Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells

Richard Bouley, Tian-Xiao Sun, Melissa Chenard, Margaret McLaughlin, Mary McKee, Herbert Y. Lin, Dennis Brown, and Dennis A. Ausiello

Program in Membrane Biology and Renal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Submitted 11 October 2002 ; accepted in final form 2 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.

polarized cell culture; tyrosine motif; µ1b adaptor motif; protein traffic


INTERACTION BETWEEN THE TYPE 2 VASOPRESSIN RECEPTOR(V2R), a member of the G protein-coupled receptor superfamily (GPCR), and the antidiuretic hormone vasopressin (AVP) plays a major role in blood volume and osmolality regulation (3, 16, 23, 44, 48, 60). The physiological effects of AVP on water transport in the kidney occur in collecting duct principal cells (13). After AVP binding to the V2R, heterotrimeric G protein-stimulated adenylyl cyclase activity leads to an elevation of intracellular cAMP and activation of protein kinase A (PKA) (2). PKA then phosphorylates the water channel aquaporin 2 (AQP2). The location of AQP2 shifts from intracellular vesicles to the apical plasma membrane of principal cells, leading to increased water permeability and subsequent transepithelial water reabsorption (13).

The sustained interaction of GPCRs with their agonists causes a time-dependent loss of response known as downregulation, which is an important mechanism for terminating GPCR signaling. Downregulation is a complex phenomenon believed to depend on ligand-induced changes in receptor conformation that allows receptor phosphorylation, desensitization (reduction in signaling upon ligand binding), internalization, and sequestration (42). Ligand-induced receptor internalization (endocytosis) can occur via different mechanisms. Whereas many GPCRs such as metabotropic glutamate, endothelin, and {beta}2-adrenergic and µ-opioid receptors are internalized in a clathrin-coated pit-dependent manner (12, 17, 63), an alternative pathway of internalization involves the interaction of GPCRs with caveolin, a specific protein associated with caveolae (14). Several GPCRs such as sphingosine 1-phosphate and muscarinic cholinergic receptors are internalized exclusively by the caveolae pathway (19, 30). Other receptors that have been shown to interact with caveolin, including {beta}2-adrenergic, B2 bradykinin, ETA endothelin, and angiotensin II receptors (15, 18, 34, 62) may utilize both the clathrin-dependent and caveolae pathways for internalization. Finally, M2 muscarinic acetylcholine and somatostatin receptors seem to be internalized exclusively through uncoated vesicles independent of either clathrin-coated pits or caveolae (40, 56).

The exact details of the internalization process of the V2R after addition of ligand are not known. The COOH-terminal tail of the V2R has been shown to be important for internalization (32). After agonist stimulation, the COOH tail of the V2R is phosphorylated and {beta}-arrestin is recruited to the receptor before the V2R-{beta}-arrestin complex is removed from the cell surface by endocytosis (11, 50). It has been suggested that ligand-induced V2R endocytosis is mediated via clathrin-coated pits (28, 54). However, the precise mechanism and the determinants that serve as positive signals for triggering internalization of the V2R have not been elucidated.

The cytoplasmic COOH tail of the V2R contains the amino acid motif NPxxY (where "x" represents any amino acid) near the end of the seventh transmembrane domain. This motif is highly conserved among GPCRs. Recent work in other GPCRs suggests that the NPxxY motif may contribute to the internalization of some, but not all, of the receptors in which it is present. Although the NPxxY motif is essential for ligand-induced internalization of {beta}2-adrenergic and neurokinin 1 receptors (5, 9), it is not required for the internalization of angiotensin II or gastrin-releasing peptide receptors (29, 41, 65). These results suggest that the role of the NPxxY motif on ligand-induced receptor internalization is receptor specific. The purpose of this study was to examine the role of the NPxxY motif in ligand-induced V2R internalization, cell surface expression, and signaling. A mutant FLAG-tagged V2R (FLAG-V2R-Y325F) in which tyrosine 325 was substituted for phenylalanine was stably expressed in LLC-PK1a epithelial cells and compared with a FLAG-tagged wild-type V2R construct. Substitution of Tyr325 by Phe325 prevented the ligand-induced internalization of V2R but did not affect the polarity of cell surface expression and did not prevent ligand binding or signal transduction by adenylyl cyclase.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and cells. AVP (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly), bovine serum albumin (BSA), 3-isobutyl-1-methyl-xanthine (IBMX), forskolin, glucose, sodium cacodylate, tyrosine, phenylalanine, acetic acid, alcohol, and inorganic salts were purchased from Sigma (St. Louis, MO). [3H]AVP (65.8 Ci/mmol) was from NEN (Boston, MA). All cell culture reagents including Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, Geneticin, Lipofectamine 2000, phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were from GIBCO BRL (Grand Island, NY). The Transwell cell culture filter chambers were from Costar (Cambridge, MA). Paraformaldehyde solution EM grade, osmium tetroxide (OsO4), glutaraldehyde, and Epon were purchased from Electron Microscopy Sciences (Washington, PA). The V2R cDNA was a gift from Dr. M. Birnbaumer (UCLA School of Medicine, Los Angeles, CA). The pcDNA3 vector was purchased from Invitrogen (Carlsbad, CA). LLC-PK1 native cells have been used in the Renal Unit for over 20 yrs. The LLC-PK1a cells were provided by Dr. Steven Krane (Arthritis Unit, Massachusetts General Hospital, Boston, MA). LLC-µ1B cells were provided by Dr. Ira Mellman (Department of Cell Biology and Ludwig Institute for Cancer Research, Yale University, New Haven, CT). These cells are LLC-PK1 cells that have been transfected to stably express the µ1B isoform of an adaptor protein that is involved in sorting some membrane proteins to the basolateral plasma membrane (20). The µ1B adaptor protein is not expressed by wild-type LLC-PK1 cells.

Receptor cDNA construction. A FLAG sequence (NH2-Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) was introduced into the extracellular NH2 terminus of the wild-type V2R (FLAG-V2R), and it was subcloned in-frame into the 5'-BamH1 and 3'-Xba1 restriction site of the pcDNA3 vector for eukaryotic cell expression. The mutation of Tyr325 to Phe325 to generate FLAG-V2R-Y325F was performed by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the resulting cDNA was sequenced to confirm the fidelity of the construct.

Transient expression of FLAG-V2R and FLAG-V2R (Y325F) in COS cells. COS cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were transfected with Lipofectamine 2000 when at 80% confluence in 96-well plates. For each well, cells were transfected with 320 ng of DNA in 0.8 µl of Lipofectamine. Transfection reagent was removed after 2 h, and cells were incubated in culture medium for a further 48 h before use in binding or in cAMP assays.

Cell culture and establishment of stable cell lines expressing FLAG-tagged V2Rs in LLC-PK1a cells. LLC-PK1a cells, which express very low levels of endogenous V2R, were cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). To obtain stable V2R wild type (LLC-FLAG-V2R)- and V2RY325F (LLC-FLAG-V2R-Y325F)-expressing cell lines, we plated LLC-PK1a cells at a density of 150,000 cells/60-mm dish, 20 h before transfection. For transfection, Lipofectamine (15 µl) with 4 µg of plasmid DNA was added, and the cells were incubated at 37°C for 4 h and washed once with serum-free DMEM. After 14-20 days of selection in medium containing 1 mg/ml Geneticin (G418), resistant colonies were isolated with cloning rings and transferred to separate culture dishes for expansion and analysis of [3H]AVP binding. For each transfection, several clones were isolated and their [3H]AVP binding distributions characterized (see Table 2). Several clones were produced that expressed each receptor and that all shared similar characteristics in terms of V2R biology, although the absolute number of receptors expressed differed among the different clones. Most of the experiments described here were performed on clones that expressed approximately the same receptor density as the endogenous wild-type V2R in native LLC-PK1 cells (see Fig. 2). One clone that expressed a higher density of FLAG-tagged wild-type receptor was used for the electron microscopy studies shown in Fig. 9 to allow better visualization of the apical receptor by immunogold labeling.


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Table 2. Ratio of [3H]AVP binding between apical and basolateral membranes of native LLC-PK1 cells, LLC-PK1a cells, and LLC-PK1 cells expressing FLAG-V2R or FLAG-V2R (Y325F)

 


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Fig. 2. Apical and basolateral distribution of 3H-labeled vasopressin ([3H]AVP) binding sites in stably transfected LLC-PK1 cell lines expressing FLAG-V2R (wild type) or FLAG-V2R (Y325F). Native LLC-PK1 cells are nontransfected cells that express endogenous porcine V2R. LLC-PK1a cells are a variant cell line that has been selected for its greatly reduced expression of endogenous V2R. All cell lines were grown for 6 days in Transwell cell culture filter chambers. They were incubated at 4°C for 3 h with 9 nM [3H]AVP on the apical (open bars) or basolateral side (filled bars). Nonspecific binding was determined in the presence of 1 µM unlabeled AVP. Columns represent means ± SE of triplicate determinations. Similar data were obtained in 3 separate experiments. AVP binding was detectable on both apical and basolateral plasma membrane domains but was more extensive on the basolateral surface. The ratio of apical-to-basolateral V2R distribution was not significantly different in any of the cell lines examined (see Table 2), but the LLC-PK1a cells showed considerably lower binding of AVP than the other cell lines.

 


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Fig. 9. Electron microscopic immunogold localization of FLAG-V2R in LLC-FLAG-V2R cells after incubation with AVP. A distinct clone of LLC-FLAGV2R cells expressing 11-fold more V2R than the clone used in the biochemical studies was used for these incubations to allow more extensive gold particle labeling of the apical cell surface. In experiments not shown, the V2R in these cells behaved otherwise identically to that expressed in the lower-expressing cell lines used in the remainder of this study. Cells were grown for 6 days on Transwell cell culture filter chambers and incubated with 10 nM AVP for 20 or 60 min. Cells on filters were fixed and incubated with anti-FLAG monoclonal antibody M5 followed by incubation with goat anti-mouse IgG conjugated to gold particles (10 nm) before preparation for electron microscopy. In the nonstimulated cell, gold particles representing the V2R were distributed over the entire cell surface (A). After 20 min of stimulation, some clusters of gold particles were evident in invaginated membrane regions (B, arrows). At higher magnification (B, inset), some of these gold-filled invaginations were shown to have a cytoplasmic coat that is characteristic of clathrin-coated pits (arrows). After 60 min of AVP treatment, very few gold particles remained on the cell surface (C). Bars, 0.5 µm (inset, 0.25 µm).

 

To compare the distribution of the Y325F mutation with another mutation that causes a severe perturbation of intracellular V2R trafficking, we developed and examined a stable LLC-PK1 cell line expressing a R113W V2R mutant. The R113W mutation is a naturally occurring mutation that causes nephrogenic diabetes insipidus and has been reported to have a functional defect in ligand binding and adenylyl cyclase stimulation, as well as an inability to reach the cell membrane in nonpolarized cells (8). As for the Y325F mutation, a FLAG sequence was introduced into the NH2 terminus of the mutant R113W V2R (FLAG-V2R-R113W), and it was cloned in-frame into the BamH1 and Xba1 restriction site of pcDNA3 for eukaryotic cell expression.

[3H]AVP binding to LLC-FLAG-V2R and LLC-FLAG-V2RY325F cells. [3H]AVP binding assays were performed on cells grown on Transwell cell culture filter chambers for 6 days. LLC-FLAG-V2R and LLC-FLAG-V2R-Y325F cells were plated at a density of 100,000 cells/filter (day 1) and grown in the culture medium described above until confluence (106 cells/filter on day 6). Briefly, 0.25 ml of ice-chilled PBS (pH 7.4, containing 0.9 mM CaCl2, 0.9 mM MgCl2, 3.5 mM KCl, and 1 mg/ml glucose), with 1 mM tyrosine, 1 mM phenylalanine, and 0.5% BSA containing the appropriate dilution of [3H]AVP, was added to each well. [3H]AVP was added to the upper or lower chamber to determine either apical (upper chamber) or basolateral (lower chamber) [3H]AVP binding. Incubation was carried out for 3 h at 4°C. Nonspecific binding was determined in the presence of excess unlabeled AVP (1 µM). Incubations were stopped by two rinses with ice-cold PBS at pH 7.4. The filters were cut out from the chambers and transferred to scintillation vials containing 500 µl of NaOH (0.1 N). After 12 h, 5 ml of scintillation fluid (Optic-Fluor; Packard, Groningen, The Netherlands) was added. The bound radioactivity was determined using the liquid-scintillation analyzer Tricarb 2200 CA from Packard. Transepithelial leak of [3H]AVP was <5% between the upper and lower chambers. [3H]AVP binding assays on LLC-µ1B cells expressing endogenous V2R were performed using the same procedure.

cAMP assays. Briefly, LLC-FLAG-V2R and LLC-FLAGV2R-Y325F cells grown for 6 days in Transwell cell culture filter chambers (or COS cells grown as described above) were pretreated for 15 min with the phosphodiesterase inhibitor IBMX (1 mM), followed by incubation with AVP (1 µM) for 10 min at 37°C on the apical side only, the basolateral side only, or both sides simultaneously. Intracellular levels of cAMP were measured with the BioTrak kit (Amersham Pharmacia Biotech, Arlington Heights, IL) as previously described (10). Each intracellular cAMP assay was performed in triplicate. A similar technique was used to study the effect of AVP on the desensitization and resensitization of the adenylyl cyclase response in cells expressing either wild-type or mutant V2R grown to confluence on 96-well plates. Both cell lines were incubated with 1 µM AVP for 20 min to induce desensitization. At the end of the incubation, the medium was removed and cells were washed with ice-cold acetic acid in PBS to remove agonist as described below. The cells were washed extensively with cold PBS. Cells to be assessed for resensitization were washed twice with DMEM, and incubation was continued in fresh serum-free medium for 45 min. The resensitization period was terminated by washing with cold PBS. Cells were reincubated with different concentrations of AVP (0.001-1,000 nM) in PBS containing IBMX (1 mM) at 37°C for 20 min. The intracellular levels of cAMP were measured with the BioTrak kit (Amersham Pharmacia Biotech) as previously described (10).

Ligand-induced V2R internalization assays. Internalization assays were performed by pretreating cells grown on Transwell filter chambers with AVP (1-1,000 nM) for 20 min at 37°C. The cells were incubated successively in ice-cold acetic acid (5 mM) in PBS for 5 and 30 min. Greater than 99.5% of the AVP attached to the cell surface was removed by this treatment, as confirmed by removal of [3H]AVP that had been added to the cells (data not shown). The acidic pH was neutralized by two consecutive washes in ice-cold PBS, pH 7.4. The washed filters were then used in binding assays as described above to determine the amount of remaining cell surface V2R. A similar series of experiments was performed after forskolin (10 µM) had been applied to the apical or basolateral cell surface for 30 min at 37°C before cold washing and [3H]AVP binding assays were carried out on both membrane domains, as described above.

Immunofluorescence staining of the FLAG epitope-tagged V2R. Cells were grown on filters for 6 days for staining of the FLAG-V2R. Both LLC-FLAG-V2R and LLC-FLAG-V2RY325F cells were either not exposed to AVP or incubated for 60 min in the presence of AVP (1 µM) before being fixed with 4% paraformaldehyde, 5% sucrose in PBS for 20 min at room temperature. FLAG-V2R (R113W) cells were fixed identically but without prior exposure to AVP. The cells were washed three times in PBS and incubated at room temperature for 1 h with an anti-FLAG monoclonal antibody (M5) (11 µg/ml; Sigma). The cells were then incubated for 1 h with donkey anti-mouse IgG conjugated to fluorescein (FITC) (12.5 µg/ml; Jackson ImmunoResearch, West Grove, PA), washed, and mounted with Vectashield (Vector Labs, Burlingame, CA). FLAG-V2R (R113W) cells were counterstained with 0.01% Evan's blue for 1 min to stain plasma membranes before being mounted in Vectashield. The cells were examined using a Bio-Rad Radiance 2000 confocal microscope.

Electron microscopy and immunogold labeling. After the sixth day of culture on filters, cells were treated on both sides simultaneously with AVP (10 nM) for 20 and 60 min in DMEM. After incubation, cells were washed twice in DMEM and then fixed for 30 min in paraformaldehyde lysine periodate (PLP) fixative prepared as previously described (10). The cells were washed three times in PBS and incubated at room temperature for 1 h with PBS/1% BSA before being incubated overnight at 4°C with anti-FLAG monoclonal antibody M5 (33 µg/ml) to detect the extracellular FLAG epitope in the V2R protein. Filters were washed in PBS and then incubated for 24 h at 4°C with goat anti-mouse IgG coupled to 10-nm gold particles (Ted Pella, Redding, CA) diluted in PBS. After being rinsed in PBS, the filters were fixed in 2% glutaraldehyde for 1 h at room temperature. The filters were rinsed in 0.1 M sodium cacodylate buffer, pH 7.4, and immersed in 1% OsO4 in cacodylate buffer for 1 h at room temperature. The filters were rinsed in cacodylate buffer, dehydrated through a graded series of ethanols to 100% ethanol, and infiltrated overnight with Epon. The filters were embedded in Epon between liquid release agent-coated glass slides and coverslips overnight at 60°C. After polymerization, small pieces of the filters were cut and reembedded in the tips of flat embedding molds. Thin sections were cut on a Reichert Ultracut E ultramicrotome, collected on Formvar-coated slot grids, and poststained with uranyl acetate and lead citrate. The sections were examined in a Philips CM 10 electron microscope.

Statistical analysis. Data are expressed as means ± SE. Statistical analyses were made using the unpaired or paired Student's t-test when applicable. Differences were considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transient expression of FLAG-V2R or FLAG-V2R (Y325F) in COS cells. FLAG-tagged V2R (FLAG-V2R) and FLAG-tagged V2R (Y325F) were transiently transfected into COS cells that lack endogenous V2R. Dose-displacement experiments were done using live cells (Table 1). Scatchard analysis showed that 1) the affinity of AVP for FLAG-V2R and FLAG-V2R (Y325F) binding sites was similar, and 2) the density of FLAG-V2R and FLAG-V2R (Y325F) at the cell surface was greater for the wild-type receptor. Incubation of COS cells transfected with FLAG-V2R or FLAG-V2R (Y325F) with AVP produced a dose-dependent intracellular cAMP accumulation in both cell types, but the increase was greater in FLAG-V2R transfected cells at any given concentration of AVP (Fig. 1A). COS cells with no transfected receptor did not show a significant increase in intracellular cAMP even in the presence of 1 µM AVP (Fig. 1A). In contrast to COS cells, the concentration dependence of cAMP accumulation was similar when AVP was applied to native LLC-PK1a epithelial cells and to cells expressing the Y325F mutant receptor (Fig. 1B).The increase of intracellular cAMP at each AVP concentration was actually about two times greater in LLC-FLAG-V2R-Y325F than in LLC-PK1a cells. Cells transfected with the wild-type V2R responded to lower concentrations of AVP, but the maximum level of cAMP achieved in these cells was approximately the same as in the cells transfected with FLAG-V2R-Y325F. Some of these differences are probably due to the variability in the density of total cell surface receptors expressed in the various cell lines (Fig. 1B). However, taken together, these results clearly show that the Y325F V2R is capable of signal transduction via cAMP in the absence of any endogenous wild-type V2R in COS cells and that cAMP generation is increased by transfection of the Y325F mutant receptor into LLC-PK1a epithelial cells.


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Table 1. [3H]AVP binding characteristics of COS cells transiently expressing FLAG-V2R or FLAG-V2R (Y325F)

 


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Fig. 1. A: intracellular cAMP levels in COS cells transiently transfected with FLAG-tagged vasopressin type 2 receptor (V2R) wild type (FLAG-V2R), a Y325F mutant V2R (FLAG-V2R-Y325F), or pcDNA3 vector. Intracellular cAMP in COS cells expressing FLAG-V2R ({bullet}) was maximal around 10 nM AVP and decreased somewhat at higher concentrations. Elevation of intracellular cAMP in COS cells expressing FLAG-V2R-Y325F ({blacksquare}) increased slowly to a reach a maximum level at ~100 nM AVP. No significant increase of intracellular cAMP in COS cells expressing the pcDNA3 vector alone was observed ({blacktriangleup}). Curves are representative of 3 independent experiments, and each measurement was carried out in triplicate. B: intracellular cAMP in native LLC-PK1a cells ({blacktriangleup}) and in cells stably transfected with FLAG-V2R ({bullet}) or FLAG-V2R-Y325F ({blacksquare}). Intracellular cAMP levels in all cells were maximal around 10 nM AVP. In this series of experiments, the absolute level of cAMP attained was greatest in cells transfected with the mutant FLAG-V2R-Y325F. Curves are representative of 3 independent experiments, and each was carried out in triplicate.

 

Characterization of [3H]AVP binding sites in LLC-FLAG-V2R and LLC-FLAG-V2R-Y325F polarized cell monolayers. LLC-PK1a cells, which express a very low level of endogenous porcine V2R, were transfected to generate cell lines expressing LLC-FLAG-V2R and LLC-FLAG-V2R-Y325F. Characterization of [3H]AVP binding sites was performed on the sixth day of culture when cells had developed into a polarized monolayer with a high transepithelial resistance (data not shown). In both LLC-FLAG-V2R and LLC-FLAG-V2RY325F cells, [3H]AVP binding was found to be time dependent, reversible, and saturable. Native LLC-PK1, LLC-PK1a, and transfected cells lines all showed both apical and basolateral binding of [3H]AVP (Fig. 2 and Table 2). Saturation binding assays confirmed the low level of AVP binding to LLC-PK1a cells compared with native LLC-PK1 cells and to LLC-PK1a cells transfected with FLAG-V2R or with the FLAG-V2RY325F mutation (Fig. 2). Scatchard analysis of both apical and basolateral [3H]AVP binding sites on LLC-FLAG-V2R cells revealed only one class of high-affinity binding site with a Kd of 4.9 ± 0.7 or 2.6 ± 0.3 nM, respectively (n = 4). The apical side showed a maximal binding capacity of 60,550 ± 15,558 sites/cell, whereas the basolateral side showed a maximal binding capacity of 190,250 ± 48,024 sites/cell, which was significantly greater than apical binding. Scatchard analysis of apical or basolateral binding sites on LLC-FLAGV2R (Y325F) cells also revealed only one class of high-affinity binding site with a Kd of 5.6 ± 0.4 or 5.4 ± 1.6 nM, respectively. Basolateral membranes showed a maximal binding capacity of 42,164 ± 9,287 binding sites/cell, which was significantly greater than the apical binding capacity of 29,493 ± 9,137 sites/cell (n = 3). In comparison, Scatchard analysis of both apical and basolateral [3H]AVP binding sites on native LLC-PK1 cells that express abundant endogenous porcine V2R also revealed only one class of high-affinity binding site with a Kd of 7.4 ± 1.4 and 4.4 ± 1.1 nM, respectively (n = 3). The apical side showed a maximal binding capacity of 22,485 ± 1,346 sites/cell, whereas the basolateral side showed a significantly greater binding capacity of 42,233 ± 4,975 sites/cell.

A similar [3H]AVP binding site distribution between the apical and the basolateral sides was found in LLC-PK1a, LLC-FLAG-V2R, and LLC-FLAG-V2R-Y325F cells (Table 2). A comparable [3H]AVP binding site distribution ratio was also observed in native LLC-PK1 cells and in the several different clones that express either FLAG-V2R or FLAG-V2R (Y325), indicating that clonal variation did not account for our findings (Table 2). This result suggests that 1) the addition of the FLAG epitope did not affect the membrane targeting of V2R, and 2) mutating the tyrosine in the NPxxY motif also did not affect the polarity of membrane targeting of the V2R. [3H]AVP binding site distribution on LLC-µ1B and LLC-PK1 cells was also examined. A tendency for the LLC-µ1B cells to have a slightly greater percentage of basolateral binding sites than wild-type LLC-PK1 cells was noticed, but this difference was not statistically significant (Fig. 3).



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Fig. 3. Apical and basolateral distribution of [3H]AVP binding sites in LLC-PK1 cell lines and the LLC-µ1B cell line. Native LLC-PK1 cells are nontransfected cells that express endogenous porcine V2R, and LLC-µ1B cells stably express the µ1B adaptor protein that is absent from native LLC-PK1 cells. The cells were incubated at 4°C for 3 h with 9 nM [3H]AVP on the apical (open bars) or basolateral side (filled bars). Columns represent means ± SD from triplicate determinations. Similar data were obtained in 3 separate experiments. AVP binding was detectable on both apical and basolateral plasma membrane domains but was more extensive on the basolateral surface. The ratio of apical-to-basolateral V2R distribution was not significantly different in either of the cell lines examined.

 

FLAG-V2R-Y325F stimulates intracellular cAMP accumulation. Intracellular cAMP accumulation in LLC-PK1a, LLC-FLAG-V2R, and LLC-FLAG-V2R-Y325F cells was measured in response to apical and/or basolateral AVP treatment (1 µM). The basal intracellular cAMP level in unstimulated cells was similar for the three cell lines (Fig. 4). The level of intracellular cAMP was increased 118-fold and 89-fold by the addition of AVP (1 µM) to either the apical or basolateral side LLC-FLAG-V2R and LLC-FLAG-V2R-Y325F cells, respectively (Fig. 4), but only about 25-fold in LLC-PK1a cells. This represents the contribution of the low amount of endogenous V2R to the overall response seen in LLC-PK1a cells. When AVP was applied simultaneously to both apical and basolateral sides of the cells, there was no significant additive effect on intracellular cAMP accumulation (Fig. 4).



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Fig. 4. Stimulation of intracellular cAMP in AVP-treated LLC-FLAG-V2R, LLC-FLAG-V2R-Y325F, and wild-type LLC-PK1a cells. Cells grown for 6 days on Transwell cell culture filter chambers were incubated for 10 min at 37°C with AVP (1 µM) on the apical (B) or basolateral side (C) or on both sides simultaneously (D). The basal intracellular cAMP levels (A) in nonstimulated cell lines were 356 ± 32 fmol/106 cells for LLC-FLAG-V2R, 413 ± 81 fmol/106 cells for LLC-FLAG-V2R-Y325F, and 494 ± 116 fmol/106 cells for LLC-PK1a cells. Columns represent means ± SD of triplicate determinations. Similar data were obtained in 3 separate experiments. AVP was effective in stimulating cAMP accumulation when applied from either the apical or basolateral surface. Simultaneous application to both surfaces resulted in no significant additive effect. The considerably lower AVP-induced cAMP elevation in nontransfected LLC-PK1a cells represents the contribution of the endogenous V2R to the overall response and confirms that upon AVP binding, the Y325F V2R is capable of increasing cAMP levels to the same extent as the transfected wild-type V2R.

 

In desensitization and resensitization assays, the mutant FLAG-V2R-Y325F was as effective as the wild-type FLAG-V2R in mediating a maximal agonist stimulation of ~35 nmol/106 cells. AVP stimulated adenylyl cyclase via its binding to the mutant or wild-type V2R (EC50: 0.38 ± 0.2 and 0.35 ± 0.09 nM, respectively, n = 3). A 20-min exposure of the LLC-FLAG-V2R or LLC-FLAG-V2R-Y325F cells to 1 µM AVP led to a ninefold rightward shift in the EC50 for agonist stimulation of adenylyl cyclase and a decrease in the maximal response (Fig. 5). To study the importance of sequestration on the resensitization process, we examined the ability of both FLAG-V2R and mutant receptors to resensitize after the removal of the desensitizing agonist. Agonist removal led to a leftward shift in the EC50 for agonist stimulation of adenylyl cyclase in both cases (Fig. 5).



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Fig. 5. Desensitization and resensitization of the adenylyl cyclase response in LLC-PK1a cells expressing wild-type and Y325F mutant V2R. Concentration-response curves are shown for adenylyl cyclase activation of wild-type (A) and mutant V2R (B) expressed in LLC-PK1a cells under control ({blacksquare}), desensitized (Desens; {blacktriangleup}), and resensitized (Resens; {bullet}) conditions. The increase in intracellular cAMP is expressed as a percentage of the maximal response obtained in the presence of 1 µM AVP on LLC-FLAG-V2R or LLC-FLAG-V2R (Y325F) cells (35 ± 27 and 34 ± 20 nmol/106 cells, respectively). Under control conditions, the EC50 values for AVP of the wild-type and mutant V2R were 0.38 and 0.35 nM, respectively. After desensitization, both receptors undergo a 9-fold rightward shift in their EC50 for agonist stimulation and a decrease in the maximal stimulation of adenylyl cyclase. After resensitization, both receptors undergo a recovery of responsiveness that results in a significant increase in the maximal stimulation of adenylyl cyclase compared with the desensitized curves. Each curve is representative of 3 independent experiments, and each was carried out in triplicate.

 

Ligand-induced internalization of FLAG-V2R-Y325F is impaired. Preincubation of LLC-FLAG-V2R with AVP led to significant loss of [3H]AVP binding sites at the cell surface due to ligand-induced receptor internalization (Fig. 6A). In contrast, preincubation of LLC-FLAG-V2R-Y325F with AVP led to very little loss of [3H]AVP binding sites at the cell surface, indicating a lack of ligand-induced receptor internalization (Fig. 6B). Interestingly, there was no significant difference in the extent or rate of ligand-induced internalization between apical and basolateral receptors. The reduction of [3H]AVP binding sites in LLC-FLAG-V2R cells was dependent on ligand binding and was not due solely to an increase in intracellular cAMP levels, because treatment of cells with forskolin, which directly stimulates adenylyl cyclase activity and leads to increased cAMP in LLC-PK1 cells, did not affect cell surface [3H]AVP binding sites in either LLC-FLAGV2R or LLC-FLAG-V2R-Y325F cells (data not shown).



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Fig. 6. Ligand-induced downregulation of [3H]AVP binding sites in LLC-FLAG-V2R (A) and LLC-V2R-Y325F cells (B) was performed as described in MATERIALS AND METHODS. Briefly, cells grown for 6 days on Transwell cell culture filters chambers were preincubated at 37°C in the presence of increasing amounts of AVP (1-1,000 nM) on either the apical or basolateral side. After 20 min of incubation, AVP was removed and cells were washed with ice-cold acidic buffer at pH 5.0 and then rewashed with ice-cold buffer at pH 7.4 before the cells were incubated at 4°C for 3 h with 9 nM [3H]AVP on either the apical or basolateral side. [3H]AVP was applied to the same side that was previously incubated with AVP, as indicated. Data are expressed as percentages relative to the total specific binding observed in untreated cells. Nonspecific binding was determined in the presence of 1 µM unlabeled AVP. The 100% values of the specific[3H]AVP binding sites were as follows: LLC-FLAG-V2R, apical 6,953 ± 187 cpm/106 cells, basolateral 9,500 ± 609 cpm/106 cells; LLC-FLAG-V2R-Y325F, apical 2,261 ± 143 cpm/106 cells, basolateral 3,307 ± 231 cpm/106 cells. Columns represent aggregated means ± SE from triplicate determinations of 5 separate experiments. A time-dependent decrease in cell surface AVP binding was seen in cells expressing the wild-type V2R, whereas considerably less downregulation occurred in cells expressing the V2R-Y325F mutant. The slight downregulation that was observed in this cell line probably represents downregulation of the small amount of endogenous V2R that is expressed by these cells.

 

Ligand-induced internalization of FLAG-V2R was confirmed by indirect immunofluorescence staining with a specific anti-FLAG monoclonal antibody (Fig. 7). Before ligand addition, FLAG-V2R was detected mainly in the plasma membrane (Fig. 7A). After incubation with AVP (1 µM) for 60 min, the FLAG-V2R staining was lost from the plasma membrane and relocated into the cytoplasm in a perinuclear compartment (Fig. 7B). In contrast, there was no observable difference in the FLAG-V2R-Y325F staining in the presence or absence of AVP (1 µM): FLAG-V2R-Y325F staining remained predominantly in the plasma membrane (Fig. 7, C and D).



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Fig. 7. Immunofluorescence localization of FLAG-V2R and FLAG-V2RY325F in LLC-PK1a cells after incubation with AVP. LLC-FLAG-V2R and LLC-FLAG-V2R-Y325F cells were grown for 6 days on Transwell cell culture filter chambers and incubated with 1 µM AVP for 0 (A and C) or 60 min (B and D) as indicated. The NH2-terminal FLAG epitope on V2R (A and B) and V2R-Y325F (C and D) was detected by incubation with a monoclonal anti-FLAG antibody (M5) followed by a secondary incubation with a polyclonal donkey anti-mouse IgG FITC-conjugated antibody. Indirect immunofluorescence staining was visualized by confocal microscopy. The results are representative of 8 independent experiments. The wild-type receptor was internalized and showed a perinuclear distribution after 60 min of AVP treatment (B), whereas the Y325F mutation remained on the plasma membrane (D). Bars, 10 µm.

 

The membrane-staining pattern of FLAG-V2RY325F suggested that receptors bearing the Y325F mutation were effectively delivered to the cell surface and that the tyrosine mutation probably did not affect the folding of the protein. Misfolded proteins are often trapped in the rough endoplasmic reticulum. Figure 8 shows the predominantly intracellular staining of the R113W mutant V2R receptor expressed in LLC-PK1 cells. This pattern is characteristic of misfolded proteins that are retained in the rough endoplasmic reticulum and illustrates the quite different behavior of the Y325F and R113W mutations with respect to their ability to be inserted into the plasma membrane.



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Fig. 8. Indirect immunofluorescence staining of LLC-PK1 cells stably transfected with a different V2R mutant in which the R113 residue is replaced by a tryptophan (W). This naturally-occurring R113W mutant results in nephrogenic diabetes insipidus, presumably due to retention of the mutant protein in the rough endoplasmic reticulum (RER). This pattern of localization is quite distinct from that seen for the Y325F mutant and most probably represents retention of the misfolded R113W V2R protein in the RER. The mutant V2R is present in a distinct perinuclear ring, representing RER/nuclear envelope staining, and is also visible as a lace-like pattern throughout the cytoplasm, again typical of RER staining. Cells were counterstained with Evan's blue to highlight the lateral plasma membranes. These membranes were poorly stained with anti-FLAG antibodies (arrows). Bar, 10 µm.

 

Electron microscopic localization of V2R in coated pits using immunogold labeling. Ligand-dependent internalization of FLAG-V2R and localization in clathrin-coated pits was confirmed using electron microscopy and immunogold labeling (Fig. 9). Before AVP treatment, FLAG-V2R was located throughout the apical cell surface of LLC-FLAG-V2R cells (Fig. 9A). After treatment with AVP for 20 min, apical FLAG-V2R was detectable in several discrete clusters that were often associated with invaginations of the plasma membrane at the base of microvilli. Microvillar staining was still present, however (Fig. 9B). At higher magnification, an electron-dense cytoplasmic coat typical of clathrin can be seen on some of the invaginations in which the gold particles are clustered (Fig. 9B, inset). One hour after treatment with vasopressin, only a few residual gold particles remained at the cell surface (Fig. 9C), because at this time point most of the V2R is located in an intracellular compartment that is not accessible to the anti-FLAG antibody when this preembedding labeling procedure is used. Ligand-dependent internalization of FLAG-V2R and localization in clathrin-coated pits was also confirmed at the basolateral side of the cells. Under baseline conditions, the FLAG-V2R gold labeling was dispersed throughout the basolateral plasma membrane (Fig. 10A). After treatment with AVP for 20 min, basolateral FLAG-V2R was detected in invaginated membrane domains characteristic of clathrin-coated pits, as described for the apical membrane (Fig. 10B, arrows). In contrast, FLAG-V2R-Y325F was not internalized in the presence of AVP. It was still dispersed on the basolateral membrane after AVP treatment and was not detected in coated pits (Fig. 10C).



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Fig. 10. Electron microscopic immunogold localization of FLAG-V2R in LLC-FLAG-V2R and V2RY325F cells after incubation with AVP. Cells were grown for 6 days on Transwell cell culture filter chambers and incubated with 10 nM AVP for 30. Cells on filters were fixed and incubated with anti-FLAG monoclonal antibody M5 followed by incubation with goat anti-mouse IgG conjugated to gold particles (10 nm) before preparation for electron microscopy. In the nonstimulated cell, gold particles representing the V2R were distributed over the basolateral cell surface (A). After 30 min of stimulation, some clusters of gold particles were evident in invaginated membrane regions (B, arrows). In the case of FLAG-V2R-Y325F, gold particles remained distributed throughout the basolateral cell surface after 30 min of AVP treatment, with no detectable clustering in clathrin-coated pits (C). These experiments were performed on the same LLC-PK1a cell clones that were used for the binding assays described in text. The higher level of basolateral expression of the V2R allowed the receptor to be detected in these cells, whereas for the apical membrane studies shown in Fig. 9, a clone having a higher level of V2R expression was used. Bars, 0.5 µm.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To study the mechanism of V2R internalization, we introduced a FLAG epitope tag into both the wild-type V2R and a V2R-Y325F mutant in which the tyrosine within the NPxxY motif was replaced by phenylalanine. Here, we report a comparative assessment of FLAG-V2R and FLAG-V2R-Y325F downregulation in LLC-PK1a cells, a variant of an established polarized kidney epithelial cell line that expresses low levels of endogenous V2R. FLAG-V2R and FLAG-V2R-Y325F were located on both apical and basolateral membranes of LLC-PK1a cells, as determined by [3H]AVP binding assays and immunocytochemical labeling techniques. A similar bipolar distribution of the V2R has previously been reported in other cultured epithelial cells such as Madin-Darby canine kidney (MDCKII) cells and canine cortical collecting tubule cells (1, 22). Furthermore, functionally relevant bipolar expression of the V2R has been reported in native collecting duct principal cells in situ (36, 49, 55). Such a heterogeneous apical/basolateral expression was also observed with other GPCRs such as angiotensin II, thyrotropin-releasing hormone, parathyroid hormone, adenosine, and atrial natriuretic factor (6, 24, 25, 43, 68).

It has been previously reported in MDCK cells that most of the V2R is on the basolateral surface by immunofluorescence staining and/or biotinylation assays (1, 61). In LLC-PK1 cells we found that ~65% of the V2R binding sites were basolateral. Similar binding assays carried out in our laboratory on MDCK cells showed that, on average, 80% of the V2R binding sites were located basolaterally (data not shown). This difference in polarized expression of the V2R in these two cell types seems to reflect the different relative amplification of apical vs. basolateral plasma membrane surface between these cell lines. The basolateral membrane surface of LLC-PK1 cells represents ~55% of the total cell surface (53). In contrast, the basolateral plasma membrane of MDCK cells represents ~80% of the total cell surface (58, 66). When this relative difference in membrane area is taken into account, the distribution of the V2R per unit area of membrane is very similar in these two lines. This distribution of the V2R between the apical and basolateral compartment reflects the influence of unknown apical and basolateral targeting motifs (27, 59). Indeed, some regions of the V2R contain signals for either apical or basolateral cell surface expression (27). In some membrane proteins, such as the LDL receptor, tyrosine-based motifs have been linked to basolateral targeting (47). However, in the case of the V2R, the tyrosine associated with the NPxxY motif does not appear to be critical in the polarized targeting process because both the wild type and the Y325F mutants behave identically with respect to their apical vs. basolateral membrane distribution. The second cytoplasmic tyrosine (Y149) localized in the second loop appears to have no detectable role in the compartmentalization of the V2R (27). This was confirmed by the examining the apical: basolateral distribution of FLAG-V2R containing a Y149F substitution in LLC-PK1a cells (31 ± 2 vs. 69 ± 2%, respectively, n = 3). Furthermore, restoration of the µ1B adaptor protein into LLC-PK1 cells (LLC-µ1B) did not significantly alter V2R targeting. This adaptor molecule, which is absent from native LLC-PK1 cells, interacts with tyrosine motifs in some proteins including the LDL receptor, resulting in basolateral targeting (20). Our data suggest that µ1B does not interact directly with the V2R to induce basolateral targeting. Taken together, therefore, available data do not support a role for either of the two candidate tyrosine motifs (Y149 or Y325) in polarized targeting of the V2R. However, some data have suggested that biosynthetic sorting and recycling pathways (postendocytotic) may not be regulated by the same signals or machinery (21, 52, 67). Of relevance to the present study is evidence that the AP1B adaptor protein via its µ1B subunit may be involved in postendocytotic basolateral protein sorting (21). Because the V2R-Y325F mutation is not internalized effectively, defective sorting in the biosynthetic pathway might be amplified by decreasing the "corrective" action of postendocytotic sorting in the recycling pathway.

The influence on signaling of the tyrosine to phenylalanine mutation seems to depend on the type of receptor examined. For example, tyrosine-mutated receptors such as neurokinin 1, gastrin-releasing peptide receptor, platelet-activating factor, and the {delta}-opioid receptor show no difference in ligand affinity (9, 39, 41, 65); the {beta}2-adrenergic receptor shows a reduced affinity (4), and the serotonin 5-HT receptor shows an increased affinity for ligand after mutation of the tyrosine residue (57). Interestingly, the influence of the mutation may be not only receptor specific but also cell type specific. Our data show that the apical and basolateral [3H]AVP binding sites for both FLAG-V2R and FLAG-V2R-Y325F have essentially the same affinity for AVP when expressed in LLC-PK1a cells. In contrast, the V2R-Y325F has a lower affinity for AVP when expressed in COS cells. This indicates that although the tyrosine within the NPxxY motif is not essential for proper agonist binding, ligand-binding affinity and cAMP generation can be modified by the cellular context in which the receptor is expressed. Because the FLAG-V2R-Y325F mutant could stimulate cAMP as effectively as FLAG-V2R in LLC-PK1a cells, we conclude that the tyrosine within the NPxxY motif of V2R is not critically involved in mediating this signal transduction process. This is supported by our data on Y325F-expressing COS cells, in which AVP treatment was able to increase intracellular cAMP levels, albeit with reduced efficiency, in the absence of any endogenous V2R.

Receptors on both the apical and basolateral membranes of LLC-PK1 cells can activate adenylyl cyclase to increase intracellular cAMP in response to AVP. Adenylyl cyclase is also stimulated by the apical or basolateral activation of another GPCR, parathyroid hormone receptor, in transfected LLC-PK1 cells (25). Interestingly, there was no additive increase in intracellular cAMP when AVP was added to both the apical and basolateral surfaces under the conditions of our experiments. This could indicate that maximal ATP substrate use is already reached upon stimulation of either the apical or basolateral receptors alone.

Both apical and basolateral cell surface [3H]AVP binding sites were reduced after pretreatment of cells expressing FLAG-V2R with AVP. The loss of cell surface binding sites implies that FLAG-V2R underwent ligand-induced internalization, as previously described in native LLC-PK1 cells and in other cell lines that express V2R, such as collecting duct cells or transiently transfected HEK-293 cells (7, 28, 35, 37, 38, 46, 51). Our results show that [3H]AVP binding sites were decreased (70% reduction) on both apical and basolateral membranes after 30 min of agonist exposure, which is consistent with other studies of V2R internalization performed on nonpolarized cells (7, 28, 46). Immunocytochemistry with FLAG-antibodies on LLC-FLAG-V2R cells confirmed that receptors were lost from the cell surface after agonist addition and that they accumulated in a perinuclear compartment. The precise nature of this compartment is unknown at present and is the subject of ongoing investigation in our laboratory.

In contrast, neither apical nor basolateral cell surface [3H]AVP binding sites were significantly reduced after pretreatment of cells expressing FLAG-V2RY325F with AVP, indicating that FLAG-V2R-Y325F did not undergo ligand-induced internalization. Immunocytochemistry using an anti-FLAG antibody in LLC-FLAG-V2R-Y325F cells confirmed that FLAG-V2RY325F remained predominantly at the cell surface (both apical and basolateral) after exposure to AVP. Our immunocytochemical data confirmed a previous report by Birnbaumer and colleagues (8) that another V2R mutation (R113W) is predominantly retained in an intracellular compartment (probably the rough endoplasmic reticulum). The staining patterns for the R113W mutation and the Y325F mutation were completely different, indicating that although the tyrosine within the NPxxY motif of the V2R is important for ligand-induced internalization, it does not cause protein misfolding that results in grossly aberrant trafficking to the cell surface. Other GPCRs, such as the {beta}2-adrenergic receptor, also require the tyrosine within the NPxxY motif for ligand-induced internalization (5).

Previous studies have suggested that the V2R is internalized by a clathrin-coated pit mechanism, based on studies using dominant negative dynamin mutants, which inhibited the internalization of V2R (11). However, caveolae-mediated endocytosis is also dynamin dependent (26, 64). Using electron microscopic immunogold-labeling to directly localize FLAG-V2R, we observed that FLAG-V2R is found throughout the apical and basolateral plasma membranes of LLC-FLAGV2R- and Y325F-expressing cells in the absence of agonist. After treatment of cells with AVP, the FLAG-V2R (but not the V2R-Y325F mutant) becomes relocated within invaginated areas of the cell surface that have the morphological features of clathrin-coated pits. Further support for clathrin-coated pit-mediated endocytosis as the mechanism of ligand-induced V2R internalization came from the use of hypertonic sucrose to inhibit V2R internalization (45). Together, these data strongly suggest that the V2R is internalized by clathrin-mediated endocytosis and that this process requires an intact NPxxY motif.

Finally, our results show that both the mutant V2RY325F and the wild-type receptor are uncoupled from cAMP production by a short exposure to AVP. These results indicate that sequestration/endocytosis is not necessary for V2R desensitization, in contrast to observations on the {beta}2-adrenergic receptor (5). The ability of both FLAG-V2R and FLAG-V2R-Y325F to resensitize after the removal of agonist also suggests that sequestration is not important in the process of resensitization of the FLAG-V2R and that this process can occur while the V2R is located at the plasma membrane. This process may be physiologically important, because the half-life for complete resensitization of the wild-type V2R after internalization is long (several hours), probably reflecting trafficking of the V2R through a complex intracellular recycling pathway before reinsertion at the cell surface (31, 33).

In summary, our data show that the NPxxY motif of the V2R is critically involved in the process of receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1 epithelial cells or in adenylyl cyclase-mediated signal transduction.


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-19406 (D. A. Ausiello) and DK-38452 (D. Brown). H. Y. Lin is the recipient of National Institutes of Health (NIH) K08 Award DK-02716. R. Bouley received fellowship support from the National Kidney Foundation (USA) and from the Heart and Stroke foundation of Canada. The Microscopy Core Facility of the MGH Program in Membrane Biology receives additional support from NIH Center for the Study of Inflammatory Bowel Disease Grant DK-43351 and NIH Boston Area Diabetes Endocrinology Research Center Grant DK-57521.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Bouley, Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: bouley{at}receptor.mgh.harvard.edu).

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.


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