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J. Biol. Chem., Vol. 279, Issue 45, 46969-46980, November 5, 2004
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From the
Departments of Pharmacology, Biochemistry, ||Physics, and ¶Center for Structural Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600
Received for publication, July 1, 2004 , and in revised form, August 10, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1.5 µM; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of 
200 nM for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The C-terminal region of the V2R has been shown to be important for agonist-mediated phosphorylation (8), receptor escape from the endoplasmic reticulum and transport to the plasma membrane (9), sequestration and endocytosis of the receptor (8), and prevention of recycling (10) of the endocytosed V2R. Consequently, we decided to explore proteins that might interact with the V2R C terminus with the intent of understanding their possible role in V2R trafficking or signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Lines—Permanent clonal MDCKII cell lines expressing HA epitope-tagged WT and mutant V2Rs were developed using the CaPO4 method as described previously (7). Briefly, 10 µg of V2pcD-N-HA and pCMV4N-V2R-AAA (individual cDNAs encoding HA epitope-tagged human WT or mutant (AAA) V2Rs, respectively) were each co-transfected with 2 µg of pRSVneo (cDNA encoding neomycin resistance) into MDCKII cells. Colonies were selected based on resistance to G418, a neomycin analog, and isolated as described previously (7). G418-resistant colonies were screened for WT or AAA V2R expression by assaying binding of the radioligand [3H]AVP. Parental and stably expressing V2R MDCKII cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C, 5% CO2. Simian kidney fibroblast (COS M6) cells were maintained in supplemented DMEM + 20 mM HEPES. The studies presented were obtained in the WT V2R, clone 61, which expresses the HA epitope-tagged WT V2R at a density of 4.3 ± 1.0 pmol/mg specific [3H]AVP binding per mg of protein, estimated in homologous competition binding studies. Studies with AAA V2R exploited two independent clonal cell lines, clone 5 (6.7 ± 1.5 pmol/mg protein) and clone 11 (3.2 ± 2.0 pmol/mg protein).
Generation of cDNAs Encoding GST-V2R-C Terminus—The cDNAs encoding the GST-FLAG-V2R-C terminus constructs in the pEBG-SrfI vector were generated via overlapping PCR extension using Pfu Turbo DNA polymerase (Stratagene). The FLAG tag was inserted immediately 5' to the V2R C-terminal sequence and downstream of the GST start site and coding sequence. The AAA V2R construct, containing the holo-V2R receptor in pCMV4, was generated using the QuickChange II site-directed mutagenesis kit (Stratagene). The cDNAs were sequenced in their entirety to confirm that the sequences were correct.
Transient Expression Studies—COS M6 cells were seeded the day prior to transfection at a density of 7.5 x 105 (60-mm dish) or 1 x 106 (100-mm dish). On the day of transfection, FuGENE 6 (Roche Applied Science) was placed dropwise into a polypropylene tube containing Opti-MEM (Invitrogen) at 37 °C and incubated for 5 min at room temperature. According to the manufacturer's instructions, 1 µg of plasmid DNA was placed into an additional tube, and the FuGENE mixture was placed dropwise on top of the DNA in a ratio of 3 µl of FuGENE to 1 µg of DNA, incubated at room temperature for 20 min, and placed onto the cells in their existing culture medium. We used 1 µg of DNA per 60-mm dish and 3 µg of DNA per 100-mm dish, respectively. The cells were maintained at 37 °C, 5% CO2 prior to assessment 48 h post-transfection.
Calmodulin-linked Agarose Pull-down Experiments—Cells were placed on ice and washed once with DPBS-CM (Dulbecco's phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4,1mM MgSO4, 0.5 mM CaCl2, pH 7.3). Cells were extracted into ice-cold dodecyl-
-D-maltoside:cholesteryl hemisuccinate buffer (DM: CHS: 4 and 0.8 mg/ml, respectively, containing 20% glycerol, 25 mM glycylglycine, 20 mM HEPES, 100 mM NaCl, 5 mM EGTA, 100 µM PMSF, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, pH 7.4), sequentially triturated with 22- and 25-gauge needles, and centrifuged at 100,000 x g for 60 min. The detergent-solubilized supernatant was precleared with BSA linked to agarose beads and incubated overnight with calmodulin-linked agarose (Sigma) that had been precleared with parental cellular lysate that did not express the V2R or V2R-C terminus. The resin was washed three times with DM:CHS wash buffer (DM:CHS-wash: 0.5 and 0.1 mg/ml, respectively, containing 25 mM glycylglycine, 20 mM HEPES, 100 mM NaCl, 5 mM EDTA, pH 7.4) and eluted into 1x Laemmli buffer for 15 min at 37 °C. Samples were run on 12% SDS-PAGE gels, transferred to PVDF, and blotted for receptor using either the mouse anti-GST (Santa Cruz Biotechnology; 1:2000) or the mouse anti-HA.11 (Babco; 1:1000) monoclonal antibodies. When binding to calmodulin, agarose was assessed as a function of calcium concentration, and the amount of free calcium present in solution was calculated using the MaxChelator program, version 2.10 (www.stanford.edu/~cpatton/maxc.html).
Estimation of V2R-C Terminus Affinity for Calmodulin—Supernatants from COS M6 cells transiently expressing either the WT or AAA GST-FLAG-V2R-C terminus were prepared as described above. The WT V2R or AAA V2R-expressing cells were solubilized by scraping 100-mm dishes (2–5) into 1.5 ml of DM-CHS extraction buffer. For some experiments, the extract of AAA V2R was concentrated further using Amicon ultracentrifugal filter devices. Serial dilutions of the WT lysate were performed to obtain a saturation binding curve for binding of the C terminus to a constant quantity of calmodulin-linked agarose, and binding to calmodulin-agarose was performed as described above. The amount of GST-C-terminal protein in the eluate and total fractions was determined quantitatively by comparison to known amounts of bacterially expressed GST. The Western blot bands were quantitated using Scion Image software.
NMR Sample Preparation and Spectroscopy—A peptide fragment of the V2R C terminus (339LLSSARGRTPPSLGPQDES357 with the cysteines normally acylated in V2R (Cys-341 and Cys-342) mutated to serine) was ordered from Genemed Synthesis and subsequently purified by reversed-phase high pressure liquid chromatography. Purity of the eluted peptide was confirmed by electrospray ionization-mass spectrometry and matrix-assisted laser desorption ionization-mass spectrometry. The final peptide was lyophilized and resuspended at 10 mg/ml in 500 mM Tris, pH 7.0. A bacterial expression vector for human calmodulin was kindly provided by Eva Thulin (University of Lund, Lund, Sweden) (11). The construct was transformed into BL21(DE3)pLysS Escherichia coli, and the bacteria were grown in M9 minimal media supplemented with 15NH4Cl. The bacteria were grown and induced with isopropyl--D-thiogalactopyranoside as described previously (11). Briefly, the growth protocol calls for serial dilution of bacteria growing at 30 °C, allowing the bacteria to double prior to each dilution. Following induction at 1 mM isopropyl--D-thiogalactopyranoside, the bacteria are incubated at 37 °C until growth stops. The protein was purified as described previously by Ca2+-dependent binding of calmodulin to phenyl-Sepharose. Samples were loaded in 1 mM CaCl2 and eluted at 1 mM EDTA. NMR samples were prepared by three steps of concentration and resuspension (100-fold dilution at each step) to thoroughly exchange purified calmodulin into 20 mM Tris, 100 mM KCl, 1 mM dithiothreitol, pH 7.0, supplemented with either 2 mM EDTA or 20 mM CaCl2. Final samples were adjusted to contain 10% D2O. The calmodulin concentration was determined by A276. Experiments were conducted on a Bruker Avance500 spectrometer equipped with a cryoprobe. Two-dimensional 15N-1H heteronuclear single quantum correlation experiments were performed at 25 °C, acquiring four scans per increment and 512 points in the indirect (t1) dimension. NMR data were processed and analyzed in XWinNMR (Bruker). For the preliminary titration mentioned in the text (data not shown), the peptide was resuspended at 18.3 mg/ml in 100 mM bis-Tris, pH 6.5, and Ca2+-calmodulin was prepared by thoroughly exchanging 15N-calmodulin into 20 mM bis-Tris, 100 mM KCl, 1 mM dithiothreitol, 20 mM CaCl2, pH 6.5.
Assessment of Transcellular Leaks of Polarized MDCKII Cells—Integrity of the cell monolayer via [3H]methoxy-inulin leak was accomplished exactly as described previously (12).
Polarized Localization of V2R at Steady State—For these studies, the appropriate number of 24.5-mm Transwells of polarized (7 days growth) WT or mutant V2R were biotinylated with NHS-Biotin (1 mg/ml) on the apical or basolateral surface as described previously (12). The cells were scraped into RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, pH 7.4) with protease inhibitors (100 µM PMSF, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin) present, and the extracts were centrifuged at 100,000 x g. The supernatant (detergent-solubilized preparation) was incubated overnight at 4 °C with streptavidin-agarose. The streptavidin-agarose resin was washed 2x with RIPA buffer with protease inhibitors and eluted with SDS sample buffer (1.6% SDS, 8.3% glycerol, 167 mM Tris, pH 6.8) for 10 min at 95 °C, and the eluate was resolved by SDS-PAGE on 12% gels. The resolved proteins, representing the fate of the V2R at the cell surface at the time of biotinylation, were transferred to PVDF, and the biotinylated, epitope-tagged V2R was identified by Western blot analysis using mouse HA.11 antibody against the HA epitope.
Cell Surface Receptor Quantitation via Intact Cell ELISA—MDCKII cells stably expressing WT V2R or AAA V2R were seeded at 1 x 104 cells per well of a 96-well plate 1–2 days prior to the experiment. The medium was replaced with serum-free DMEM 20 min before the assay. Cells were treated with either vehicle alone or with 1 µM AVP and placed in the incubator at 37 °C and 5% CO2 throughout the drug treatment. The vehicle was 50 mM Tris, pH 7.4, 3 mM MgCl2, 1 mM EDTA, pH 8.0, 0.1% BSA, 1 mg/ml bacitracin. After treatment, the medium was removed, and the cells were fixed with 4% paraformaldehyde containing 0.12 M sucrose in DPBS-CM for 20 min at room temperature. Cells were washed twice with DPBS-CM for 5 min each and treated with 3% BSA in DPBS-CM to block nonspecific antibody binding for 30 min at 37 °C. Cells were incubated with 1:500 of anti-rat HA (Roche Applied Science) in 3% BSA in DPBS-CM for 1 h at 37 °C and washed three times with DPBS-CM. Cells were incubated with 1:100 horseradish peroxidase-conjugated goat anti-rat (Amersham Biosciences) in 3% BSA in DPBS-CM for 1 h at 37 °C. The colorimetric substrate, o-phenylenediamine dihydrochloride (Pierce, 1 mg/ml), was added and incubated for 30 min to 1 h at room temperature. The reaction was stopped with the addition of an equal volume of 1 M sulfuric acid. The absorbance at 490 nm was determined for each well using a SpectraMAX 190 microtiter plate reader (Molecular Devices). Four replicates of each treatment condition were performed per experiment.
Immunolocalization of the Wild Type and Mutant V2R—Stably expressing WT V2R MDCKII cells were grown in clear 12-mm Transwell chambers and maintained for 6–9 days prior to fixing with 4% paraformaldehyde for 20 min. Paraformaldehyde-fixed cells were washed in DPBS-CM, rinsed with 50 mM NH4Cl in DPBS-CM for 15 min, and permeabilized with 0.2% Triton X-100 in DPBS-CM for 15 min at room temperature. Permeabilized cells were washed once with DPBS-CM and incubated with DPBS-CM containing 2% BSA for 30 min. The Transwells were carefully cut away from their plastic support and placed into a 12-well plate for antibody labeling. The cells were incubated with a 1:500 dilution of rat anti-HA monoclonal primary antibody for 1 h at room temperature. After rinsing cells three times in DPBS-CM (10 min/wash), a 1:1000 dilution of Alexa488-conjugated goat anti-rat IgG in DPBS-CM with 2% BSA was added, and the cells were incubated in the dark for 1 h at room temperature. Cells were then washed prior to mounting on glass slides with Aqua-Poly/Mount and covered with a glass coverslip. Slides were stored in the dark until examination with a Zeiss LSM 510 confocal laser scanning inverted microscope in the Vanderbilt Cell Imaging Core Facility.
For the AVP-mediated internalization studies, polarized cells were treated either with vehicle alone or with 1 µM AVP, which was added to both the apical and basolateral chambers of the Transwells, and incubated at 37 °C and 5% CO2 throughout the drug treatment. After treatment, the medium was removed, and the cells were fixed and labeled for immunofluorescence examination as described.
Competition Binding—To assess the potency of AVP in competing for the WT V2R and AAA V2R, [3H]AVP binding assays were performed. Confluent 100-mm dishes of MDCKII cells stably expressing the WT V2R or AAA V2R were placed on ice and washed once with DPBS-CM. The cells were scraped into 15:5:5 buffer (15 mM HEPES, 5 mM EGTA, 5 mM EDTA, pH 7.6) containing 100 µM PMSF and centrifuged at 18,000 rpm in an SS-34 rotor for 15 min. The pellet was resuspended in 15:5:5 buffer by sequential trituration with 22- and 25-gauge needles. Centrifugation and resuspension was repeated one time. Finally, the pellet was resuspended in 50 mM Tris, pH 7.4, 3 mM MgCl2, 1 mM EDTA, pH 8.0, 0.1% BSA, and 1 mg/ml bacitracin, which was included to protect the radioligand from proteolysis. Membranes were incubated, with shaking, for 1 h at room temperature in the presence of 1.4 nM [3H]AVP. The incubation was terminated by harvesting on a Brandel cell harvester. Nonspecific binding was defined as that binding detected in the presence of 10 µM AVP. The samples were counted using a Packard 1600 TR Tri-Carb scintillation counter. Kd and Bmax values were calculated from these homologous competition binding experiments as described by Limbird and Motulsky (13).
cAMP Accumulation—Basal or AVP-mediated cAMP accumulation in intact cells was measured by assessing the conversion of [3H]ATP into [3H]cAMP in cells prelabeled with [3H]adenine to permit synthesis of [3H]ATP intracellularly. MDCKII cells were seeded into 24-well plates at 1 x 105 cells per well. Twenty four hours later, the cells were labeled overnight (12–16 h at 37 °C) in supplemented DMEM containing 6 µCi/ml of [3H]adenine (PerkinElmer Life Sciences or MP Biomedicals). On the day of the assay, cells were placed on a 37 °C plate and rinsed with DPBS-CM, and serum-free DMEM supplemented with 20 mM HEPES was added. Cells were treated with AVP for 15 min, with vehicle alone to obtain basal cAMP levels, or with 10 µM forskolin to assess direct activation of adenylyl cyclase. Reactions were terminated by adding 750 µl of an ice-cold solution containing 12% trichloroacetic acid, 2 mM cAMP, 2 mM ATP, and [14C]cAMP (1400 cpm/ml) to permit an assessment of recovery of [3H]cAMP in subsequent purification steps. Cells were then placed on ice for 10 min, after which the reactions were transferred to glass test tubes using disposable transfer pipettes. Each well was washed with 120 µl of DMEM supplemented with 20 mM HEPES and combined with its respective harvested sample for a final volume of 2 ml. After neutralization with 120 µl of 5 N NaOH, cellular debris was pelleted by centrifugation (3000 rpm, 10 min at 4 °C). The supernatants were subjected to sequential Dowex and alumina column chromatography to isolate [3H]cAMP (14). Recovery of [3H]cAMP from other triturated nucleotides during this procedure was corrected based on the recovery of [14C]cAMP tracer in the stop solution.
Measurement of Intracellular Ca2+—We used a FlexStation II (Molecular Devices) fluorometric imaging plate reader to measure changes in intracellular Ca2+. MDCKII cells stably expressing the WT V2R or AAA V2R were plated in 96-well clear bottom black microplates (Corning Glass) at 1 x 105 cells per well and incubated overnight at 37 °C and 5% CO2. On the day of the assay, cells were loaded with Calcium 3 dye (FLIPR Calcium 3 assay kit, Molecular Devices) for 1 h at 37 °C in Hanks' buffered saline solution (HBSS), pH 7.4, containing 0.1% BSA and 2.5 mM probenecid, in the presence (Invitrogen catalog number 14065-056) of 1.26 mM calcium. After loading, cells were washed three times with 1x HBSS, pH 7.4, containing 0.1% BSA and 2.5 mM probenecid lacking calcium (Invitrogen catalog number 14185-052, supplemented with 0.9 mM magnesium). Cells were placed in 1x HBSS with or without calcium as indicated and placed in the FlexStation II for the assay. The machine was used in Flex mode, and the fluorescent intensity was measured from the bottom with the excitation at 485 nm and emission at 525 nm for 90 s at 25 °C. Trituration upon addition of compound during the FlexStation assay had no effect on the position of the AVP concentration-response curve for the WT V2R.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 1, the full-length V2R also interacts with calmodulin in vitro in a calcium-dependent fashion. Detergent extracts from MDCKII cells expressing WT V2R were incubated with calmodulin-linked agarose. Calcium-dependent binding of the V2R occurred at pH 7.6, and even greater binding was observed at pH 6.8, which more closely resembles intracellular pH. Binding could be detected at 1 µM free [Ca2+], based on calculations using the MaxChelator program as detailed under "Experimental Procedures." Evaluation of lower concentrations of calcium could not be achieved with confidence, based on predictions of the MaxChelator program. In some experiments, as in Fig. 1A, binding of precursor forms (22) of the V2R to calmodulin also was detected.
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5% of that for WT V2R C terminus at equivalent protein concentrations (see below for affinity estimated for the WT V2R C terminus). The in vitro interaction with GST-V2R, like for the holo-V2R (Fig. 1), was indeed found to be calcium-dependent, because elution of the V2R C terminus from calmodulin-agarose could be affected by the addition of 10 mM EDTA (Fig. 2D). Together, these data demonstrate that the interaction of the V2R C terminus with calmodulin-linked agarose depends on the presence of calcium and an essential RGR motif in the V2R C terminus.
To confirm further the observed interaction between the V2R C-terminal RGR motif and calmodulin, we performed a series of NMR experiments using uniformly 15N-enriched calmodulin and a peptide fragment of the V2R C terminus containing the RGR motif (RGRm). For these experiments, RGRm was titrated into solutions of calmodulin, in both the absence (190 µM calmodulin) and presence (150 µM calmodulin) of calcium. The peaks in these two-dimensional spectra arise from backbone and side chain amide N-H groups, providing a minimum of one probe for each residue in the protein. The position of each peak is highly sensitive to both the structure and influences from the surrounding environment.
Fig. 3A shows a selected region of the two-dimensional 15N-1H heteronuclear single quantum correlation NMR spectra obtained for Ca2+-loaded calmodulin in the absence (black) and presence (red) of 1.5 molar eq of the RGRm peptide, whereas Fig. 3B shows the corresponding spectra for the apoprotein. The observation that many peaks shift position when the peptide is present in Fig. 3A but not Fig. 3B reveals that RGRm binds to calmodulin in a Ca2+-dependent manner, corroborating the findings in Fig. 2D.
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1:1 indicated that the dissociation constant (Kd) must be at least 100-fold lower than the protein concentration of 150 µM. Thus, the Kd of RGRm for calmodulin is 1.5 µM. An estimate of the affinity of the entire V2R C terminus was obtained by examining the concentration-dependent binding of the GST-V2R-C terminus to calmodulin-agarose by using the methods described in Fig. 2C. The estimated Kd of calmodulin for the WT V2R C terminus was 200 nM (evaluated in three independent experiments). We were not able to rigorously estimate a Kd value for the AAA V2R C terminus due to limitations in the amount of mutant protein available to achieve concentrations where we could detect binding. The AAA V2R, Like the WT V2R, Localizes to the Basolateral Surface of Polarized MDCKII Cells—To address the functional consequence of mutating the V2R C-terminal region on receptor localization, we stably expressed the AAA V2R in Madin-Darby canine kidney (MDCKII) cells. This model system allows the study of trafficking of surface proteins to polarized cellular surfaces, and we have examined the trafficking of WT and mutant V2R X-linked nephrogenic diabetes insipidus alleles in these cells previously (22). As shown in Fig. 4A, both the WT and AAA V2R are localized at the cell surface (Fig. 4A, upper panels, XY scan) of polarized stably expressing MDCKII cells, based on recognition of the HA epitope introduced into the N terminus of each of these receptors; a Z scan of these cells reveals that both the WT and AAA V2R are enriched at the lateral subdomain (Fig. 4A, lower panel, Z scan) of polarized cells.
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AVP-induced AAA V2R Internalization Parallels That of the WT V2R—We examined the AVP-induced internalization of the AAA V2R versus WT V2R by using a cell surface ELISA. Calmodulin has been shown to play a role in the endocytosis of the serotonin 5-HT1A receptor, which is important for receptor-mediated extracellular signal-regulated kinase activation (27). The V2R is known to undergo agonist-induced internalization, without recycling to the cell surface (10). In Fig. 5A, both the WT and AAA V2R remained localized at the cell surface during the 90-min incubation of these studies, whereas treatment with 1 µM AVP led to an accelerated rate of V2R loss from the surface that was indistinguishable for the WT and AAA V2R. Parallel immunofluorescence studies (Fig. 5B) revealed that WT and AAA V2R have similar internalization profiles at 30 and 90 min, with the appearance of intracellular puncta. Because the AAA V2R appeared more diffuse upon internalization in these studies, we exploited a complementary approach, i.e. antibody feeding, to assess any possible differential fates of WT versus AAA V2R following internalization. In these studies, surface receptor was bound to anti-HA antibody by pretreating the cells for 60 min at 4 °C, and cells subsequently were treated with agonist. By using this approach, the cell surface pool of WT V2R and AAA V2R internalized to intracellular puncta that were visually indistinguishable (data not shown), arguing against a different fate of WT versus AAA V2R post-internalization. A second AAA V2R clone stably expressed in MDCKII cells was tested and displayed similar profiles (data not shown). These data indicate that the AAA V2R internalizes in response to AVP with kinetics and a morphological profile comparable with that of the WT V2R.
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As shown in Fig. 7, the WT and AAA V2R displayed similar concentration dependence in eliciting cAMP accumulation in response to increasing concentrations of AVP, with EC50 values of 6.0 and 1.5 nM respectively. We performed a nonparametric, two-tailed t test which yielded a p value of 0.49, indicating that these EC50 values are not significantly different. These data indicate that mutation of the V2R C terminus to create the AAA V2R does not perturb receptor coupling to Gs, which is the transducer of V2R activation by AVP to elevations in cAMP production.
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WT V2R Stimulation of Increases in Intracellular Calcium Cannot Be Mimicked by the AAA V2R—In the isolated rat inner medullary collecting duct (4), the V2R has been demonstrated to stimulate an increase in intracellular calcium in response to AVP, which is necessary for the enhanced water permeability at the apical surface of renal cells (28). To test the ability of the AAA V2R to increase intracellular calcium, we utilized the FlexStation II (Molecular Devices) to measure real time changes in intracellular calcium, as described under "Experimental Procedures." The WT V2R demonstrated a dose-dependent increase in intracellular calcium in response to AVP stimulation. This rise in calcium was not observed for WT V2R-expressing cells pretreated with 10 µM of the V2R antagonist SR121463B or for MDCKII parental cells not expressing heterologous V2R (Fig. 8A).
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We next tested the source of the increase in intracellular calcium. Fig. 8C illustrates that the removal of extracellular calcium blocked the AVP-induced increase in intracellular calcium for the WT V2R. Also, chelation of intracellular calcium by BAPTA-AM inhibited the AVP-induced rise in intracellular calcium by the WT V2R. These data are consistent with the interpretation that both extra- and intracellular stores of calcium contribute to AVP-stimulated increases in [Ca2+]i. Our preliminary studies of the effects of antagonists of calcium mobilization (e.g. TMB-8) on AVP-induced calcium elevations were confounded by profound changes in basal [Ca2+]i, as observed previously in tests of calmidazolium (29), the calmodulin antagonist, on modulation of [Ca2+]i; consequently, we did not pursue this avenue of investigation further.
We tested whether the addition of membrane-permeant cAMP analogs could stimulate an increase in intracellular calcium in WT V2R-expressing MDCKII cells. Fig. 8D illustrates that neither dibutyryl-cAMP nor 8-bromo-cAMP stimulated an increase in intracellular calcium. Similarly, the addition of 10 µM forskolin also did not elicit a calcium response, even though forskolin caused a readily measured increase in cAMP accumulation in all cell lines evaluated (Fig. 7). As a positive control, we observed that ATP stimulated calcium rises in the same experiment (data not shown), presumably via purinergic receptors (30), indicating that the target cells evaluated were capable of mobilizing intracellular calcium. Taken together, these data illustrate that the accumulation of cAMP and increase in intracellular calcium appear to be occurring independently of one another in response to AVP stimulation of the V2R in MDCKII cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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200 nM, consistent with data obtained for other GPCR peptides and calmodulin (Table I). For the V2R, mutation of the calmodulin-interacting region blocks elevations in intracellular calcium in response to AVP. Other calmodulin-interacting domains also may exist in the V2R, as the first intracellular loop contains a second putative binding site predicted by sequence homology (31) and association of the holo-AAA V2R with calmodulin-agarose has been detected in preliminary studies (data not shown).
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signaling. For the 5-HT1A receptor, calmodulin modulates receptor endocytosis required for subsequent extracellular signal-regulated kinase activation in that system (27). In our studies, we learned that the interacting domain for calmodulin includes the RGR sequence in the C terminus. However, as for other GPCRs that interact with calmodulin, this motif does not correspond exactly to classical calmodulin-interacting motifs, such as the IQ or the 1-8-14 motifs (24).
The V2R increases cAMP in virtually all target cells evaluated. The V1R is classically responsible for elevations in [Ca2+]i, but the V2R also has been implicated in elevating intracellular calcium concentrations in response to AVP in some target cells (28). We similarly observed that the WT V2R causes rapid elevations in [Ca2+]i, and this AVP-evoked enhancement of [Ca2+]i is not observed for the AAA V2R. Despite the complete loss of AVP-stimulated elevation of [Ca2+]i by the AAA V2R, we observed virtually no change in coupling to Gs and activation of adenylyl cyclase, or to mitogen-activated protein kinase (data not shown) for the AAA V2R when compared with the WT V2R. These findings reveal that AVP stimulation of [Ca2+]i in MDCKII cells occurs independently of these other second messenger systems. This interpretation is corroborated by our findings that neither cell-permeant cAMP analogs nor forskolin, which causes dramatic increases in cAMP (Fig. 7), elevate [Ca2+]i in any of our MDCKII clonal cell lines. This dissociation of the cAMP and calcium pathways is in agreement with data from others (35, 36) evaluating AVP responses in isolated rat inner medullary collecting ducts but is in contrast to the findings of Chou et al. (4) who also using isolated rat inner medullary collecting ducts as a model system.
Another interesting aspect of our findings is the different concentration-response relationship between AVP stimulation of cAMP accumulation versus elevation of [Ca2+]i. The AVP concentration-response curve for increased intracellular calcium of the WT V2R appears to be almost threshold in nature and is not amplified like the cAMP response (compare occupancy, Fig. 6, with cAMP response, Fig. 7.) The blockade of AVP-stimulated calcium elevation by both removal of extracellular calcium and chelation of intracellular calcium would be consistent with a mechanism for calcium mobilization involving both extra- and intracellular stores. Future studies will hopefully reveal the in vivo physiological significance of this novel calcium elevation in response to high V2R occupancy.
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FOOTNOTES |
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** To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Robinson Research Bldg., Rm. 464, Nashville, TN 37232-6600. Tel.: 615-322-6379; Fax: 615-322-6378; E-mail: lee.limbird{at}vanderbilt.edu.
1 The abbreviations used are: V2R, V2 vasopressin receptor; AVP, arginine vasopressin; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; CaM, calmodulin; GPCR, G protein-coupled receptor; HA, hemagglutinin; MDCKII, Madin-Darby canine kidney II; NHS, sulfo-N-hydroxysuccinimide; DPBS, Dulbecco's phosphate-buffered saline; RGRm, V2R C-terminal RGR motif; WT, wild type; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; TEMED, N,N,',N'-tetramethylethylenediamine; 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HBSS, Hanks' buffered saline solution; PMSF, phenylmethylsulfonyl fluoride.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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AAA (344–346) stably expressed in MDCKII cells was compared with the WT V2R by confocal microscopy. Cells were grown for 7 days on clear 12-mm Transwells at 37 °C, fixed with 4% paraformaldehyde, and immunoisolated as described under "Experimental Procedures." The rat anti-HA monoclonal antibody was detected by Alexafluor-488-conjugated goat anti-rat IgG secondary antibody. Localization of the HA epitope tag on the N terminus of the WT V2R and AAA V2R was analyzed on a Zeiss LSM 510 laser confocal microscope, where the XY is presented in the upper panel of each image. Z scans shown in the lower panel display a laser-sectional side view of the V2R-expressing MDCKII cells. The yellow line across each XY scan represents the laser sectioning that resulted in the Z scan (A). MDCKII cells stably expressing the WT V2R or AAA V2R were grown for seven to 8 days on 24.5-mm Transwells. The data shown are from a representative experiment performed at least three times. B, polarized MDCKII cells were surface-biotinylated and subjected to streptavidin-agarose chromatography, SDS-PAGE, and Western blot analysis for epitope-tagged WT V2R and AAA V2R, n = 3, as described under "Experimental Procedures." The data shown are from a representative experiment, with the average receptor distribution as 19% apical, 81% basolateral for the WT V2R, and 22% apical and 78% basolateral for the AAA V2R.
)or with AVP (
), and the AAA V2R was treated with vehicle (
). B, AVP-mediated V2R internalization viewed with immunofluorescence. The localization of the WT V2R and AAA V2R in polarized MDCKII cells was observed at time 0 and following stimulation with 1 µM AVP. Cells were grown on 12-mm clear Transwells (Costar) for 6 days to achieve polarization of these stably expressing clonal cell lines. The medium was replaced with serum-free medium 20 min before the experiment. Cells were incubated for 0, 30, or 90 min at 37 °C in the absence (time 0) or presence (30 and 60 min) of AVP. The medium was aspirated, and cells were immediately fixed with 4% paraformaldehyde and processed for examination of immunofluorescence as described under "Experimental Procedures." The data shown are from one experiment, where three fields of view were imaged, performed twice. Similar findings also were observed when the same question was addressed by feeding anti-HA antibody at the cell surface and watching the entry of the HA-tagged V2R over time as described under "Experimental Procedures."
; n = 5), and for clone 11 of the AAA V2R (
), a V2R antagonist. Parental MDCKII cells (224) not heterologously expressing the V2R did not increase intracellular calcium in response to any concentration of AVP tested. Data are from multiple independent experiments performed in duplicate: WT V2R (n = 8); AAA V2R (clone 5, n = 6); AAA V2R (clone 11, n = 4); parental cell line (n = 3). B, the AAA V2R is unable to increase intracellular calcium at any concentration of AVP. Both clone 5 (