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
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ABSTRACT |
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polarized cell culture; tyrosine motif; µ1b adaptor motif; protein traffic
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
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
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 -arrestin is
recruited to the receptor before the V2R-
-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
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.
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MATERIALS AND METHODS |
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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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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.
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RESULTS |
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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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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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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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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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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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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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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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DISCUSSION |
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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 -opioid receptor show no difference
in ligand affinity (9,
39,
41,
65); the
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 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
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.
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DISCLOSURES |
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FOOTNOTES |
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REFERENCES |
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2. Ausiello DA, Holtzman EJ, Gronich J, and Ercolani L. Cell signalling. In: The Kidney: Physiology and Pathophysiology (2nd ed.), edited by Seldin DW and Giebisch G. New York: Raven, 1995, p. 645-706.
3. Bakris G, Bursztyn M, Gavras I, Bresnahan M, and Gavras H. Role of vasopressin in essential hypertension: racial differences. J Hypertens 15: 545-550, 1997.[ISI][Medline]
4. Barak LS, Menard L, Ferguson SS, Colapietro AM, and Caron MG. The conserved seven-transmembrane sequence NP(X)2,3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor. Biochemistry 34: 15407-15414, 1995.[ISI][Medline]
5. Barak LS,
Tiberi M, Freedman NJ, Kwatra MM, Lefkowitz RJ, and Caron MG. A highly
conserved tyrosine residue in G protein-coupled receptors is required for
agonist-mediated beta 2-adrenergic receptor sequestration. J Biol
Chem 269:
2790-2795, 1994.
6. Becker BN,
Cheng HF, Burns KD, and Harris RC. Polarized rabbit type 1 angiotensin II
receptors manifest differential rates of endocytosis and recycling.
Am J Physiol Cell Physiol 269:
C1048-C1056, 1995.
7. Birnbaumer M,
Antaramian A, Themmen AP, and Gilbert S. Desensitization of the human V2
vasopressin receptor. Homologous effects in the absence of heterologous
desensitization. J Biol Chem
267: 11783-11788,
1992.
8. Birnbaumer M, Gilbert S, and Rosenthal W. An extracellular congenital nephrogenic diabetes insipidus mutation of the vasopressin receptor reduces cell surface expression, affinity for ligand, and coupling to the Gs/adenylyl cyclase system. Mol Endocrinol 8: 886-894, 1994.[Abstract]
9. Bohm SK, Khitin
LM, Smeekens SP, Grady EF, Payan DG, and Bunnett NW. Identification of
potential tyrosine-containing endocytic motifs in the carboxyl-tail and
seventh transmembrane domain of the neurokinin 1 receptor. J Biol
Chem 272:
2363-2372, 1997.
10. Bouley R,
Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA, and Brown D.
Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane
insertion of aquaporin 2 in renal epithelial cells. J Clin
Invest 106:
1115-1126, 2000.
11. Bowen-Pidgeon D, Innamorati G, Sadeghi HM, and Birnbaumer M.
Arrestin effects on internalization of vasopressin receptors. Mol
Pharmacol 59:
1395-1401, 2001.
12. Bremnes T,
Paasche JD, Mehlum A, Sandberg C, Bremnes B, and Attramadal H. Regulation
and intracellular trafficking pathways of the endothelin receptors.
J Biol Chem 275:
17596-17604, 2000.
13. Brown D and Nielsen S. Cell biology of vasopressin action. In: Brenner and Rector's the Kidney (6th ed.), edited by Brenner BM. Boston: Saunders, 2000, p. 575-594.
14. Carman CV,
Lisanti MP, and Benovic JL. Regulation of G protein-coupled receptor
kinases by caveolin. J Biol Chem
274: 8858-8864,
1999.
15. Chun M,
Liyanage UK, Lisanti MP, and Lodish HF. Signal transduction of a G
protein-coupled receptor in caveolae: colocalization of endothelin and its
receptor with caveolin. Proc Natl Acad Sci USA
91: 11728-11732,
1994.
16. Cowley AW Jr, Cushman WC, and Quillen EW Jr, Skelton MM, and Langford HG. Vasopressin elevation in essential hypertension and increased responsiveness to sodium intake. Hypertension 3: I93-I100, 1981.[Medline]
17. Dale LB,
Bhattacharya M, Seachrist JL, Anborgh PH, and Ferguson SS.
Agonist-stimulated and tonic internalization of metabotropic glutamate
receptor 1a in human embryonic kidney 293 cells: agonist-stimulated
endocytosis is beta-arrestin1 isoform-specific. Mol
Pharmacol 60:
1243-1253, 2001.
18. De Weerd WF and
Leeb-Lundberg LM. Bradykinin sequesters B2 bradykinin receptors and the
receptor-coupled G alpha subunits Gq and
G
i in caveolae in DDT1 MF-2 smooth muscle cells.
J Biol Chem 272:
17858-17866, 1997.
19. Dessy C, Kelly
RA, Balligand JL, and Feron O. Dynamin mediates caveolar sequestration of
muscarinic cholinergic receptors and alteration in NO signaling.
EMBO J 19:
4272-4280, 2000.
20. Folsch H, Ohno H, Bonifacino JS, and Mellman I. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99: 189-198, 1999.[ISI][Medline]
21. Gan Y, McGraw TE, and Rodriguez-Boulan E. The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane. Nat Cell Biol 4: 605-609, 2002.[ISI][Medline]
22. Garcia-Perez A and Smith WL. Apical-basolateral membrane asymmetry in canine cortical collecting tubule cells. Bradykinin, arginine vasopressin, prostaglandin E2 interrelationships. J Clin Invest 74: 63-74, 1984.[ISI][Medline]
23. Hashimoto J, Imai Y, Minami N, Munakata M, and Abe K. Effects of vasopressin V1 and V2 receptor antagonists on the development of salt-induced hypertension in Dahl rats. J Cardiovasc Pharmacol 26: 548-554, 1995.[ISI][Medline]
24. Hashimoto Y, Ozaki J, Yasuhara M, Hori R, Suga S, Itoh H, Nakao K, and Inui K. Functional evidence for an apical ANP receptor in LLC-PK1 kidney epithelial cells. Eur J Pharmacol 268: 443-445, 1994.[Medline]
25. Hayes G, Forgo J, Bringhurst FR, Segre G, and Murer H. Expression of parathyroid hormone receptors in MDCK and LLC-PK1 cells. Pflügers Arch 430: 636-644, 1995.[ISI][Medline]
26. Henley JR,
Krueger EW, Oswald BJ, and McNiven MA. Dynamin-mediated internalization of
caveolae. J Cell Biol 141:
85-99, 1998.
27. Hermosilla R and Schulein R. Sorting functions of the individual cytoplasmic domains of
the G protein-coupled vasopressin V2 receptor in Madin Darby canine
kidney epithelial cells. Mol Pharmacol
60: 1031-1039,
2001.
28. Hocher B, Merker HJ, Durr JA, Schiller S, Gross P, and Hensen J. Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis. Biochem Biophys Res Commun 186: 1376-1383, 1992.[ISI][Medline]
29. Hunyady L, Bor
M, Baukal AJ, Balla T, and Catt KJ. A conserved NPLFY sequence contributes
to agonist binding and signal transduction but is not an internalization
signal for the type 1 angiotensin II receptor. J Biol
Chem 270:
16602-16609, 1995.
30. Igarashi J and
Michel T. Agonist-modulated targeting of the EDG-1 receptor to
plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role
of caveolin-1 in sphingolipid signal transduction. J Biol
Chem 275:
32363-32370, 2000.
31. Innamorati G,
Le Gouill C, Balamotis M, and Birnbaumer M. The long and the short cycle.
Alternative intracellular routes for trafficking of G-protein coupled
receptors. J Biol Chem 276:
13096-13103, 2001.
32. Innamorati G,
Sadeghi H, Eberle AN, and Birnbaumer M. Phosphorylation of the V2
vasopressin receptor. J Biol Chem
272: 2486-2492,
1997.
33. Innamorati G,
Sadeghi HM, Tran NT, and Birnbaumer M. A serine cluster prevents recycling
of the V2 vasopressin receptor. Proc Natl Acad Sci USA
95: 2222-2226,
1998.
34. Ishizaka N,
Griendling KK, Lassegue B, and Alexander RW. Angiotensin II type 1
receptor: relationship with caveolae and caveolin after initial agonist
stimulation. Hypertension 32:
459-466, 1998.
35. Jans DA and Hemmings BA. cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells. FEBS Lett 281: 267-271, 1991.[ISI][Medline]
36. Jurzak M, Jans DA, Haase W, Peters R, and Fahrenholz F. Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections. Exp Cell Res 203: 182-191, 1992.[ISI][Medline]
37. Kim JK, Summer SN, and Schrier RW. Arginine vasopressin receptor internalization and recycling in rat renal collecting tubules. J Recept Res 14: 139-152, 1994.[ISI][Medline]
38. Kirk KL.
Binding and internalization of a fluorescent vasopressin analogue by
collecting duct cells. Am J Physiol Cell Physiol
255: C622-C632,
1988.
39. Kramer HK, Andria ML, Kushner SA, Esposito DH, Hiller JM, and Simon EJ. Mutation of tyrosine 318 (Y318F) in the delta-opioid receptor attenuates tyrosine phosphorylation, agonist-dependent receptor internalization, and mitogen-activated protein kinase activation. Brain Res Mol Brain Res 79: 55-66, 2000.[ISI][Medline]
40. Krisch B,
Feindt J, and Mentlein R. Immunoelectronmicroscopic analysis of the
ligand-induced internalization of the somatostatin receptor subtype 2 in
cultured human glioma cells. J Histochem Cytochem
46: 1233-1242,
1998.
41. Laporte SA, Servant G, Richard DE, Escher E, Guillemette G, and Leduc R. The tyrosine within the NPXnY motif of the human angiotensin II type 1 receptor is involved in mediating signal transduction but is not essential for internalization. Mol Pharmacol 49: 89-95, 1996.[Abstract]
42. Lefkowitz RJ. G protein-coupled receptors. III. New roles
for receptor kinases and beta-arrestins in receptor signaling and
desensitization. J Biol Chem
273: 18677-18680,
1998.
43. LeVier DG,
McCoy DE, and Spielman WS. Functional localization of adenosine
receptor-mediated pathways in the LLC-PK1 renal cell line. Am J
Physiol Cell Physiol 263:
C729-C735, 1992.
44. Lolait SJ, O'Carroll AM, McBride OW, Konig M, Morel A, and Brownstein MJ. Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature 357: 336-339, 1992.[ISI][Medline]
45. Lutz W and
Kumar R. Hypertonic sucrose treatment enhances second messenger
accumulation in vasopressin-sensitive cells. Am J Physiol Renal
Fluid Electrolyte Physiol 264:
F228-F233, 1993.
46. Lutz W, Sanders M, Salisbury J, and Kumar R. Internalization of vasopressin analogs in kidney and smooth muscle cells: evidence for receptor-mediated endocytosis in cells with V2 or V1 receptors. Proc Natl Acad Sci USA 87: 6507-6511, 1990.[Abstract]
47. Matter K, Hunziker W, and Mellman I. Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71: 741-753, 1992.[ISI][Medline]
48. Mimura Y, Ogura T, Kataoka H, Oishi T, Asano N, Kishida M, Yamauchi T, Ogawa N, and Makino H. Alterations of renal V1 and V2 receptors in spontaneously hypertensive rats and DOCA-salt hypertensive rats using computerized quantification for macro-autoradiogram. Res Commun Mol Pathol Pharmacol 95: 43-56, 1997.[ISI][Medline]
49. Nonoguchi H, Owada A, Kobayashi N, Takayama M, Terada Y, Koike J, Ujiie K, Marumo F, Sakai T. and Tomita K. Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts. J Clin Invest 96: 1768-1778, 1995.[ISI][Medline]
50. Oakley RH,
Laporte SA, Holt JA, Barak LS, and Caron MG. Association of beta-arrestin
with G protein-coupled receptors during clathrin-mediated endocytosis dictates
the profile of receptor resensitization. J Biol Chem
274: 32248-32257,
1999.
51. Oakley RH,
Laporte SA, Holt JA, Caron MG, and Barak LS. Differential affinities of
visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled
receptors delineate two major classes of receptors. J Biol
Chem 275:
17201-17210, 2000.
52. Odorizzi G and
Trowbridge IS. Structural requirements for basolateral sorting of the
human transferrin receptor in the biosynthetic and endocytic pathways of
Madin-Darby canine kidney cells. J Cell Biol
137: 1255-1264,
1997.
53. Pfaller W, Gstraunthaler G, and Loidl P. Morphology of the differentiation and maturation of LLC-PK1 epithelia. J Cell Physiol 142: 247-254, 1990.[ISI][Medline]
54. Pfeiffer R, Kirsch J, and Fahrenholz F. Agonist and antagonist-dependent internalization of the human vasopressin V2 receptor. Exp Cell Res 244: 327-339, 1998.[ISI][Medline]
55. Ravid R, Swaab DF, and Pool CW. Immunocytochemical localization of vasopressin-binding sites in the rat kidney. J Endocrinol 105: 133-140, 1985.[Abstract]
56. Roseberry AG and Hosey MM. Internalization of the M2 muscarinic acetylcholine receptor
proceeds through an atypical pathway in HEK293 cells that is independent of
clathrin and caveolae. J Cell Sci
114: 739-746,
2001.
57. Rosendorff A, Ebersole BJ, and Sealfon SC. Conserved helix 7 tyrosine functions as an activation relay in the serotonin 5HT(2C) receptor. Brain Res Mol Brain Res 84: 90-96, 2000.[ISI][Medline]
58. Salas PJ, Misek DE, Vega-Salas DE, Gundersen D, Cereijido M, and Rodriguez-Boulan E. Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity. J Cell Biol 102: 1853-1867, 1986.[Abstract]
59. Saunders C,
Keefer JR, Bonner CA, and Limbird LE. Targeting of G protein-coupled
receptors to the basolateral surface of polarized renal epithelial cells
involves multiple, non-contiguous structural signals. J Biol
Chem 273:
24196-24206, 1998.
60. Sawyer WH and Manning M. The development of potent and specific vasopressin antagonists. Kidney Int Suppl 26: S34-S37, 1988.[Medline]
61. Schulein R, Lorenz D, Oksche A, Wiesner B, Hermosilla R, Ebert J, and Rosenthal W. Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells. FEBS Lett 441: 170-176, 1998.[ISI][Medline]
62. Schwencke C, Okumura S, Yamamoto M, Geng YJ, and Ishikawa Y. Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane. J Cell Biochem 75: 64-72, 1999.[ISI][Medline]
63. Segredo V, Burford NT, Lameh J, and Sadee W. A constitutively internalizing and recycling mutant of the µ-opioid receptor. J Neurochem 68: 2395-2404, 1997.[ISI][Medline]
64. Simpson JC, Smith DC, Roberts LM, and Lord JM. Expression of mutant dynamin protects cells against diphtheria toxin but not against ricin. Exp Cell Res 239: 293-300, 1998.[ISI][Medline]
65. Slice LW, Wong
HC, Sternini C, Grady EF, Bunnett NW, and Walsh JH. The conserved NPXnY
motif present in the gastrin-releasing peptide receptor is not a general
sequestration sequence. J Biol Chem
269: 21755-21761,
1994.
66. Von Bonsdorff CH, Fuller SD, and Simons K. Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J 4: 2781-2792, 1985.[Abstract]
67. Whistler JL,
Enquist J, Marley A, Fong J, Gladher F, Tsuruda P, Murray SR, and Von Zastrow
M. Modulation of postendocytic sorting of G protein-coupled receptors.
Science 297:
615-620, 2002.
68. Yeaman C,
Heinflink M, Falck-Pedersen E, Rodriguez-Boulan E, and Gershengorn MC.
Polarity of TRH receptors in transfected MDCK cells is independent of
endocytosis signals and G protein coupling. Am J Physiol Cell
Physiol 270:
C753-C762, 1996.