Institut National de la Santé et de la Recherche Médicale (S.T., T.D., B.M., M.T., C.B.), Unité 469, 34094 Montpellier Cedex 5, France; Department of Cell Biology (M.A.A., R.J.), Institut Cochin, 75014 Paris, France; and Département de Biochimie (S.T., A.B., M.B.), Université de Montréal, Montréal, Québec H3C 3J7, Canada
Address all correspondence and requests for reprints to: Michel Bouvier, Département de Biochimie, Faculté de Médecine, P.O. Box 6128, Succ. Centre-Ville, Montréal, Québec H3C 3J7, Canada. E-mail: michel.bouvier{at}umontreal.ca.
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ABSTRACT |
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INTRODUCTION |
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The observation that heterodimerization of GbR1 and GbR2 is a prerequisite for the cell surface expression of GbR1 (15) has been taken as evidence suggesting that dimerization occurs before the receptors reach the cell surface and probably as early as the ER. However, this may not be a general phenomenon because GABAb and taste receptors (16, 17) are the only two examples where obligatory heterodimerization has been demonstrated. Whether heterodimerization could also represent an early event for other GPCR thus remains an open question.
In the present study, we investigated the possibility of homo- and heterodimerization among the vasopressin (VR)/oxytocin (OTR) receptor family and used this model to investigate dimer ontogeny and regulation. Arginine vasopressin (AVP) and oxytocin (OT) are two neurohypophysial hormones that differ from each other only by two amino acids. They mediate their physiological actions through their interactions with at least four closely related receptor subtypes (18): the vasopressin V1aR, V1bR, and V2R and the OTR. Although specific physiological functions have been attributed to each hormone, such as vasopressor effect and urine concentration for AVP and uterotonic and milk-ejecting activities for OT, they both show relatively high affinity for the four receptor subtypes. Interestingly, V1aR and OTR are coexpressed in several tissues such as the myometre (19, 20) and some regions of the central nervous system (21), whereas V1aR is found also with V2R in the collecting duct (22, 23, 24, 25). Taken with the high level of sequence homology between the receptor subtypes (4050%), such colocalization would be compatible with the occurrence of heterodimerization in native tissues.
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RESULTS AND DISCUSSION |
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To identify the various receptor species, Western blot analyses were carried out with the anti-HA antibody in cells expressing the HA-V1aR and treated or not with tunicamycin to inhibit glycosylation. In the absence of tunicamycin (Fig. 1A, lane 1), four species were detected. The Mr (relative molecular mass) 45,000 (m) and 90,000 (d) species were shown to represent the precursor core-glycosylated monomeric and dimeric V1aR forms because they were sensitive to endoglycosidase H (endoH) treatment (data not shown). The Mr 75,000 (M) and 150,000 (D) species were found to be resistant to endoH and thus correspond to the mature monomer and dimer. Inhibition of carbohydrate addition by tunicamycin reduced the number of immunoreactive bands, leaving only one monomeric and one dimeric species (Fig. 1A
, lane 2) further confirming that the heterogeneity observed in control conditions reflects distinct maturation states of the glycoproteins.
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Coimmunoprecipitations were then carried out in cells coexpressing different receptor pairs. For cells coexpressing HA-V1aR (Fig. 1A) and either Myc-V1aR (lane 4), Myc-OTR (lane 5), or Myc-V2R (lane 6), clear HA immunoreactivity was detected after total immunoprecipitation with the anti-Myc antibody indicative of intermolecular interactions between the differentially epitope-tagged receptors. This suggests the existence of V1aR homodimers as well as V1aR-V2R and V1aR-OTR heterodimers. A faint nonspecific band at Mr 50,000 was detected in mock-transfected cells (Fig. 1A
, lane 3) and no specific HA immunoreactivity was detected in cells expressing only the Myc-epitope receptor forms (data not shown), confirming the selectivity of the antibody used. In all cases, diffuse bands corresponding to the monomeric and dimeric immature as well as dimeric mature species were detected. Interestingly, almost no monomeric mature form was seen. This difficulty to detect the V1aR mature monomeric form in the coimmunoprecipitation assays is consistent with the resistance of many GPCR dimers to sodium dodecyl sulfate (SDS) denaturation (13).
Coimmunoprecipitations carried out in cells coexpressing HA-OTR (Fig. 1B) and either Myc-OTR (lane 2), Myc-V1aR (lane 3), or Myc-V2R (lane 4) yielded similar results. Indeed, immunoreactive HA-OTR could be detected after immunoprecipitation of the Myc-tagged OTR, V1aR, or V2R, confirming the existence of V1aR-OTR heterodimers and indicating that OTR can also form homodimers and V2R-OTR hetero-dimers. In these experiments, the monomeric immature (Mr 38,000) and mature (Mr 55,00065,000) forms were mainly detected indicating that the dimeric species were more efficiently denatured by SDS. The existence of glycosylation heterogeneity (27) most likely explains the presence of two distinct mature receptor bands.
The coimmunoprecipitations did not result from spurious interactions due to overexpression of the different receptors because no coimmunoprecipitation could be observed between the unrelated HA-GbR2 (Fig. 1C) and Myc-V1aR (lane 2) or Myc-OTR (lane 3). In contrast, significant coimmunoprecipitation could be observed between HA-GbR2 and Myc-GbR1b, two receptors known to form heterodimers (lane 4) (3, 4, 5).
Taken together, these results suggest that V1aR, V2R, and OTR are able to form specific homo- and heterodimers. Interestingly, stable molecular interactions were observed for both the fully glycosylated and the immature forms of the receptor, suggesting that dimerization may occur early during the receptor secretory pathway.
Assessment of V1aR, V2R, and OTR Dimerization in Vivo by Bioluminescence Resonance Energy Transfer (BRET)
To determine whether the intermolecular interactions detected in the coimmunoprecipitation assays truly reflected dimerization occurring in vivo, BRET studies were performed in living cells. For this purpose, wild-type human V1aR, V2R, and OTR were tagged at their C terminus with Renilla-luciferase (Rluc) or enhanced yellow fluorescent protein (YFP). Fusion to Rluc and YFP did not significantly impair the binding or signaling properties of the V1aR, OTR, and V2R as assessed by their ability to promote hormone-stimulated inositol phosphate accumulation (V1aR and OTR) or cAMP accumulation (V2R), respectively (Table 1). The occurrence of dimerization was assessed by determining the transfer of energy between the Rluc and YFP fusion constructs expressed in HEK 293T cells upon addition of the luciferase substrate, coelenterazine h. For this purpose, cells expressing physiologically relevant levels for each receptor combination were used (
200 fmol of receptor/mg of protein at a Rluc/YFP ratio of
0.3). As shown in Fig. 2
, significant BRET was observed for all pairs considered (V2R-Rluc/V2R-YFP, V1aR-Rluc/V1aR-YFP, OTR-Rluc/OTR-YFP, V1aR-Rluc/V2R-YFP, V1aR-Rluc/OTR-YFP, and V2R-Rluc/OTR-YFP). The specificity of the interactions is demonstrated by the lack of significant energy transfer detected under similar experimental conditions when V1aR-Rluc, V2R-Rluc, or OTR-YFP was coexpressed with GbR2-YFP and GbR2-Rluc, respectively. BRET detection was not influenced by the relative position of the donor and acceptor on the receptor pairs because identical results were obtained for the reciprocal constructs (data not shown). These results demonstrate that V1aR, V2R, and OTR can form both homo- and heterodimers in living cells.
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Relative Affinity of Protomers within Homo- and Heterodimers
In an attempt to determine the relative affinity of receptors for each other, BRET saturation curves were carried out. For this, Rluc construct was maintained constant, whereas the concentration of the YFP partner was gradually increased. Representative BRET saturation curves obtained for V1aR-Rluc/V1aR-YFP, OTR-Rluc/OTR-YFP, and V1aR-Rluc/OTR-YFP are presented in Fig. 5. The saturation of the BRET signal observed corresponds to the engagement of all available Rluc constructs in energy transfer with their YFP counterparts and thus represents the highest BRET signal achievable for a given pair. The difference in the maximum BRET signals observed for the different combination cannot be used as a quantitative measure of the relative number of dimers formed for each pair. Indeed, maximal BRET level is not only a function of the dimer numbers but also depends on the distance between the energy donors and acceptors as well as their relative orientation within the dimers. However, the concentration of the YFP construct required to reach the half-maximum BRET, i.e. 50% of maximum energy transfer, and defined here as the BRET50, provides a measure of the relative affinity of the partners for each other. No significant difference between the BRET50 was observed for any of the pairs considered (independently of the partner orientations), indicating that the VR and OTR had a similar affinity for each other than for themselves (Table 2
). As previously shown in Fig. 2
, no energy transfer was observed between V1aR, V2R or OTR, and GABA GbR2 (Fig. 5
).
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Assessment of the Ligand Binding and Signaling Properties of the Heterodimers vs. Homodimers
Although still limited in numbers, some studies have suggested that the formation of GPCR heterodimers could have important implication on receptor pharmacology or signaling. For instance, it was reported that heterodimerization between and
opioid receptors leads to a complex that is unable to bind either
or
selective agonists alone. However, when the two types of ligand were added simultaneously, binding to the heterodimer was regained indicating the occurrence of cooperativity (6). Since this original observation, complex cooperative behaviors in both binding and signaling were observed for µ/
opioid receptor (30), AT1 angiotensin/B2 bradykinin (9), and CCR2/CCR5 chemokine (31) receptor heterodimers. In the case of the CCR2/CCR5 and µ/
opioid receptors, heterodimerization was also found to change the coupling selectivity toward the G proteins (7, 31). To evaluate whether heterodimerization within the VR/OTR family could affect their binding or signaling properties, the binding properties of the natural ligand vasopressin was assessed in cells expressing each of the receptor individually or in combination. As shown in Fig. 6
, A and B, coexpression of the receptors had no significant influence on the binding of [3H]-vasopressin, indicating that heterodimerization did not affect the affinity of the receptors for the hormone or that only the homodimers retained binding capacity. It should be noted, however, that the affinity of vasopressin for the three receptor subtypes expressed alone is virtually identical. This may obscure modest changes that could result from the heterodimerization.
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Although our data do not exclude the possibility that the heterodimers could engage a different signaling pathway that was not studied herein, they suggest that each receptor subtype maintains at least some of its pharmacological properties within the dimer. The absence of Gs stimulation by the V1aR and OTR selective agonist may indicate that only one G protein at a time can be recruited to the receptor heterodimer and that the identity of the G protein is determined by the activated receptor protomer. This would be consistent with the recent suggestion that only one G protein may be activated by the GABAbR1/GABAbR2 heterodimer (32). Also consistent with this notion, structural considerations concerning the receptor/G protein interface indicate that a single G protein heterotrimer can be nicely accommodated under one receptor dimer (33).
Subcellular Distribution of the VR and the OTR Dimers
The observation from the coimmunoprecipitation studies that V1aR, V2R, and OTR homo- and heterodimers were obtained for both mature and immature forms begs the question of their site of formation. In fact, constitutive dimerization as well as the existence of immature receptor dimers may suggest, as it is the case for other oligomeric proteins (34, 35, 36), that assembly may occur as part of the biosynthetic process. BRET approach on intact cells cannot provide direct information on the cellular location of the detected interactions. Therefore, the subcellular distribution of the dimers was assessed after fractionation over sucrose gradients of cells coexpressing the Rluc/YFP constructs (Fig. 7). Each fraction was subjected to fluorescence/luminescence analysis and to BRET measurements. The presence of immunoreactive Na+/K+-ATPase and calnexin was used as an index of plasma membrane and ER enrichment, respectively (Fig. 7B
). As shown in Fig. 7
, A and C, the fluorescence and luminescence signals originating from OTR-YFP, V2R-YFP, V1aR-Rluc, and V2R-Rluc, respectively, were found in both plasma membrane- and ER-enriched fractions consistent with the expected distribution for a plasma membrane protein. Similar distributions were obtained with the V1aR-YFP and OTR-Rluc construct (data not shown). BRET ratio monitored with V1aR-Rluc/V1aR-YFP, OTR-Rluc/OTR-YFP or V1aR-Rluc/OTR-YFP (Fig. 7A
) and V2R-Rluc/V2R-YFP, V1aR-Rluc/V2R-YFP or V2R-Rluc/OTR-YFP (Fig. 7C
) had similar patterns. Significant BRET could be detected in both plasma membrane- and ER-enriched fractions, indicating that dimers existed in these two compartments. Although the separation was not perfect and the ER marker contaminated the tail fractions of the plasma membrane peak, BRET could clearly be observed in both ER and plasma membrane fractions that were devoid of the other compartment marker. The similar BRET levels observed despite the lower level of receptor detected in the ER is not surprising because BRET is a ratiometric measurement that reflects the proportion of Rluc constructs engaged in dimerization and not the absolute amount of dimers. As previously reported in Fig. 2
, no BRET could be observed between V1aR-Rluc and GbR2-YFP or between OTR-Rluc and GbR2-YFP, confirming the specificity of the interactions. These results support the notion that V1aR, V2R, and OTR homo- and heterodimers exist as early as the ER and can also be found at the cell surface. Consistent with this notion, the presence of stable homo- and heterodimers at the cell surface was also demonstrated using coimmunoprecipitation experiments carried out on whole cells (data not shown). The apparent difference in the proportion of immature receptor species (presumably localized in the ER) detected in the Western blot analysis (Fig. 1
), and the fraction of receptor found in the ER-enriched fractions in the sucrose gradient experiments may be due to the fact that complex sugars could partially mask the epitope located at the N terminus of the receptor, thus decreasing their immunoreactivity. However, one cannot exclude the possibility that the N-terminally tagged receptor could have a somewhat lower trafficking efficacy (37).
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Effect of Ligands on Receptor Dimerization
The initial composition of the dimers formed in the ER would reflect the affinity of the protomers for each other and their relative abundance. Once at the cell surface, the preformed dimers could either become the target for dynamic regulation by ligands or maintain their composition depending on the stability of the complex. To distinguish between these two possibilities, we next determined whether ligand could modulate the VR and OTR dimerization. For this purpose, BRET ratio measurements were performed after treatments with saturating concentrations of either V1aR, OTR or V2R agonists (AVP and OT) and antagonists (HO-LVA, OTA, and SR121463a, respectively) for 10 min in cells coexpressing the Rluc/YFP construct pair V1aR/V1aR, V2R/V2R, OTR/OTR, V2R/V1aR, V1aR/V2R, V1aR/OTR, and OTR/V2R. BRET signals were not affected by neither the agonists (Fig. 8A) nor the antagonists (data not shown) used. This lack of increase did not result from the saturation of the BRET signal because no effect of the ligands was observed even for YFP/Rluc ratios that generated submaximal BRET. Longer ligand exposure (up to 30 min) was also without effect on the BRET measured (data not shown). This lack of ligand promoted change in BRET contrasts with the increase in BRET signal observed between the melatonin receptor MT2-Rluc and MT2-YFP upon agonist exposure.
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The fact that ligand-independent BRET signals were observed for each pair considered not only suggests the existence of constitutive dimers for all members of the VR/OTR family but also indicates that ligand binding is without effect on their oligomerization state. The constitutive nature of the dimers is consistent with crystallographic studies revealing that the N-terminal ligand-binding domain of the metabotropic glutamate receptor is dimeric whether in the presence or the absence of its natural ligand glutamate (39). More recently, the N-terminal portion of the Wnt receptor, frizzled, was also shown to crystallize as a constitutive dimer (40).
It should be emphasized that the BRET levels observed is a function of the efficacy of energy transfer that is given by the following equation: Efficacy = R06/(R06 +R6), where R0 is the Förster distance at which the efficacy is equal to 0.5 and R is the distance between the donor and acceptor (41). It follows that the changes in distance have a dramatic effect around the R0 but can be undetectable for distances significantly smaller than R0 where the transfer efficacy is reaching its maximum (1). In fact, changes in distance can be assessed accurately only when the distance between donor and acceptor lies between 0.5R0 and 1.5R0. The lack of effect of the ligands on the BRET detected in the present study could therefore indicate that the distance between Rluc and YFP within the V1aR, V2R, and OTR homo- and heterodimers is already smaller than 0.5R0 and cannot be accurately detected. Alternatively, the ligand promoted changes in conformation may not be readily transmitted to the carboxyl end of the receptor where the fluorophores are located. With these considerations in mind, it becomes easier to explain why ligand-promoted changes in energy transfer [BRET or fluorescence resonance energy transfer (FRET)] are found in some studies (10, 11, 42, 43, 44, 45) but not in others (46, 47), depending on the receptors considered.
The receptor expression levels used to study constitutive and ligand-regulated dimerization is also an important parameter that needs to be taken into consideration. In the present study, the constitutive BRET and the lack of ligand-mediated effect for the AVP/OT receptor family was confirmed at expression levels comparable to those observed in tissues endogenously expressing these receptors. This therefore further supports the notion that constitutive homo- and heterodimerization may occur in vivo.
CONCLUSION
Taken together, these results indicate that as was suggested for the GABAb receptor, the V1aR, V2R, and OTR form homo- and heterodimers as early as the ER. These oligomeric complexes do not appear to be dynamically regulated by ligand, suggesting that the receptors exist as constitutive dimers that remain stably associated during the activation process. The similar relative affinities that the VR/OTR family members show for each others indicates that the proportion of each homo- and heterodimer will vary as a function of their relative expression levels. This has obvious consequences on the possible regulation of the homo- and heterodimerization process in cells coexpressing the different subtypes. Because no obvious difference in the pharmacological or signaling properties could be found between the homo- and the heterodimers, additional work will be needed to determine whether constitutive heterodimerization of these receptors can have functional implications.
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MATERIALS AND METHODS |
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Construction of Receptor Plasmids
The V1aR, V2R, and OTR different hybrid protein constructs were generated from the cDNA of the human receptors subcloned in pRK5 using the GeneEditor In Vitro Site-Directed Mutagenesis System (Promega Corp., Madison, WI) or QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
Primers were made to introduce an epitope tag (Myc or HA) at the amino terminus of the V1aR, V2R, and OTR receptor constructs.
V1aR-YFP, V2R-YFP, OTR-YFP.
Sense and antisense primers were designed to introduce an AgeI site at the 3' end and to remove the receptors stop codon. An XbaI site was also inserted at the 3' end of OTR, immediately downstream of the AgeI site. The pEYFP vector (CLONTECH Laboratories, Inc., Palo Alto, CA) was cut with AgeI/XbaI to excise the YFP coding region so that it could be inserted into the pRK5-V1aR, pRK5-V2R, or pRK5-OTR vector.
V1aR-Rluc.
pRK5-V1aR-Rluc was constructed using the NheI/XbaI sites to excise Rluc from the pRL-CMV vector (Promega Corp.) and insert it in frame in pRK5-V1aR vector.
V2R-Rluc, OTR-Rluc.
AgeI site was created at the beginning of the Rluc coding region, and the AgeI/XbaI fragment was inserted in frame into the pRK5-V2R or pRK5-OTR vector.
GbR1-Rluc, GbR2-Rluc.
The human GbR1 or GbR2 coding sequence was amplified out of its original vector with two XbaI sites containing primers to generate a stop codon-free fragment that then could be subcloned in-frame into the XbaI site of the pCDNA-3.1-hRluc vector.
GbR2-YFP, Myc-GbR1b, and HA-GbR2.
pCDNA-3 plasmids encoding these constructs were generously provided by GlaxoSmithKline (Stevenage, UK).
ß-Arrestin2-YFP.
The rat ß-arrestin2-YFP was constructed as previously described (41).
ß-Arrestin2-Rluc.
The rat ß-arrestin2 coding sequence was amplified out of its original vector and subcloned in-frame into the pCDNA-3.1-hRluc vector.
All constructs were verified by direct DNA sequencing.
Cell Culture and Transfection
HEK 293T cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine, at 37 C in a humidified atmosphere at 95% air and 5% CO2. Transient transfections were performed using the calcium phosphate precipitation method (48). Cells were seeded at a density of 5 x 105 or 2 x 106 cells per six-well plates or 100-mm Petri dishes, respectively, and transfected with the indicated plasmids.
Total and Cell Surface Coimmunoprecipitations
Seventy-two hours post transfection, cells were washed three times with cold PBS and incubated on ice for 1 h in blocking buffer (PBS containing 0.2% BSA), followed by incubation with anti-Myc (9E10) or anti-HA (12CA5) antibody 1/250 in blocking buffer on ice for 1 h. After four washes with blocking buffer, cells were lysed 30 min in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM iodoacetamide, 5 µg/ml leupeptin; 10 µg/ml benzamidine; and 5 µg/ml Soybean Trypsin inhibitor) and centrifuged at 12,000 x g for 30 min at 4 C. Protein concentration of lysates was determined with the DC assay kit (Bio-Rad Laboratories, Inc.) using BSA as standard, and the same total quantity of proteins was used in each immunoprecipitation reaction. For the total immunoprecipitation, lysates were incubated overnight at 4 C with anti-Myc or anti-HA antibody 1/500 and 50 µl of protein G-Sepharose. For the surface immunoprecipitation, only protein G-Sepharose was added. The immunoprecipitation complexes were collected by centrifugation and washed four times with cold RIPA buffer containing 150, 250, 350, and 150 mM NaCl, respectively. The final pellet was resuspended in sample buffer (60 mM Tris-HCl, pH 7.4; 2% SDS; 15% glycerol; and 50 mM dithiothreitol) and heated at 95 C. Proteins were then resolved by SDS-PAGE before being transferred to nitrocellulose. The rabbit polyclonal anti-HA (Y11) 1/3000 was used to detect the receptors, and the immunoreactivity was revealed using horseradish peroxidase-coupled antirabbit antibody 1/10,000.
Deglycosylation Assays
Deglycosylation of immunocomplexes was carried out with endoH or PNGaseF. Protein G-Sepharose/antibody/antigen complexes were washed with 50 mM sodium phosphate (pH 7.5) and incubated for 20 h at 37 C in deglycosylation buffer (50 mM sodium phosphate, 0.5% N-dodecyl-ß-D-maltoside, 50 mM EDTA, 0.2 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml benzamidin, 2 mM 110 phenantroline, 1% ß-mercaptoethanol, 0.1% SDS) in the presence or absence of endoH 40 mU/ml or PNGaseF 20 U/ml at pH 5.5 or pH 7.5, respectively. Reactions were stopped by adding 5x sample buffer and heating the samples at 95 C.
Inhibition of glycosylation was performed with Tunicamycin at a final concentration of 2 µg/ml. The reagent was added just before the transfection and the treatment was maintained until the immunoprecipitation experiment.
Inositol Phosphate Assays
Twenty to 24 h after transfection, cells transiently expressing the V1aR or OTR constructs were labeled with myo-[2-3H]inositol (1020 Ci/mmol; NEN Life Science Products) at a final concentration of 1 µCi/ml. After a 24-h labeling period, cells were equilibrated at 37 C in PBS for 1 h and then incubated for 10 min with 10 mM LiCl before being stimulated for 15 min with increasing concentrations of AVP or OT (from 10-12 to 10-6 M). The reaction was stopped with perchloric acid and increases in intracellular inositol phosphate levels were determined by anion-exchange chromatography as previously described (49).
cAMP Assays
To study the functional properties of the BRET fusion V2R, cAMP accumulation was measured using the TRACE/HTRF technology (CIS-Bio International, Marcoule, France) (50). Briefly, cells were harvested 48 h after transfection, resuspended in PBS supplemented with 1 mM isobutylmethylxanthine, and distributed in a 96-well plate (7000 cells per well). Cells were then stimulated with increasing concentrations of AVP (from 10-13 to 10-6 M). cAMP accumulations were determined using the homogeneous time resolved fluorescence cAMP kit and measuring the fluorescence intensities of the anti-cAMP-cryptate and anti-cAMP-XL665 conjugates at 620 and 665 nm, respectively.
For the study of the V2R-V1aR and V2R-OTR heterodimer signaling properties, cAMP accumulation assays were performed by metabolically labeling the cells with 3H-adenine (NEN Life Science Products, Beverly, MA) for 6 h and purifying total cAMP over Dowex/alumina sequential chromatography as previously described (51).
BRET Assays
Forty-eight hours after transfection, cells were washed twice with PBS and detached with PBS containing 5 mM EDTA. Cells were then resuspended in PBS containing 1 g/liter glucose and distributed in a 96-well microplate (100,000 cells per well). Coelenterazine h was added to a final concentration of 5 µM, and readings were collected using a multi-detector plate reader FUSION (Packard Instrument Co., Meriden, CT), allowing the sequential integration of the signals detected in the 440- to 500-nm and 510- to 590-nm windows. The BRET ratio is defined as [(emission at 510590) - (emission 440500) x Cf]/(emission at 440500), where Cf corresponds to (emission at 510590)/(emission at 440500) for the -Rluc construct expressed alone in the same experiment. Total fluorescence and luminescence signals were also determined for all samples using a Fluorocount and Luminocount (Packard) to assess the level of expression of the YFP and Rluc fusion constructs.
Cell Membrane Preparations and Radioligand Binding Assays
Forty-eight hours after transfection, cells were washed twice with PBS and detached with PBS containing 5 mM EDTA. Cells were then resuspended in PBS containing 1 g/liter glucose and BRET experiments were carried out. The remaining cells were resuspended in lysis buffer (15 mM Tris-HCl, pH 7.4; 2 mM MgCl2; and 0.3 mM EDTA), polytron homogenized and centrifuged at 100 x g for 5 min at 4 C. Supernatants were recovered and centrifuged at 44,000 x g for 20 min at 4 C. Pellets were washed in membrane buffer (50 mM Tris-HCl, pH 7.4; and 5 mM MgCl2), and centrifuged. Membranes were resuspended in an appropriate volume of membrane buffer and protein concentration was determined. Membranes were used immediately or stored at -80 C. Binding assays were performed at 30 C for 1 h using [3H]-AVP as the radioligand. Nonspecific binding was determined in the presence of 10 µM unlabeled AVP. Affinities for [3H]-AVP were determined in saturation binding assay using concentrations ranging from 0.1620 nM. Affinities for other ligands were determined from competition experiments using 2 nM [3H]-AVP as the radioligand. The concentration of the unlabeled ligands varied from 10 pM to 1 µM. The ligand binding data were analyzed by nonlinear regression using Prism 3.0 (GraphPad Software, Inc., San Diego, CA).
Flow Cytometry
Forty-eight hours after transfection, cells were washed twice with PBS and detached with PBS containing 5 mM EDTA. Cells were then resuspended in PBS (1,500,000 cells/ml) and sorted by fluorescence-activated cell sorting (FACS) set up to detect the YFP fluorescence. The fluorescence, the luminescence, as well as the BRET signal were then measured for the initial total cell population and each resulting fraction (30,000 cells per well).
Cellular Fractionation
Forty-eight hours post transfection, cells were washed three times with cold PBS, scraped off, and lysed with cold hypotonic lysis buffer (20 mM HEPES, pH 7.4; 2 mM EDTA; 2 mM EGTA; 6 mM magnesium chloride; 1 mM PMSF; 5 µg/ml leupeptin; 10 µg/ml benzamidine; and 5 µg/ml soybean trypsin inhibitor). The lysate was homogenized with 30 strokes of a tight-fitting dounce. Cellular debris and unlysed cells were removed by centrifugation at 1000 x g for 5 min at 4 C. The supernatant was collected and supplemented with 2 M sucrose to achieve a final concentration of 0.2 M sucrose. A discontinuous sucrose step gradient (0.5, 0.9, 1.2, 1.35, 1.5, and 2 M) was made using lysis buffer. Cell lysate was applied to the top of the gradient and samples were centrifuged for 16 h at 130,000 x g. Fractions were then subjected to fluorescence/luminescence analysis and to BRET measurements as described before. Identification of plasma membrane and ER enriched fractions was achieved by Western blot using rabbit polyclonal anti-Na+/K+-ATPase (Sigma, St. Louis, MO) and anticalnexin (StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) antibodies, respectively.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AVP, Arginine vasopressin; BRET, bioluminescence resonance energy transfer; BRET50, 50% of the maximum BRET; endoH, endo-ß-N-acetylglucosaminidase H; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; GbR1, GABAbR1 receptor; GbR2, GABAbR2 receptor; GPCR, G protein-coupled receptor; HA, hemagglutinin; HEK 293T, human embryonic kidney 293T cells; Mr, relative molecular mass; OT, oxytocin; OTR, oxytocin receptor; PMSF, phenylmethylsulfonyl fluoride; PNGaseF, peptide-N-glucosidase F; Rluc, Renilla-luciferase; SDS, sodium dodecyl sulfate; V1aR, V1a vasopressin receptor; V1bR, V1b vasopressin receptor; VR, vasopressin receptor; V2R, V2 vasopressin receptor; YFP, enhanced yellow fluorescent protein.
Received for publication June 26, 2002. Accepted for publication December 17, 2002.
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REFERENCES |
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