well as AVP binding and stimulation of adenylyl cyclase activity (G. Innamorati and M. Birnbaumer, unpublished) in agreement with the data reported by Liu and Wess (10). These data suggested that there was no specific sequence requirement for refolding to take place because, other than the presence of the putative palmitoylation sites, the composition of this segment is quite different between the two receptors. Since the G345ter truncation of the V2R showed ligand-binding affinity and G protein coupling comparable to the those of the wild type receptor, it is apparent that these functions of the receptor are not influenced by the last 27 amino acid residues of the V2R, in agreement with the data obtained with the V2R\/V1a chimera.

It is not likely that the observed lack of maturation of the V2R-R337ter was due to the absence of the palmitoylation sites. Our data regarding V2R palmitoylation (H. M. Sadeghi, G. Innamorati, M. Dagarag, and M. Birnbaumer, submitted) demonstrated that elimination of palmitoylation sites in full-length and truncated proteins decreased, but did not eliminate, maturation and cell surface expression of the receptor. Truncated receptors of this superfamily that do not contain palmitoylation sites have been observed on the plasma membrane (6, 11, 12). Osawa and Weiss (4) reported a rhodopsin receptor truncated upstream from the palmitoylation sites that was expressed in the plasma membrane. While deletion of an additional five amino acids resulted in total loss of cell surface expression, replacement of each one of these five amino acids by alanine did not eliminate cell surface expression, proving that the effect was not due to the identity of the amino acids but to the length of the segment. Total deletion of the carboxy terminus resulted in loss of cell surface expression of EtA, PTH/PTH-related peptide, and neurokinin-2 receptors (13, 14, 15), suggesting that a portion of the C terminus is required for proper folding of these receptor proteins. The difference in migration in SDS-PAGE observed for the precursor and the mature nonglycosylated V2R (Fig. 3AGo) should be ascribed to differences in folding of the peptide backbone since it is independent of N-linked glycosylation.

In COS.M6 cells, the wild type V2R produced predominantly the immature form rather that the mature receptor. In the case of V2R-R337ter, only the immature receptor was produced. Determination of the half-life of the immature V2R-R337ter protein, illustrated in Fig. 4Go, revealed that this receptor form is degraded slower that the wild type precursor. The immature wild type V2R had a t1/2 of 3.0 ± 0.3 h, whereas the t1/2 of the immature R337ter V2R was 8.5 ± 1.4 h (n = 3). The existence of a proofreading system in the ER has been suggested as a mechanism that starts the degradation of improperly folded proteins, as in the case of mutant forms of the cystic fibrosis conductance transmembrane regulator (16). If the immature receptors were available for degradation at the ER, it is likely that one would observe no difference in half-life between the wild type and the mutant receptors. The results suggested that degradation of the immature receptor takes place after the protein has exited the ER, and that the transit through this compartment is slower for the mutant precursor. Because the half-life of the immature wild type V2R in stably transfected cells is short, approximately 20 min (17), in transiently transfected cells overproduction of immature receptor protein most likely attenuates the rate of processing and degradation of this receptor form.



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Figure 4. Half-Life of V2R in Transiently Transfected COS.M6 Cells

A representative experiment showing the pattern obtained with wild type and R337ter V2R after 1 h metabolic labeling with [35S]methionine/cysteine, in the subsequent chase period in transiently transfected COS.M6 cells. Complementary DNAs encoding the nonglycosylated form of the receptor were transfected to facilitate visualization and quantitation of the mature form of V2R. The proteins were immunoprecipitated, eluted with 2x sample buffer, and analyzed by SDS-PAGE, and the dried gel was exposed for 12 h to a Kodak film after it was soaked in Amplify. A faint band of mature receptor is visible only in the extracts from cells expressing the full-length protein. The half-life of the immature form of the wild type and R337ter were 3.0 ± 0.3 and 8.5 ± 1.4 h, respectively (n = 3). The data are presented as the mean ± SEM.

 
We concluded that the receptor truncated at codon 337, although containing all seven transmembrane regions, failed to complete the refolding process that produces mature V2R from its precursor form. To define the minimum length of this segment required for proper protein maturation, a series of truncated receptors progressively longer than R337ter were expressed. As is summarized in Table 2Go, we found that a minimum length of 340 amino acids was required to obtain a functional receptor localized to the cell surface. Figure 5AGo illustrates the appearance of a detectable mature band of receptor protein when the length of the protein changed from 339 to 340 amino acids. As shown in Fig. 5BGo, addition of amino acid 341 enhanced significantly the amount of mature receptor detected. Expression of the receptor protein containing cysteine (wild type) or serine at position 341 revealed that this enhancement was not caused by the appearance of an acceptor site for palmitoylation. The data in Fig. 5Go also illustrate that whereas no difference in the migration of the mature forms containing 340 or 345 amino acids was detected, the appearance of amino acid 340 promoted a conformational change in the protein that was detectable under the denaturing conditions of SDS-PAGE. This conformational change was concomitant with the detection of ligand binding and coupling activity. We were unable to conclude whether the immature receptor forms trapped inside the cell possess AVP-binding activity. The high level of nonspecific binding and the unfavorable specific activity of the tritiated ligand prevented the detection of binding in detergent extracts of cells expressing the wild type receptor, thus precluding our attempts to identify the binding activity of the truncated immature forms.


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Table 2. Summary of the Correlation between the Number and Identity of the Amino Acids That Follow Arginine 337 and the Appearance of a Mature Functional Truncated V2 Vasopressin Receptor

 


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Figure 5. Minimum Length Requirement for Receptor Maturation

Metabolic labeling with [35S]methionine/cysteine, immunoprecipitation of the full-length and truncated V2Rs expressed in COS.M6 cells, and analysis of the samples were performed as described for Fig. 3Go. Panel A, The arrows point to the mature receptors detected in cells expressing the truncated V2Rs. Although the dried gel was exposed to film for 48 h at -70 C, no mature bands were detected in lanes containing proteins shorter than 340 amino acids. Panel B, The arrows point to the mature receptors detected in cells expressing the full-length (372 amino acids) and the truncated V2Rs. The dried gel was exposed to film for 12 h at -70 C. The abundance of the V2R342t containing either cysteine or serine as amino acid 341 is illustrated.

 
Figure 6Go illustrates the acquisition of receptor function by the addition of amino acids 340 and 341. Due to the low number of binding sites detected in intact cells, the appearance of receptor-mediated stimulation of adenylyl cyclase activity was the most sensitive assay for the presence of an active receptor. Addition of amino acid 341, either a cysteine or serine, resulted in higher receptor numbers per cell and, as a consequence, significantly higher levels of stimulation of adenylyl cyclase activity. The maximal activity observed and the EC50 values were very close to those of the G345ter truncated receptor. The increase in receptor number associated with the presence of cysteine 341 suggested that palmitoylation plays a role in yielding higher number of receptors per cell, but it is not required to obtain a functional V2R. Figure 7Go illustrates the results revealing that the identity of amino acid 340 modifies the ability of the receptor to undergo this final transition. Leucine 340 was substituted by alanine, cysteine, or histidine in V2R341ter. When tested for function, it was found that leucine and cysteine in that position sustained the formation of an active receptor protein, with cysteine enhancing receptor abundance, while histidine and alanine impaired this phenomenon. These results were matched by the ability of each construct to produce detectable amounts of mature receptor, as assessed by metabolic labeling, and suggest a possible requirement for hydrophobicity at this position, provided either by leucine or perhaps by palmitoylation of the cysteine.



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Figure 6. Minimum Length Requirement for Receptor Maturation

Adenylyl cyclase activity was determined as described in Materials and Methods in homogenates of HEK 293 cells expressing transiently the truncated and full-length receptor proteins. AVP stimulation of adenylyl cyclase activity was determined in homogenate of cells expressing the truncated V2Rs. A parallel experiment measured for each construct the number of receptors per cell expressed in this transfection. The values were: 340ter, not detectable; 341ter, 0.2 x 105; 342ter, 6.8 x 105; 342ter/341S, 2.3 x 105; and 345ter, 15.0 x 105 receptors per cell. Basal and 100 nM VIP-stimulated adenylyl cyclase activity in the homogenates tested were: 1.3 ± 0.1 and 27.3 ± 0.6 pmol/min/mg cAMP accumulated.

 


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Figure 7. V2R Maturation and the Identity of Amino Acid 340

Homogenate of cells expressing V2R341ter bearing different amino acids at position 340 were tested for their ability to mediate AVP stimulation of adenylyl cyclase activity and compared with the activity of 345ter. A parallel experiment measured the number of receptors per cell expressed by each construct in this transfection. The values were: 340L, 0.4 x 105; 340A, not detectable; 340H, not detectable; 340C, 1.5 x 105; and 345ter, 15 x 105 receptors per cell. Basal and 100 nM VIP-stimulated adenylyl cyclase activity in the homogenates tested were: 1.5 ± 0.1 and 25.4 ± 0.9 pmol/min/mg cAMP accumulated.

 
In summary, we established for the V2R the minimum length of the peptide chain after transmembrane VII that is required for this protein to achieve its mature conformation. This transformation was detected as a change in migration in SDS-PAGE and the simultaneous appearance of a functional receptor on the cell surface. These data point to the existence in the ER of a quality control process for proteins that is distinct from the calnexin/calreticulin system, which depends on protein glycosylation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
High-glucose DMEM (DMEM-HG), HBSS, Dulbecco’ PBS (D-PBS), penicillin/streptomycin, 0.5% trypsin/5 mM EDTA, and FBS were from GIBCO (Grand Island, NY); methionine/cysteine-free DMEM was from ICN (Costa Mesa, CA); cell culture plasticware was from COSTAR (Cambridge, MA); AVP, (-) isoproterenol, and isobutylmethylxanthine were from Sigma (St. Louis, MO); forskolin was from Calbiochem (San Diego, CA); [3H]arginine vasopressin, specific activity 60–80 Ci/mmol, and EXPRE35S35S-Express Protein Labeling Mix, specific activity >1,000 Ci/mmol were from Du Pont-New England Nuclear (Boston, MA); [3H]cyclic 3',5'-AMP was from ICN Biochemicals (Irvine, CA); Amplify was from Amersham (Arlington Heights, IL). All other reagents were from Sigma, St.Louis, MO.

Construction of Mutant V2Rs
All cDNA mutations of the V2R, as well as the addition of the sequence encoding the HA epitope (YPYDVPDYA) at the N or C terminus of the wild type and mutant V2Rs, were introduced into the human V2R cDNA using a PCR-based approach (18). Stop codons were introduced at amino acids 337 and 345 to encode V2R R337ter and G345ter, respectively. The resulting cDNA constructs were sequenced fully by the dideoxy chain termination method of Sanger et al. (19) and cloned into the expression vector pcDNA3 (Invitrogen, Boston, MA) for expression in eukaryotic cells.

Cell Culture and Expression in Cells
COS.M6 cells were grown in DMEM-HG, supplemented with 10% heat-inactivated FBS, penicillin (50 U/ml), and streptomycin (50 µg/ml). For transient transfection, COS.M6 cells, kept below 75% confluence, were plated at a density of 0.5 x 106 cells per 100-mm dish and transfected the following day by a modification of the method of Luthman and Magnusson (20). Briefly, after rinsing with HBSS, each dish received 800 µl of HBSS, pH 7.05, containing 3 µg plasmid DNA mixed with 0.5 mg/ml diethylaminoethyl-Dextran. After 20 min at room temperature, 100 µM Chloroquine in DMEM containing 2% FBS was added. After 3 h at 37 C the cells were exposed to 10% dimethyl sulfoxide in HBSS for 2 min, rinsed twice with DMEM-HG without additives, and returned to growth medium at 37 C.

Metabolic Labeling with [35S]Methionine/Cysteine and Immunoprecipitation
Proteins were labeled in 100-mm dishes by a modification of the method published by Keefer and Limbird (21). Forty eight hours after transfection, cells were starved for 1 h in methionine/cysteine-free DMEM and then labeled for 1 or 2 h with 2 ml of the same medium containing 100 µCi of EXPRE35S35S-Express Protein Labeling Mix/plate. After the medium was removed, 5 ml DMEM-HG with 10% FBS were added, and the cells were incubated at 37 C for the times indicated in the text (chase period). Cells were then rinsed, washed twice with ice-cold D-PBS, scraped from the plate, and collected by centrifugation. The cell pellet from each plate was disrupted 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 phenylmethylsulfonylfluoride, 1 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin). Homogenization was achieved by drawing the cells through needles of decreasing gauge (20G, 25G) fitted into a 3-ml plastic syringe. Cell extracts were then clarified by mixing them with 50 µl of a 50% slurry of prewashed Protein A-Sepharose in the same buffer. Prewashed Protein A-Sepharose was prepared by addition of 1.0 ml of 25 mg/ml BSA in RIPA buffer and mixing for 1 h, followed by two washes with RIPA buffer alone. For immunoprecipitation, an antibody raised against a portion of third intracellular loop of human V2R (AntiV2#2, peptide VPGPSERPGGRRRGR) was added to the clarified extracts at a concentration of 10 µg/ml and incubated overnight at 4 C. 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 and washed three times for 4 min on ice with RIPA buffer. The samples were then eluted using 80 µl of 2x sample buffer with 10% ß-mercaptoethanol. For endoglycosidase H treatment, samples were eluted with 80 µl of 100 µM peptide 2 in RIPA buffer for 30 min at room temperature and, after addition of 1 mU of the enzyme, the eluates were incubated at room temperature for 1 h. After the samples were mixed with an equal volume of 2x sample buffer containing 10% ß-mercaptoethanol, they were electrophoresed in 10% SDS-polyacrylamide gels. Radioactive bands were visualized by treating the gel with Amplify and exposing the dried gels to Kodak-Xomat film at -70 C for the indicated times. For determination of the relative intensity of the obtained band, densitometric measurements were performed using the Bio-Rad Imaging Densitometer model GS-670 (Bio-Rad, Richmond, CA).

ELISA
COS.M6 cells were transiently transfected with the wild type V2R or the receptor containing the HA epitope at the C terminus, HA(C), or N terminus, HA(N). Twenty four hours after transfection, cells were plated at a density of 5 x 105 cells per well in a polylysine-coated 96-well plate. The next day, the medium was removed, 1 µg 12CA5 antibody/100 µl DMEM containing 10% FBS was added, and the plate was incubated at 37 C for 1 h followed by two rinses with PBS without Ca2+/Mg2+ (PBS w/o). Cells were then fixed with 4% formaldehyde in PBS w/o. Wells were incubated with a 1:2,500 dilution of horseradish peroxidase-conjugated sheep anti-mouse IgG in PBS (HRP-IgG, Amersham, Arlington Heights, IL) for 2 h at 37 C and then rinsed twice with PBS w/o. The enzymatic reaction was carried out using H2O2 as substrate and o-phenylenediamine (2.5 mM in 0.1 M phosphate-citrate buffer, pH 5.0) as substrate. The reaction was stopped with 30 µl of 3 M HCL, and the color was measured at 490 and 650 nm in a microplate reader model Vmax (Molecular Devices Corporation, Sunnyvale, CA).

[3H]AVP Binding to Intact Cells
Twenty four hours after transfection, cells were plated in 24-well plates at a density of 0.5–1.0 x 105 cells per 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 ice cold D-PBS with 2% BSA and 20 nM [3H]AVP in the presence (nonspecific) or absence (total) of 10 µM AVP (18). Plates were incubated for 2 h on 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 radioactivity. After 30 min at 37 C, the fluid from the wells was transferred to scintillation vials containing 3.5 ml of Beckman ULTIMA-FLO M (Packard, Meriden, CT) scintillation fluid for radioassay.

Adenylyl Cyclase Activity in Cell Homogenates
Adenylyl cyclase activity was assayed as previously described (18). The medium contained in a final volume of 50 µl 0.1 mM [{alpha}-32P]ATP (1–5 x 106 cpm), 4 mM MgCl2, 10 µM GTP, 1 mM EDTA, 1 mM [3H]cAMP (~10,000 cpm), 2 mM isobutylmethylxanthine, a nucleoside triphosphate-regenerating system composed of 20 mM creatine phosphate, 0.2 mg/ml (2,000 U/mg) creatine phosphokinase, 0.02 mg/ml myokinase (448 U/mg), and 25 mM Tris-HCl, pH 7.4. Hormones (diluted in 1% BSA) were present at the concentrations indicated in Figs. 2Go, 6Go, and 7Go. Reactions were stopped by the addition of 100 µl of a solution containing 40 mM ATP, 10 mM cAMP, and 1% SDS. The cAMP formed was isolated by a modification (22) of the standard double chromatography method of Salomon et al. (23). 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 picomoles of cAMP formed per min per mg of homogenate protein, or percent maximal vasoactive intestinal peptide (VIP) response. Protein concentration was determined by the method of Lowry et al. (24) using BSA as standard.


    FOOTNOTES
 
Address requests for reprints to: Mariel Birnbaumer, Department of Anesthesiology, University of California Los Angeles Medical Center, BH-612 CHS, 10833 Leconte Avenue, Los Angeles, California 90024-1778.

This work was supported in part by NIH Grant DK 41–244 (to M.B.).

Received for publication September 24, 1996. Revision received January 17, 1997. Accepted for publication January 28, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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