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J Biol Chem, Vol. 275, Issue 4, 2381-2389, January 28, 2000

Structural Implication for Receptor Oligomerization from Functional Reconstitution Studies of Mutant V2 Vasopressin Receptors^*

Angela Schulz, Robert Grosse, Günter Schultz, Thomas Gudermann, and Torsten Schöneberg

From the Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, D-14195 Berlin, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1-3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The G-protein-coupled receptors (GPCRs)¹ constitute the largest protein superfamily found in nature, and conservative estimations based on genome data from Caenorhabditis elegans suggest that about 5% of the human genome encodes for GPCRs (1). Therefore, mutational alteration of GPCR function is reflected in an ever growing number of diseases caused by mutations within GPCR genes. It has been demonstrated that single amino acid substitutions and receptor truncations are responsible for several hereditary and acquired diseases such as retinitis pigmentosa, familial male precocious puberty, hyper- and hypothyroidism, or X-linked nephrogenic diabetes insipidus (2).

Based on findings that GPCRs are composed of multiple folding units (3), we recently demonstrated that functionally inactive V2 vasopressin receptors (V2-Rs) containing clinically relevant mutations in the C-terminal third of the receptor can be functionally rescued by coexpression with a C-terminal V2-R fragment (4, 5). The application of this approach for gene therapy purposes appears promising because reconstitution of receptor function is predicted to occur only in cells where the mutant receptor is expressed endogenously, and, therefore, specific cell targeting is not required. The observed ability of the C-terminal V2-R fragment to interact with different mutant V2-Rs is consistent with several recent reports, suggesting that GPCRs can form dimers. In the last few years, a large body of evidence has evolved demonstrating the formation of homo and heterodimers in class I and III GPCRs (6-15). As shown for epitope-tagged ₂-adrenergic and muscarinic receptors, the association is highly specific for a given receptor subtype giving rise only to homodimers (16, 17). In addition to investigations in transient expression systems, in situ studies with the dopamine D3 receptor and rhodopsin suggest the coexistence of receptor monomers and oligomeric complexes under physiological circumstances (18, 19).

Two structural models of dimer formation have been proposed (20). One dimeric structure, referred to as the "contact dimer," is based on the two-dimensional electron diffraction map of rhodopsin (21). Two tightly packed bundles of seven transmembrane domains (TMDs) are positioned next to each other. The contact interface between the two monomeric receptors is assumed to be located between the lipid-orientated transmembrane receptor portions. The so-called "domain-swapped dimer" has been proposed to explain the reconstitution phenomenon observed with truncated (4, 22, 23) and chimeric GPCRs (24). In this dimer structure, the two receptor molecules fold around a hydrophilic interface by exchanging their N-terminal (TMDs 1-5) and C-terminal (TMDs 6-7) folding domains. The domain-swapped dimer model is supported by reconstitution studies with two chimeric ₂-adrenergic and m3 muscarinic receptors (24).

Based on the domain-swapped dimer model and the functional data from reconstitution experiments, we hypothesized that receptors mutationally altered in the N-terminal folding domain (TMDs 1-5) can be rescued by coexpressing a nonmutated N-terminal receptor fragment. However, all attempts to restore function of mutant V2-Rs failed despite a noncovalent interaction between the two molecules as shown by ELISA and coimmunoprecipitation studies. When the extracellular disulfide bond known to be essential for GPCR function was disrupted by mutation, the function of mutant receptors was restored upon fragment coexpression. The results of our study favor a model in which GPCRs specifically associate by lateral interaction rather than by a domain-swapped mechanism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Mutant V2 Vasopressin Receptors and Plasmid DNAs-- All V2-R mutations (see Fig. 1) were introduced into V2-R-pcDps (4), a mammalian expression vector containing the entire coding sequence of the human V2-R, using a polymerase chain reaction-based site-directed mutagenesis and restriction fragment replacement strategy (25). For immunological detection of the various V2-R constructs, a stretch of nucleotides coding for a nine-amino acid epitope (YPYDVPDYA) (26) derived from the influenza virus hemagglutinin protein (HA tag) was inserted after the initiating Met codon. The wild-type human cholecystokinin type A receptor (CCK_A-R; a generous gift from Dr. S. A. Wank, NIH) was subcloned into pcDps. In addition to the wild-type V2-R (HA-V2-R), all missense-mutated V2-R constructs, the C-terminally truncated V2-Rs, the CCK_A-R (HA-CCK_A-R), and the rat m3 muscarinic receptor (HA-m3-R) (27) were tagged with an N-terminal HA epitope. The identity of the various constructs and the correctness of all polymerase chain reaction-derived sequences were confirmed by restriction analysis and direct DNA sequencing according to standard methodology by using an automated sequencer (Applied Biosystems Inc.).

To monitor the transfection efficiency and for control purposes in ELISA studies, a mammalian expression plasmid (pEGFP-C1 vector, CLONTECH, Palo Alto, CA) for the green fluorescent protein (GFP) was used.

Cell Culture, Transfection, and Functional Assays-- COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified 7% CO₂ incubator. For transient transfection of COS-7 cells, a calcium phosphate coprecipitation method (28) was applied. Thus, cells were split into 12-well plates (2 × 10⁵ cells/well) and transfected with a total amount of 5 µg of plasmid DNA/well. After 48 h cells were prelabeled with 2 µCi/ml of [³H]adenine (31.7 Ci/mmol, NEN Life Science Products) and incubated overnight. For cAMP assay, transfected cells were washed once in serum-free Dulbecco's modified Eagle's medium containing 1 mM 3-isobutyl-1-methylxanthine (Sigma), followed by incubation in the presence of the indicated arginine vasopressin (AVP; Sigma) concentrations for 1 h at 37 °C. Reactions were terminated by aspiration of the medium and addition of 1 ml of 5% trichloric acid. The cAMP content of cell extracts was determined by anion exchange chromatography as described (29).

For radioligand binding studies, cells were harvested 72 h after transfection (20 µg of plasmid DNA/100-mm dish), and saturation binding assays were performed using membrane homogenates. Incubations were carried out for 1 h at 22 °C in a 0.25-ml volume with six different concentrations (1.25-100 nM) of [³H]AVP (64 Ci/mmol; NEN Life Science Products). Nonspecific binding was defined as binding in the presence of 10 µM AVP. Binding data were analyzed by a nonlinear curve-fitting procedure using the computer program GraphPad Prism (GraphPad Software, San Diego, CA).

ELISAs-- To estimate cell surface expression of receptors carrying an N-terminal HA-tag, we developed an indirect cellular ELISA (4), hereafter referred to as "surface ELISA." Briefly, COS-7 cells were seeded into 48-well plates, transfected, fixed without disrupting the cell membrane, and incubated with a biotin-labeled anti-HA monoclonal antibody (12CA5, Roche Molecular Biochemicals). Bound anti-HA antibody was detected with the help of a peroxidase-labeled streptavidin conjugate (Sigma).

To further assess the amounts of full-length HA-tagged V2-Rs and to demonstrate the association of V2-R constructs, a previously developed "sandwich ELISA" was used (5). In brief, 3 days after transfection (12 µg of plasmid DNA/60-mm dish), COS-7 cells were harvested, and cell pellets were resuspended in 150 µl of lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1% desoxycholate, 1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). Cell debris was removed by centrifugation, and supernatants were used for ELISAs. Microtiter plates were coated with a polyclonal rabbit antibody directed against a peptide corresponding to the C-terminal 29 amino acids of the human V2-R (kindly provided by Dr. Paul Goldsmith, NIH; 5 µg/ml in PBS). After incubation at 4 °C for 16 h, plates were blocked with 10% fetal bovine serum in PBS. Cell lysates were added and incubated at 37 °C for 2 h. Plates were washed three times with PBS containing 0.05% Triton X-100 (PBS-T). The biotin-labeled monoclonal anti-HA antibody (12CA5; 1 µg/ml PBS-T) was added, and plates were incubated at 37 °C for 2 h. Plates were washed with PBS-T and incubated with an 1:5,000 dilution of peroxidase-conjugated streptavidin for 1 h at 37 °C. After removal of excess unbound conjugate, H₂O₂ and o-phenylenediamine (2.5 mM each in 0.1 M phosphate-citrate buffer, pH 5.0) were added to serve as substrate and chromogen, respectively. After 15 min the enzyme reaction (carried out at room temperature) was stopped by the addition of 1 M H₂SO₄ containing 0.05 M Na₂SO₃, and color development was measured bichromatically at 492 and 620 nm using an ELISA reader (Titertek Multiskan MCC/340, Flow Laboratories, Inc. McLean, VA).

To compare the expression levels of epitope-tagged receptor constructs including truncated V2-Rs, HA-CCK_A-R, and HA-m3-R an indirect ELISA, referred to as "total ELISA," was developed. Transfected COS-7 cells were harvested and lysed as described above (see sandwich ELISA). Lysates were prediluted 1:4 with PBS, and microtiter plates (MaxiSorp plate; Nunc A/S, Roskilde, Denmark) were coated with 200 µl of receptor lysate/well. After incubation at 4 °C for 16 h, plates were blocked with 10% fetal bovine serum in PBS. Plates were washed three times with PBS-T. Then the biotin-labeled monoclonal anti-HA antibody (12CA5; 10 µg/ml PBS-T) was added, and plates were incubated at 37 °C for 2 h. Bound anti-HA antibody was detected by using a peroxidase-labeled streptavidin conjugate as described above for the sandwich ELISA.

Immunoprecipitation-- COS-7 cells were transfected with various V2-R constructs (12 µg of plasmid DNA/60-mm dish) as described above. About 72 h later, cells were washed twice with PBS and treated with 150 µl of lysis buffer. After vigorous vortexing, followed by removal of cell debris by centrifugation, 150 µl of PBS-T and 20 µg/ml of the anti-V2-R antibody were added to the supernatants containing solubilized receptor protein. Following incubation of samples at 4 °C for 2 h at constant rotation, 60 µl of 10% (w/v) protein A-Sepharose beads (Sigma) were added, and samples were incubated overnight at 4 °C. Sepharose beads were pelleted (12,000 × g for 3 min) and washed twice with 1 ml of washing buffer A (600 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 1% Nonidet P-40) and twice with 1 ml of washing buffer B (300 mM NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.4). Pellets were boiled with 40 µl of SDS sample buffer, and SDS-polyacrylamide gel electrophoresis (10%) was performed. After electrotransfer the nitrocellulose filter was probed with a biotin-labeled anti-HA monoclonal antibody (12CA5, 1 µg/ml in PBS/0.1% Tween 20). The peroxidase-labeled streptavidin conjugate (1:5,000 in PBS/0.1% Tween 20) and the ECL system (Amersham Pharmacia Biotech) were used to detect the bound anti-HA antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutant V2-Rs Cannot Be Rescued by Coexpression of N-terminal Receptor Fragments and Mutated V2-Rs-- We have recently shown that mutant V2-Rs harboring missense or nonsense mutations within the last third (TMDs 6-7) of the receptor molecule can be functionally rescued by supplying a receptor fragment spanning the mutated receptor portion (4). The targeted expression of specific receptor polypeptides may lead to novel strategies in the treatment of diseases caused by inactivating GPCR mutations (5). Because of the "subunit character" of GPCRs, we hypothesized that the function of receptors carrying inactivating mutations within TMDs 1-5 can also be restored by coexpression with N-terminal receptor fragments. In initial experiments, clinically relevant mutant V2-Rs (HA-R137H, HA-S167L) were expressed alone and with a fragment (HA-E242X) truncated within the third intracellular loop (Fig. 1). In accord with the fact that both missense mutations were found in patients with nephrogenic diabetes insipidus, AVP administration resulted only in minor increases in intracellular cAMP levels (about 2-fold) as compared with the wild-type HA-V2-R (Fig. 2A).

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Structure of mutant V2 vasopressin receptors used in this study. The cDNA of V2-R was modified by site-directed mutagenesis (see "Experimental Procedures") to introduce missense mutations (C112A, R137H, S167L, R181C, C192A, and Y280C) at the indicated amino acid positions (upper panel). The lower panel shows the C-terminally truncated (HA-W71X to HA-R337X) and the N-terminally truncated (E242tail and L292tail) receptor fragments. An HA epitope tag was added to the N terminus of the wild-type V2-R (HA-V2-R) and all mutant V2-R constructs except of the tail fragments. The positions of the seven transmembrane domains (TMDs 1-7) are indicated.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Coexpression of mutant V2 vasopressin receptors with E242X. To test the functional consequence of coexpression of selected HA-V2-R mutants with the C-terminally truncated HA-E242X construct, COS-7 cells were transfected and incubated without agonist (A, open bars) or 100 nM AVP (A, filled bars) and increasing AVP concentrations (B). Intracellular cAMP levels were determined in an accumulation assay as described under "Experimental Procedures." Data (B) are presented as a percentage of maximum cAMP response (Emax values; Table II). All data are expressed as the means ± S.E. of two independent experiments, each carried out in triplicate (A) and duplicate (B).

Interestingly, coexpression with HA-E242X did not significantly increase the signaling efficacy of the two mutant V2-Rs. Similar negative results were obtained by coexpression of HA-R137H with a full-length V2-R containing an inactivating mutation (HA-Y280C) in TMD 6 (data not shown). Radioligand binding and ELISA studies that were performed in parallel revealed a dramatic reduction in cell surface expression levels of HA-R137H and HA-S167L (<15% of HA-V2-R) but comparable whole cell expression levels (Table I). Control coexpression experiments with the complementary N- and C-terminal folding domains (HA-E242X + E242tail) showed the expected reconstitution of receptor function (Fig. 2A) (4). We speculated that intracellular retention of the mutant receptors may somehow interfere with functional receptor reconstitution. Therefore, an additional nephrogenic diabetes insipidus-causing missense mutation (R181C) was chosen that is characterized by proper plasma membrane expression but a right-shifted concentration-response curve (Tables I and II and Fig. 2B). However, coexpression of HA-R181C with HA-E242X or HA-Y280C resulted neither in a significant increase in specific AVP-binding sites (data not shown) nor in a shift of the concentration-response curve to lower AVP concentrations comparable with those found with the HA-V2-R or HA-V2-R plus HA-E242X (Fig. 2B and Table II).

V2-R Oligomerization Requires TMDs 3-5 and TMD 6-- Direct interaction between the mutant full-length receptor and the supplied receptor fragment is a necessary prerequisite for functional reconstitution. Because the function of mutant V2-Rs was not restored after coexpression of HA-E242X, sandwich ELISA and immunoprecipitation studies were performed to demonstrate a direct interaction of N-terminal receptor fragments with the full-length V2-R.

To quantify the association of HA-E242X with nontagged full-length V2-Rs, a sandwich ELISA was used (5). Because nontagged V2-Rs contained only the C-terminal epitope and all truncated receptors the N-terminal HA epitope but not the C-terminal epitope, a positive signal in the sandwich ELISA can only be achieved if both proteins form a complex. Lysates from COS-7 cells cotransfected with the full-length HA-V2-R and V2-R (known to form dimers (16) and to guarantee an 1:1 dilution of HA-V2-R) served as positive control in all experiments (Table III). To control the specificity of this interaction, HA-m3-R and HA-CCK_A-R were coexpressed with nontagged V2-R. OD readings less than 25% of the positive control were observed highlighting the specificity of V2-R oligomerization. First, nontagged versions of R137H and R181C were coexpressed with HA-E242X. As shown in Table III, both mutant V2-Rs (R137H and R181C) showed a significant association with HA-E242X, indicating that a missing interaction between the mutant full-length receptors and the supplied fragment was not responsible for the lack of functional reconstitution. Interestingly, R181C displayed a similar ability to interact with HA-E242X as compared with the wild-type V2-R. Transfection of R137H, R181C, V2-R, and HA-E242X alone gave no OD readings in the sandwich ELISA (Table I).

Because the chosen missense mutations did not interfere with fragment/receptor interaction we next attempted to investigate the structural requirement necessary for association by using the wild-type V2-R. Thus, the HA-V2-R was systematically truncated (Fig. 1) and studied in sandwich ELISAs. The HA-tagged R337X construct not containing most of the C terminus of the receptor was coexpressed with nontagged V2-R. OD readings were similar to those obtained with the positive control indicating a strong interaction between both receptors. No significant interactions (OD value, <25% of HA-V2-R) were observed with constructs containing only TMDs 1-2 (HA-W71X and HA-R113X; Table III). However, all fragments encompassing at least TMDs 1-3 significantly (HA-V206X) or even fully (HA-E242X to HA-L312X) interacted with V2-R when compared with HA-R337X (Table III).

To exclude the possibility that the lack of interaction observed with some constructs was due to drastically reduced expression levels, we developed an indirect ELISA to quantify all HA-tagged receptor constructs. Following direct coating of microtiter plates with lysates from transfected COS-7 cells, total cellular expression levels of the various HA-tagged receptor constructs were compared by using a monoclonal anti-HA antibody in an indirect ELISA. All constructs showed similar or slightly increased total cellular expression levels as compared with the HA-V2-R (Table III). This observation was in accord with the band intensities observed in immunoprecipitation studies and Western blot studies (see below and Fig. 3). Only HA-W71X, HA-R113X, and HA-V206X displayed expression levels that were reduced by 20-30% (Table III). In addition, correct membrane insertion of HA-W71X, HA-R113X, HA-E242X, and HA-R337X was studied by an indirect cell surface ELISA. As previously shown for W71X and R337X (30, 31), all truncated mutant V2-Rs were correctly inserted into the plasma membrane, but cell surface expression levels were less than 10% as compared with the HA-V2-R (Table I). Cotransfection of HA-R337X and V2-R did not increase the cell surface amount of HA-R337X (5% of HA-V2-R) as compared with HA-R337X alone (11% of HA-V2-R; Table I).

View larger version (63K): [in this window] [in a new window]   Fig. 3.   Immunoprecipitation of mutant V2 vasopressin receptors. COS-7 cells were transfected with plasmids (12 µg of plasmid DNA/60-mm dish) coding for the various V2-R constructs and the HA-m3-R. All constructs except V2-R and E242tail contained an N-terminal HA epitope. The DNA ratio of cotransfected constructs was 1:1. For immunoprecipitation cell lysates were incubated with an antibody directed against the C-terminal portion of the V2-R. SDS-polyacrylamide gel electrophoresis was conducted under reducing conditions. After electroblotting, HA-tagged constructs were detected with the help of a monoclonal anti-HA antibody (see "Experimental Procedures"). A prominent nonspecific band at 55 kDa, probably the heavy chain of the antibody, was present in all lanes. Positions of molecular mass markers (in kDa) are shown on the left. One experiment out of three with similar results is presented.

To verify the results found with the ELISAs, coimmunoprecipitation and Western blot studies were conducted. Lysates from COS-7 cells cotransfected with the nontagged V2-R and HA-tagged truncated V2-R constructs (HA-E242X and HA-R337X) were incubated with a polyclonal antibody directed against the V2-R C terminus, and immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions. After electroblotting HA-tagged constructs were detected with the help of a monoclonal anti-HA antibody. As shown in Fig. 3, the HA-tagged wild-type V2-R migrated as a broad band ranging from 38 to 42 kDa. SDS-resistant V2-R dimers and oligomers were found at higher molecular ranges (~80 and ~120 kDa). A prominent nonspecific band at 55 kDa, probably the heavy chain of the antibody, was present in all lanes. Because of a deleted C terminus (HA-E242X and HA-R337X) or the absence of an HA tag (V2-R), the constructs HA-E242X, HA-R337X, and V2-R were not detectable in Western blot analysis when expressed alone. Upon coexpression of both truncation mutants with V2-R, an additional band at 29 or 35 kDa representing HA-E242X or HA-R337X, respectively, was detected (Fig. 3). Similar results were obtained using only the C-terminal fragment, E242tail. Interestingly, coexpression experiments with HA-R337X revealed additional bands that correspond in size to putative dimers (~70 kDa) and oligomers (~100 kDa; Fig. 3, seventh and eighth lanes). Based on the calculated molecular mass of HA-R337X and taking into account that coexpression with both full-length V2-R and E242tail gave a similar band pattern, the higher molecular mass forms are likely to represent HA-R337X homodimers and homooligomers on the Western blot. Homodimer formation was not seen with the HA-E242X construct. The HA-R337X homodimers must be associated with the V2-R or E242tail prior to coimmunoprecipitation to be detectable on Western blots. Because the HA-R337X mutant receptor is trafficking-deficient (Table I) (31, 32), HA-R337X dimer formation occurs intracellularly and independently of receptor function or presence of agonist. As shown in Fig. 3, HA-m3-R could not be coimmunoprecipitated with V2-R, indicating the high specificity of V2-R/fragment interaction.

Immunoprecipitation studies indicated that the C-terminal folding unit (E242tail) composed of only TMDs 6-7 showed a strong association with HA-R337X and HA-E242X (Fig. 3). In contrast, an N-terminal construct (HA-R113X) encompassing only two TMDs (TMDs 1-2) displayed no specific interaction with the V2-R. To further characterize structural domains that participate in oligomerization, E242tail (TMDs 6-7) and L292tail (TMD 7) were coexpressed with C-terminally truncated V2-R constructs and tested in sandwich ELISA. A significant interaction was observed only for E242tail (TMDs 6-7) with HA-E242X and HA-R337X (Table III), indicating an essential role of TMD 6 in receptor/fragment association. The expression and proper plasma membrane insertion of L292tail was verified by immunofluorescence studies (Fig. 4). Using a C-terminal anti-V2-R antibody, membrane fluorescence was only observed in permeabilized COS-7 cells (Fig. 4D), whereas nonpermeabilized cells showed no specific signals (Fig. 4C). Similar results were previously obtained with the E242tail fragment (4).

View larger version (108K): [in this window] [in a new window]   Fig. 4.   Expression and cellular localization of the V2-R and the L292tail fragment. COS-7 cells were transfected (5 µg of plasmid DNA/well) with the human V2 vasopressin receptor (A and B) and the L292tail fragment (C and D). After 72 h, immunofluorescence studies were performed on nonpermeabilized (A and C) and permeabilized (B and D) cells, which were incubated with a polyclonal antibody directed against the V2-R C terminus. One experiment out of two with similar results is shown.

Functional Rescue of Mutant V2-Rs following Disruption of the Extracellular Disulfide Bond-- In contrast to previous studies demonstrating an interaction and a functional reconstitution of missense mutations in TMD 6 by coexpressing the E242tail fragment (4, 5), HA-E242X did not restore function of inactivating mutations within TMDs 1-5, despite a specific interaction with the wild-type V2-R and mutant V2-Rs. These latter findings were in conflict with the hypothesis that the coexpressed N-terminal folding domain (TMDs 1-5) and the C-terminal folding unit of the mutant receptor can form a functional receptor (domain-swapping model). One possible explanation for these conflicting results suggests multiple contact sites located within the N-terminal folding unit that interact with a distinct interface in the C-terminal receptor portion, probably TMD 6. We speculated that the N-terminal interface is not necessarily destabilized by the chosen missense mutations (R137H, S167L, and R181C). In case of mutations within TMD 6, the interaction between the N- and C-terminal folding units is disrupted, allowing rescue by a domain-swapping mechanism.

A disulfide bond connecting the first and second extracellular loops is an important structural element present in most GPCRs (Fig. 1). It is likely that this disulfide bond significantly contributes in maintaining the tertiary structure of the N-terminal folding domain and, therefore, stabilizes the interface of the N-terminal folding domain for association with the C-terminal folding domain. We reasoned that disruption of the disulfide bond may disturb the tertiary structure of N-terminal folding domain and would facilitate potential rescue by using a coexpression strategy. To test this hypothesis, two mutant V2-Rs were generated in which the disulfide bond-forming cysteine residues were replaced by alanine (C112A and C192A). Both V2-R mutants were expressed in COS-7 cells and tested in cAMP accumulation assays. Agonist stimulation of HA-C112A resulted in a 7.6-fold increase in intracellular cAMP levels, whereas HA-C192A was almost unresponsive (1.8-fold) to 10 µM AVP (Table II). As shown in Fig. 5A, both mutants exhibited a shift in concentration-response curves toward higher AVP concentrations when compared with the HA-V2-R (Fig. 2B), and in binding studies saturation was not achieved. Because radioligand binding studies revealed no saturable AVP-binding sites at 100 nM ³[H]-AVP, ELISA studies were performed to estimate expression levels. The cell surface expression levels of the two cysteine mutants were reduced to 40%, but total cellular expression levels were similar as for HA-V2-R (Table I). These data indicate that V2-R malfunction following disulfide bond disruption is based on improper ligand binding and trafficking. Because the second cysteine within the second extracellular loop (Cys¹⁹⁵; Fig. 1) is not conserved among the members of the vasopressin/oxytocin receptor subgroup and alanine replacement did not significantly influence V2-R signaling, it is unlikely that Cys¹⁹⁵ participates in disulfide bond formation.²

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Functional reconstitution of mutant V2 vasopressin receptors by coexpression with HA-E242X. COS-7 cells were transfected with the indicated V2 vasopressin receptor constructs and stimulated with increasing AVP concentrations (10 pM to 10 µM). An accumulation assay was used to determine intracellular cAMP levels (see "Experimental Procedures"). Data (means ± S.E.) are expressed as a percentage of maximum cAMP response (Emax values; Table II) of three to four independent experiments, each carried out in duplicate.

Both cysteine mutants (HA-C112A and HA-C192A) were then coexpressed with HA-E242X. Aside from a reduction of the E_max value (5.8-fold) because of cotransfection, no significant changes in AVP-induced cAMP formation were observed for HA-C112A. In contrast, cotransfection of HA-C192A and HA-E242X resulted in an increased E_max value (2.5-fold) and a biphasic concentration-response curve (Table II and Fig. 5A), suggesting the coexistence of two V2-R populations, one with functional properties of HA-C192A and one with wild-type V2-R properties. To appreciate this gain-of-function, it is important to note that these coexpression experiments (e.g. HA-C192A + HA-E242X) were controlled by transfecting identical DNA amounts of individual constructs (C192A) that were not diluted with empty vector DNA.

Coexpression of two angiotensin II type 1 receptors containing inactivating mutations in the N-terminal folding unit (TMD 3 and TMD 5) restored ligand binding abilities but did not rescue the capability of G_q coupling (6). Using a similar approach we coexpressed the two cysteine V2-R mutants (HA-C112A and HA-C192A). We speculated that in case of domain rearrangement between the two mutant receptors this experimental setup would allow the formation of an intermolecular disulfide bond in a trans-complementary fashion. However, coexpression of HA-C112A and HA-C192A did not result in high affinity [³H]AVP-binding sites (data not shown), and a shift in the concentration-response curve (Fig. 5A and Table II) making the formation of a functionally relevant intermolecular disulfide bond unlikely.

Next, we constructed two double mutants (HA-C112A/R181C and HA-R137H/C192A) by cloning the C112A and C192A mutations into the HA-R181C and HA-R137H constructs, respectively. Both double mutants showed neither AVP-induced cAMP formation at 10 µM AVP nor specific [³H]AVP binding. ELISA studies revealed a reduced cell surface expression (60% of HA-V2-R) but unchanged total cellular expression for HA-C112A/R181C. The expression of HA-R137H/C192A was significantly reduced in both surface and sandwich ELISAs (Tables I and II). Interestingly, coexpression of both HA-C112A/R181C and HA-R137H/C192A with HA-E242X resulted in a gain-of-function (2.2-fold over basal; Table II) comparable with those found with HA-E242X and E242tail (Fig. 2). EC₅₀ values were similar to the wild-type V2-R (Table II and Fig. 5B) supporting a domain-swapped receptor rearrangement. For control purposes two HA-E242X constructs (HA-C112A/E242X and HA-C192A/E242X) were generated harboring either C112A or C192A in addition to the truncation at codon position 242. In contrast to HA-E242X, none of these truncated mutants was able to rescue the function of HA-C112A/R181C and HA-R137H/C192A (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously used a coexpression approach to functionally rescue clinical relevant V2-R mutations, mainly receptor truncations, with a C-terminal receptor fragment (4, 5). However, most of the mutations found in nephrogenic diabetes insipidus patients are missense mutations distributed preferentially in the transmembrane segment and the outer loop regions. To test the general feasibility of this potential therapeutic strategy, we extended the coexpression approach to missense mutations found in the first two-thirds of the V2-R. The ability of functional complementation of mutant GPCRs upon coexpression with receptor fragments (reviewed in Ref. 3) favors the hypothetical domain-swapped dimer model (Fig. 6, A and B) in which the ring-like TMD bundle is preserved after substitution for the mutant folding unit (Fig. 6D). In this study we tested the hypothesis of whether V2-Rs containing mutations in the N-terminal receptor portion (TMDs 1-5) can be rescued by a coexpressed nonmutated folding unit (TMDs 1-5). Specifically, we coexpressed a V2-R fragment truncated in the third intracellular loop (HA-E242X) to gain function of mutant full-length V2-Rs (HA-R137H, HA-S167L, and HA-R181C) by generating a partially domain-swapped dimer as shown in Fig. 6D. Functional assays showed that complementation was achieved neither with intracellularly retained V2-Rs (HA-R137H and HA-S167L) nor with properly transported (HA-R181C) mutant V2-Rs (Fig. 2). Therefore, these data are not consistent with a model in which the N-terminal receptor fragment substitutes for the mutant folding domain by forming a domain-swapped array.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Proposed structure of V2 vasopressin receptor dimers. TMDs of GPCRs form a ring-like structure in a counter-clock wise fashion as view from extracellular (A). GPCRs are composed of at least two independent folding domains (TMDs 1-5 and TMDs 6-7) that are connected by the third intracellular loop (arrow: i3 loop). Accumulating evidence suggests that wild-type GPCRs can exist in dimeric complexes, and two structural models of dimer formation have been suggested (16). The contact interface of so-called swapped dimers is recruited from the rearrangement of two independent folding domains of the individual receptor monomers (B). The ring-like TMD arrangement is still guaranteed by complementary exchanging the folding domains. In contact dimers, a lateral interaction of the individual receptor molecules is assumed (C). Coexpression studies have shown (Fig. 3 and Table III) that C-terminally truncated receptor fragments can associate with the full-length wild-type receptor (D and E). Because there is no support for domain-swapped arrays (B and D) from functional studies of coexpressed mutant V2-Rs and truncated or missense mutated V2-Rs (see "Results"), an oligomer formation by lateral interaction is more likely (C and E). Mutational disruption of the highly conserved extracellular disulfide bond (arrow: disulfide bond) destabilizes the TMD structure (F and G) and allows receptor reconstitution in a domain-swapping fashion upon fragment coexpression (H) (see "Discussion").

Functional complementation of two identical full-length receptors harboring different missense mutations in the N- and C-terminal domains would be the most convincing evidence demonstrating the existence of homodimers in a domain-swapped structure (Fig. 6B). However, all our efforts to rescue the function of receptors containing missense mutations in the N-terminal domain (R137H, S167L, and R181C) by coexpression with a V2-R mutant harboring the mutation in the C-terminal domain (Y280C) were unsuccessful. These data are consistent with findings from coexpressed N-terminal receptor fragments (see above) and also argue against a domain-swapped receptor array.

Because there was no functional evidence for a domain-swapped structure, a direct receptor/fragment interaction, which is a necessary prerequisite for functional reconstitution, had to be demonstrated. Using different techniques (coimmunoprecipitation, Western blot analysis, and ELISA) we clearly showed that the coexpressed fragment (HA-E242X) is associated with the full-length V2-R even in the presence of inactivating mutations (R137H and R181C). Additionally, high molecular weight bands of the V2-R in Western blot analysis (Fig. 3) support the existence of V2-R oligomer formation as shown in previous studies (16, 33).

Systematic analysis of the structural requirements for receptor oligomerization revealed that constructs truncated downstream of TMD 3 can associate with the full-length receptor in a specific manner (Fig. 3 and Table III). Our data also implicate a minor role of TMDs 1-2 in receptor complexation. Despite high cellular expression levels and a correct plasma membrane insertion, no significant interaction of the full-length V2-R and fragments containing only TMD 1 (HA-W71X) or TMDs 1-2 (HA-R113X) was observed. These findings agree with a recent study showing that a V2-R construct truncated within the i3 loop but lacking TMDs 1-2 was able to specifically inhibit V2-R function, probably by specific interaction with the wild-type receptor (33). Summarizing our data at this point, we have experimentally demonstrated that N-terminal receptor fragments specifically interact with full-length V2-Rs even in the presence of inactivating mutations. Because functional reconstitution was not achieved, the observed interaction is unlikely due to a domain exchange but is rather caused by a lateral interaction (Fig. 6, C and E).

The association of the N- and C-terminal folding domains in a monomeric receptor is ensured by covalent linkage via the third intracellular loop in addition to specific intramolecular contact sites. Rearrangement of the compact TMD bundle in monomers and formation of a homodimer in a domain-swapping fashion must have an energetic advantage to occur spontaneously. As shown in previous studies (5), interaction of even noncovalently linked N- and C-terminal domains appears to be very strong and can only be disrupted by sample boiling. Therefore, it is reasonable to assume that a single missense mutation must create a major disturbance within one contact interface, energetically favoring an interaction with the second receptor monomer or coexpressed receptor fragment via domain swapping as proposed in Fig. 6 (B and D). This assumption is supported by our previous studies showing that a coexpressed C-terminal domain is able to restore the function of missense mutations in TMD 6 (4, 5). Based on the current GPCR model (34), TMD 6 is thought to play a pivotal role in global GPCR structure, making multiple contacts to TMDs 1-5 (Fig. 6). Our data are consistent with this notion that a single mutation within the N-terminal domain does not interfere with a proper receptor core assembly. According to this model, mutational disruption of distinct interaction sites involving TMD 6 may disturb proper assembly of the N- and C-terminal receptor domains, allowing functional reconstitution by coexpression of receptor fragments. In a very recent study Jakubik and Wess (35) used a sandwich ELISA to determine essential residues within the N- and C-terminal folding domains of the muscarinic m3 receptor, maintaining the specific interaction between both receptor subunits. This study showed that several missense mutations known to alter ligand binding abilities of muscarinic receptors did not interfere with fragment association, whereas three highly conserved proline residues thought to have an impact on the -helical TMD structure are essential for proper receptor assembly.

Most GPCRs contain a conserved pair of extracellular cysteine residues linking the first and second extracellular loops via a disulfide bond (Figs. 1 and 6). Functional analyses of mutant GPCRs in which these cysteine residues were replaced by other amino acids have shown that this disulfide bond may be critical for receptor function (36-40). We speculated that mutational disruption of the disulfide bond may disturb the tertiary structure of the N-terminal folding domain and may allow for a domain exchange. Replacement of the cysteine residues by alanine in HA-C112A and HA-C192A resulted in a shift of the agonist-response curves to higher AVP concentrations and reduced plasma membrane expression levels (Fig. 5A and Table I). These data indicated that disruption of the disulfide bond in the V2-R does not influence the ability of the receptor to activate G_s protein but interferes with high affinity ligand binding and receptor trafficking. Similar observations were made with mutant muscarinic receptors, showing that the disulfide bond is required for efficient cell surface expression but not for G_q-protein activation (40). Despite the disruption of the disulfide bond, the receptor core structure appears to be still intact, allowing receptor function. As a consequence of the latter findings, coexpression of HA-C112A with HA-E242X did not result in reestablishing normal V2-R function. Surprisingly, a biphasic concentration-response curve was determined when HA-C192A was coexpressed with HA-E242X (Fig. 4A). This implicates the coexistence of two functional receptor forms, the HA-C192A and a complex assembled from HA-C192A and HA-E242X with wild-type V2-R function. The obvious discrepancy in their abilities to be functionally rescued by HA-E242X may result from an unknown function of Cys¹⁹² additional to its participation in disulfide bond formation. This assumption is supported by the fact that, in contrast to C112A, alanine substitution of Cys¹⁹² almost abolished agonist-induced cAMP formation (Table II).

The findings that disruption of the extracellular disulfide bond did not completely prevent receptor function and that expression of HA-C192A together with HA-E242X partially restored wild-type function prompted us to propose a receptor model in which this structural alteration forces the mutant receptor into an equilibrium of a properly (Fig. 6G) and an improperly assembled receptor (Fig. 6F). Only the misfolded receptor can be reconstituted in a competitive fashion as shown in Fig. 6H. By introducing additional mutations (R181C and R137H) into C112A and C192A, respectively, the equilibrium is shifted to the misfolded receptor stage as indicated by complete loss of function (Table II). However, coexpression of both HA-C112A/R181C and HA-R137H/C192A with HA-E242X restored wild-type receptor function (Fig. 5B and Table II).

In summary, we have shown that the V2 vasopressin receptor as an example of class I GPCRs forms oligomeric complexes with both full-length and truncated V2-R constructs. The lack of functional reconstitution of missense mutations within TMDs 1-5 upon coexpression with mutant receptors supports an oligomer structure by lateral interaction rather than by a domain-swapping mechanism. The domain-swapping mechanism can only be operative after major structural alteration of the folding domain interface.

	ACKNOWLEDGEMENTS

We are grateful to Dr. S. A. Wank (NIH, Bethesda, MD) for providing the CCK_A receptor cDNA and Dr. P. Goldsmith (NIH) for supplying an affinity-purified polyclonal antibody raised against the C terminus of the human V2 vasopressin receptor. We thank Dr. J. Wess for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany. Tel.: 49-30-8445-1861; Fax: 49-30-8445-1818; E-mail: schoberg@zedat.fu-berlin.de.

² A. Schulz and T. Schöneberg, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; AVP, arginine vasopressin; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; PBS, phosphate-buffered saline; TMD, transmembrane domain; V2-R, V2 vasopressin receptor; CCK_A-R, cholecystokinin type A receptor; m3-R, rat m3 muscarinic receptor; GFP, green fluorescent protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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