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• W2041097342 abstract "The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in β-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the “long cycle.” The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane. The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in β-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the “long cycle.” The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane. Vasopressin (1Krisch B. Feindt J. Mentlein R. J. Histochem. Cytochem. 1998; 46: 1233-1242Crossref PubMed Scopus (34) Google Scholar) is a small peptide hormone recognized by three different G protein-coupled receptors (GPCRs)1 of which the V1a and V1b subtypes are coupled by Gq/11 and the V2 is coupled to Gs. Interaction with the agonist instantly leads to GPCR activation, phosphorylation, desensitization, and sequestration, followed by the return of the dephosphorylated receptors to the cell surface. In a few exceptions, the internalized receptor is degraded in the lysosomal compartment. The pathway by which GPCRs are recycled is under intense study, and an increasing number of elements specific for the receptor or the cell type have been identified, although little is known about how the different pathways and organelles are involved. The majority of GPCRs internalize via clathrin-coated pits, although some, such as the somatostatin 2 receptor, have been found mostly in uncoated vesicles (1Krisch B. Feindt J. Mentlein R. J. Histochem. Cytochem. 1998; 46: 1233-1242Crossref PubMed Scopus (34) Google Scholar). Furthermore, in the case of the β2adrenergic receptor (β2-AR), the endothelin receptor, and the cholecystokinin receptor, an additional internalization mechanism has been described involving caveolin (2Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google Scholar, 3Chun M. Liyanage U.K. Lisanti M.P. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11728-11732Crossref PubMed Scopus (326) Google Scholar, 4Roettger B.F. Rentsch R.U. Pinon D. Holicky E. Hadac E. Larkin J.M. Miller L.J. J. Cell Biol. 1995; 128: 1029-1041Crossref PubMed Scopus (204) Google Scholar). Once internalized in the different types of vesicles, different receptors can remain separate or join in endosomal compartments (see Refs. 5Gaudriault G. Nouel D. Dal Farra C. Beaudet A. Vincent J.P. J. Biol. Chem. 1997; 272: 2880-2888Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar and 6Cao T.T. Mays R.W. von Zastrow M. J. Biol. Chem. 1998; 273: 24592-24602Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar and Fig.1). Proteins that are differentially distributed in the intracellular organelles and are present as different isoforms, such as dynamin, arrestins, and Rab GTPases (7Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar), could be critical in establishing the traffic pattern followed by a given GPCR in a given cell type. Once endocytosed, trans-membrane proteins can return to the cell surface by at least two different ways: directly from sorting endosomes via the “short cycle” or indirectly traversing the perinuclear recycling endosomes that constitute the “long cycle” (8Trischler M. Stoorvogel W. Ullrich O. J. Cell Sci. 1999; 112: 4773-4783Crossref PubMed Google Scholar). The C terminus of many GPCRs has been recognized as the major regulatory domain of the protein controlling the efficiency of internalization and other phenomena like desensitization. The human V2 vasopressin receptor (V2R) contains multiple phosphorylation sites, since progressive truncations in this region gradually reduced the level of phosphorylation. Receptor desensitization, internalization, and recycling were affected differently, depending on which stretch of amino acids was missing. Phosphorylation sites between positions 345 and 361 (of the 371 V2R amino acids; see Fig.2) appear to regulate the interaction with the internalization machinery, since their removal modifies the extent of receptor internalization (9Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Crossref PubMed Scopus (68) Google Scholar). These phosphorylation sites together with others further downstream, specifically serine and threonine from positions 345–364, create a signal regulating the traffic of the receptor back to the plasma membrane (10Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). 2C. Le Gouill, G. Innamorati, and M. Birnbaumer, manuscript in preparation. Although the last three amino acids of the V2R (TSS) are also targets of phosphorylation, no functional changes were detected after their elimination, leaving the role of this cluster yet to be determined. The fate of the intracellularly retained V2R is not immediate lysosomal degradation, since the internalized receptor remains intact for several hours (11Innamorati G. Sadeghi H. Birnbaumer M. J. Recept. Signal. Transduct. Res. 1999; 19: 315-326Crossref PubMed Scopus (30) Google Scholar), creating a puzzle about the identity of the organelle accumulating the receptor. We previously postulated that a deficient phosphorylation step could be responsible for the lack of recycling; however, it remained unclear whether in HEK cells the receptor was deviated to an organelle that is not part of the recycling pathway or whether it was simply trapped somewhere along the route normally leading to the cell surface. The pathway followed by the V2R was compared using confocal microscopy to that of other receptors known to recycle in HEK 293 cells, such as the V1a and the β2-adrenergic receptors (12Innamorati G. Sadeghi H. Birnbaumer M. J. Biol. Chem. 1998; 273: 7155-7161Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar) with the purpose to identify possible differences in their intracellular localization and to determine at which point the recycling and nonrecycling pathways physically diverged. Tissue culture supplies and media were from Life Technologies, Inc. Tritiated AVP was from PerkinElmer Life Sciences; nonradioactive AVP was from Sigma. The following antibodies were utilized: anti-HA monoclonal 12CA5 and anti-c-Myc monoclonal 9E10 from the ATCC; anti-c-Myc polyclonal from Upstate Biotechnology, Inc. (Lake Placid, NY); an anti β-arrestin 2 polyclonal developed against amino acids 333–410 of rat β-arrestin 2 fused to glutathioneS-transferase was a generous gift of Dr R. Lefkowitz (Duke University Medical School); an anti-Rab11 polyclonal developed against amino acids 88–103 was a generous gift of Dr. D. D. Sabatini (New York University School of Medicine). V1a/V2 and the V2/V1a receptor chimeras, and the S363A-V2R, were prepared using a PCR-based approach. The resulting constructs were sequenced and cloned into the vector pcDNA3 (Invitrogen) for expression in mammalian cells. The junctions of the chimeras were located at the palmitoylated cysteines of the V1a and V2 receptors shown in Fig. 2. HEK 293 cells were plated at a density of 2.5 × 106 cells/150-mm dish and transfected the following day with 14 ml of a mixture of 100 μm chloroquine and DEAE-dextran (0.25 mg/ml) containing 3 μg of plasmid DNA in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. After 2 h at 37 °C, the solution was removed, and the cells were treated for 1 min at room temperature with 10% dimethyl sulfoxide in Dulbecco's phosphate-buffered saline (D-PBS), rinsed twice with D-PBS, and returned to the 37 °C incubator in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. The clones expressing the HA-tagged V1a and V2 receptors have been previously characterized (12Innamorati G. Sadeghi H. Birnbaumer M. J. Biol. Chem. 1998; 273: 7155-7161Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar). The clone expressing the V2R-S363A was obtained as previously described (10Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). The clone expressing β2-AR was a generous gift from Dr. M. von Zastrow (University of California at San Francisco). To examine receptor recycling, cells were plated 24 h after transfection in polylysine-coated 24-well plates (1.5–2.5 × 105cells/well). The following day, cells were treated with 100 nm AVP (or vehicle) for 20 min at 37 °C to promote V2R sequestration. The hormone remaining on the cell surface was removed by two washes with D-PBS, two washes with 150 mm NaCl plus 5 mm acetic acid, and three washes with D-PBS, all at 4 °C. Fresh Dulbecco's modified Eagle's medium plus 10% fetal bovine serum was added, and the cells were returned to the 37 °C incubator. Hormone binding was measured at the indicated time of recovery, and the cells were washed twice with ice-cold D-PBS before adding 0.5 ml of an ice-cold binding mixture containing 25 nm [3H]AVP in D-PBS with 2% bovine serum albumin, 0.5 mm CaCl2, and 2 mmMgCl2. After a 2-h incubation in the cold room, the binding mixture was removed by aspiration, the cells were rinsed twice with ice-cold D-PBS, and 0.5 ml of 0.1 m NaOH was added to extract bound radioactivity. After 30 min at 37 °C, the fluid from each well was transferred to a scintillation vial containing 3.5 ml of Ultima-Flo M scintillation fluid (Packard) for radioassay. Nonspecific binding was determined under the same conditions in the presence of 10 μm unlabeled AVP. Each experimental point was assayed in triplicate; the data presented are the mean ± S.E. of four experiments. Transfected cells were seeded on glass coverslips and treated as described in the figure legends. Receptors with an extracellular epitope were labeled by anti-Myc or anti-HA antibodies unmodified or directly coupled to Alexa 488 (Molecular Probes, Inc., Eugene, OR) under nonpermeabilizing conditions. Treatments with antibodies or with hormone were performed in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum supplemented with 10 mm Na-HEPES, pH 7.4, at the temperatures indicated in the figure legends. Cells were fixed for 30 min with a solution of 4% paraformaldehyde pH 7.4 in D-PBS. When necessary, nonpermeabilized cells were incubated with the primary polyclonal antibody for 2 h at room temperature, followed by a 30-min incubation with the secondary antibody at room temperature. The samples were analyzed by confocal laser-scanning microscopy utilizing the Carl Zeiss Laser Scanning System LSM 510. Previous experiments showed that serines located at the C terminus are essential for the existence of a domain retaining the internalized V2R inside the cell. Substituting the C terminus with the homologous portion of the V1a receptor (V1aR) resulted in a chimeric V2/V1a receptor that was still coupled to Gs (15Liu J. Wess J. J. Biol. Chem. 1996; 271: 8772-8778Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). After efficient ligand-promoted internalization, the chimeric receptor recycled to the cell surface with an even higher efficiency than the wild type V1aR (Figs. 2 and 10). The complementary V1a/V2R chimera, created by substituting the V2R C terminus for the tail of the V1aR, coupled to Gq and was internalized to the same extent as either wild type receptor; however, its return to the cell surface was greatly impaired, leaving the receptor trapped inside the cell as shown in Fig. 2. Thus, the predisposition of the V1a receptor to recycle was suppressed by a retention domain entirely confined to the C terminus. The exact amino acid composition of the signal remains to be defined; single point mutations like serine 357 to alanine also produced a fully recycling and partially (90% of WT) phosphorylated receptor,2 expanding the domain beyond the three serines (362) initially identified (10Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). In general, preventing the full phosphorylation of the retention domain yields a recycling V2R that traffics in a manner that is indistinguishable from the V1aR or any other recycling receptor when analyzed by radioligand binding assay. To learn about the nature of the subcellular structures trapping the V2R, its intracellular course was examined by immunocytochemistry. The pathways followed by epitope-tagged V1aR and V2R were compared employing two previously characterized stably transfected HEK 293 clones (12Innamorati G. Sadeghi H. Birnbaumer M. J. Biol. Chem. 1998; 273: 7155-7161Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar). As shown in Fig. 3, no endocytosis was observed at 4 °C for either AVP receptor in the absence or presence of hormone. As illustrated in Fig. 3, increasing the temperature to 16 °C allowed the formation of agonist-induced clusters of intense immunoreactivity likely to correspond to clathrin-coated pits filled with receptors still at the cell surface, as reported for the β2-AR (6Cao T.T. Mays R.W. von Zastrow M. J. Biol. Chem. 1998; 273: 24592-24602Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The subsequent endocytic process was strictly temperature-dependent for the V1a and the V2 receptors, since it was blocked below 20 °C even in the presence of saturating concentrations of ligand. In the absence of AVP, increasing the temperature to 37 °C prompted a slow constitutive internalization component for both receptor subtypes as illustrated in the middle panel of Fig. 3 for the V1a receptor. This hormone-independent endocytosis was not triggered by the presence of the antibody, since the number of [3H]AVP binding sites on the cell surface remained unchanged before and after exposure of the cells to the monoclonal antibody for 1 h at 37 °C (data not shown). For the V1a and V2 receptors, the fraction internalized at 37 °C was dramatically increased by the presence of 100 nm AVP. After 60 min of hormone treatment, the majority of the V2R was concentrated in a very large endosomal compartment in close proximity to the nucleus, whereas the V1aR was distributed in smaller cytosolic endosomes as shown in Figs. 3 and 4. The phenomenon was analyzed in further detail by time lapse confocal microscopy; the video showed the formation of small vesicles containing V2R that were initially dispersed throughout the periphery of the cell and later moved toward a single “aggregation point.” To directly compare the distribution of the recycling V1aR and the nonrecycling V2R in the same cell, both receptors were tagged with different epitopes, transiently cotransfected, and visualized after a 60-min treatment with 100 nm AVP at 37 °C. Fig.5 shows the images obtained with two representative cells. Similar to the results obtained with stable clones (shown above), the internalized V2R accumulated in a perinuclear vesicular aggregate. By overlaying the staining corresponding to the V1a and the V2 receptors, some degree of colocalization was observed at the plasma membrane and in a number of vesicles present only at the periphery of the perinuclear aggregate. The intracellular localization of the vasopressin receptors was compared with that of the transferrin (Tfn) receptor (TfnR), a membrane protein with a well characterized intracellular pathway. Stably transfected cells expressing the V1aR or the V2R were incubated at 37 °C for 1 h, in the presence of fluorescently labeled Tfn and 100 nm AVP, fixed, and observed with the confocal microscope. As shown in Fig. 6, some staining corresponding to either the V1a or the Tfn receptors was scattered throughout the cytosol, while a small proportion of the V1aR was localized to the periphery of a bulky assembly in the proximity of the nucleus, where the majority of the TfnR was concentrated. The V1aR appeared excluded from the inner core of this compartment that could therefore represent a subsequent step reached by the TfnR after traversing common early endosomes, possibly the same endosomes where, as shown in Fig. 5, the V1aR merged with the V2R. In contrast, most of the V2R was found in the same compartment where the majority of TfnR accumulated. An endosomal subcompartment called the pericentriolar or perinuclear recycling compartment (PNRC) has been described as an organelle formed by tubulo-vesicular structures that gather recycling molecules directed to the plasma membrane once they have been separated from their cargo headed for the lysosomes (16Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (420) Google Scholar). Rab11, a small GTP-binding protein, has been found mainly associated with the PNRC by Trischler et al. (8Trischler M. Stoorvogel W. Ullrich O. J. Cell Sci. 1999; 112: 4773-4783Crossref PubMed Google Scholar) and others. As illustrated in Fig.7, staining with a polyclonal antibody specific for Rab11 (17Ren M. Xu G. Zeng J. De Lemos-Chiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Crossref PubMed Scopus (397) Google Scholar) demonstrated an extensive colocalization with the V2R in the large juxtanuclear structure thus identified as the PNRC. In parallel experiments, the distribution of the V1a and the β2-adrenergic receptors was compared with that of Rab11. For the β2-AR, only marginal colocalization was observed in vesicles appearing as smaller endosomes surrounding the PNRC. The V1aR was practically absent from the PNRC. To examine whether trapping of the V2R inside the cell was related to targeting to the PNRC, the endocytic pathway of a recycling mutant V2R (V2R-S363A) was characterized. Compared with the WT receptor, this mutant was expressed at the same level, phosphorylated 90% as much, and fully active in terms of G protein coupling (10Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). Similarly to the V1a and V2 receptors, the V2R-S363A was constitutively internalized at a low rate when incubated at 37 °C in the absence of hormone (Fig. 8). Exposure to 100 nm AVP increased its internalization and resulted in an intracellular distribution that resembled the WT V2R rather than the V1a or the β2-adrenergic receptors. Most of the internalized V2R-S363A converged to a single large area in the proximity of the nucleus, where it colocalized with Rab11 in the PNRC. The staining of the V2R-S363A in the PNRC seemed less dense than the WT staining, possibly due to the continuous recycling of the protein. Likewise, the mutant receptor was also detected in many small peripheral fluorescent endosomes. It has been suggested that a strong and long term interaction with β-arrestin protects the phosphorylated sites from phosphatases and is responsible for the nonrecycling of the V2R (13Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). Thus, a lasting colocalization of the receptor with this adaptor protein would have been expected only with the WT and not with the recycling V2R-S363A mutant. To verify this prediction, a polyclonal antibody raised against the carboxyl terminus of β-arrestin was used to examine its location after ligand-mediated internalization. An immunoblot against an HEK cell lysate, shown in Fig. 9 A, indicated that the antibody detected both isoforms of the endogenous protein. Surprisingly, as depicted in Fig. 9 B, after 60 min of hormone treatment, β-arrestin was present in the PNRC region, where it colocalized with both the WT and V2R-S363A receptors. In parallel experiments, the V1a and the β2-adrenergic receptors failed to colocalize with arrestin that was found dispersed throughout the cytosol, as previously described for many receptors (7Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar). Similar to the V1aR, most of the GPCRs expressed in HEK 293 cells return to the cell surface after ligand-induced internalization. This process is likely to involve repeated recycling as demonstrated under similar conditions for the β2-AR (18Morrison K.J. Moore R.H. Carsrud N.D. Trial J. Millman E.E. Tuvim M. Clark R.B. Barber R. Dickey B.F. Knoll B.J. Mol. Pharmacol. 1996; 50: 692-699PubMed Google Scholar). In contrast, no recycling was observed for the human V2R expressed in the same cell line, as well as in COS M6, Madin-Darby canine kidney, and HeLa cells. Since the internalized protein remained intact for hours, direct targeting to lysosomes was excluded (11Innamorati G. Sadeghi H. Birnbaumer M. J. Recept. Signal. Transduct. Res. 1999; 19: 315-326Crossref PubMed Scopus (30) Google Scholar). To compare the intracellular distribution of internalized V2R to a recycling receptor, epitope-tagged V1a and V2 vasopressin receptors expressed in HEK cells were examined by immunofluorescence microscopy. The spontaneous and the ligand-promoted internalization of both receptors were temperature-dependent processes. This is common among GPCRs, but it is not typical of all endocytic processes; for example, Cao et al. (6Cao T.T. Mays R.W. von Zastrow M. J. Biol. Chem. 1998; 273: 24592-24602Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) reported receptor-mediated Tfn internalization in HEK 293 cells at 16 °C, while agonist-bound β2-AR remained on the cell surface. Ligand-mediated internalization at 37 °C induced the colocalization of the two AVP receptors only in small endosomes. After 1 h in the presence of hormone, a progressive accumulation of the proteins in different intracellular compartments was observed: the V1aR in vesicles scattered throughout the cytoplasm and the V2R in an agglomeration located in the vicinity of the nucleus. The partial colocalization was not surprising, since the V1a and the V2 receptors are likely to be internalized via the same endosomes by a process that is sensitive to dominant negative arrestins and to the GTPase-defective dynamin (13Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). The pits collecting the AVP receptors are therefore likely to be the same as those gathering the β2-AR. The latter has also been found in caveolae, like the vasoactive intestinal peptide 1 and the endothelin b receptors. However, internalization of the V2R via caveoli was unlikely, according to Pfeiffer et al. (19Pfeiffer R. Kirsch J. Fahrenholz F. Exp. Cell Res. 1998; 244: 327-339Crossref PubMed Scopus (36) Google Scholar), who failed to detect colocalization of the receptor with caveolin, and according to Chini et al., 3B. Chini, personal communication. who reported the susceptibility of the V2R to Triton X-100 extraction. Using fluorescent AVP as a marker, a previous study examined the intracellular traffic of naturally expressed rat V1aR in A10 cells and pig V2R in LLCPK1 cells. Despite the restrictions in interpretation imposed by the heterogeneity of the cell lines, the intracellular fluorescent pattern of the internalized receptors was also distinct, and the location of both receptors was strikingly similar to what we observed in HEK 293 cells (20Lutz W. Sanders M. Salisbury J. Kumar R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6507-6511Crossref PubMed Scopus (46) Google Scholar). Opposite to our observations, this study described a nonrecycling V1aR and a recycling V2R. This discrepancy could be caused by the absence of the serine triplet in this protein (21Gorbulev V. Buchner H. Akhundova A. Fahrenholz F. Eur. J. Biochem. 1993; 215: 1-7Crossref PubMed Scopus (117) Google Scholar), yet in tissues that naturally express the human V2R, specific factors or phosphatases could turn off the “switch” created by a transient phosphorylation-dependent retention signal (10Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar) and allow receptor recycling. Accordingly, dephosphorylation has recently been shown to be a requirement for the exocytosis of internalized β2-AR in human epidermoid carcinoma cells A431 (22Shih M. Lin F. Scott J.D. Wang H.Y. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and δ opioid receptor in human neuroblastoma SK-N-BE cells (23Hasbi A. Allouche S. Sichel F. Stanasila L. Massotte D. Landemore G. Polastron J. Jauzac P. J. Pharmacol. Exp. Ther. 2000; 293: 237-247PubMed Google Scholar). By colocalizing the internalized human V2R expressed in HEK cells with TfnR, our experiments confirmed recent data reported by Fahrenholz and co-workers (19Pfeiffer R. Kirsch J. Fahrenholz F. Exp. Cell Res. 1998; 244: 327-339Crossref PubMed Scopus (36) Google Scholar). Additionally, by utilizing the small GTPase Rab11 as a marker (24Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1084) Google Scholar), our data established the identity of the organelle containing the receptor after AVP treatment as the PNRC. This compartment is known as an intermediate station for the recycling TfnR and other receptors like the EGF and insulin receptors. Thus, the two AVP receptors followed alternative pathways, previously described by other investigators as the “short cycle,” in which receptor molecules return to the plasma membrane directly from the sorting endosomes (25Hopkins C.R. Gibson A. Shipman M. Miller K. Nature. 1990; 346: 335-339Crossref PubMed Scopus (316) Google Scholar), and the “long cycle,” where the receptors appeared in large vesicles and tubules forming the PNRC in close proximity to the microtubule organizing center (16Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 26Daro E. van der Sluijs S.P. Galli T. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9559-9564Crossref PubMed Scopus (281) Google Scholar, 27McGraw T.E. Greenfield L. Maxfield F.R. J. Cell Biol. 1987; 105: 207-214Crossref PubMed Scopus (163) Google Scholar, 28Tooze J. Hollinshead M. J. Cell Biol. 1992; 118: 813-830Crossref PubMed Scopus (56) Google Scholar). As presented in Fig. 1, rather than parallel internalization pathways, functional and morphological data have suggested the existence of a complex endocytic network that internalizes different receptors by a variety of specialized vesicles following different routes that can converge or diverge at different points. Differential distribution of internalized GPCRs has been demonstrated in more than one case when comparing the degradative versus recycling pathways (29Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 30Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 31Bremnes T. Paasche J.D. Mehlum A. Sandberg C. Bremnes B. Attramadal H. J. Biol. Chem. 2000; 275: 17596-17604Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Reaching the PNRC did not assure to the V2R a passageway to the cell surface. As suggested earlier, this could be a consequence of the artificial expression system; however, expression in HEK cells did not induce “misrouting” of internalized V2R to an organelle from which it could not exit. The recycling V2R-S363A mutant proved indeed that the receptor could exit the PNRC once the anchoring domain had been inactivated by dephosphorylation. Dephosphorylation of the β2-AR is initiated in early endosomes (32Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) or perhaps even earlier at the plasma membrane (18Morrison K.J. Moore R.H. Carsrud N.D. Trial J. Millman E.E. Tuvim M. Clark R.B. Barber R. Dickey B.F. Knoll B.J. Mol. Pharmacol. 1996; 50: 692-699PubMed Google Scholar, 32Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 33Zhang J. Ferguson S.S. Barak L.S. Aber M.J. Giros B. Lefkowitz R.J. Caron M.G. Recept. Channels. 1997; 5: 193-199PubMed Google Scholar, 34Lin F. Wang H. Malbon C.C. J. Biol. Chem. 2000; 275: 19025-19034Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), whereas the location of the phosphatases acting on the AVP receptors is unknown. The fact that the recycling mutants are dephosphorylated more efficiently than the wild type links the two events but does not provide a cause-effect relationship between them. If the recycling receptors encounter some of the phosphatases after the PNRC, the blockage will prevent the WT V2R from reaching these enzymes and completing the dephosphorylation process initiated earlier. Alternatively, the loss of phosphate at certain sites might be required, while still in the endosomal compartment, to facilitate the continuation of the process. The cleavage of multiple phosphate groups is unlikely to happen as a single step, and a hierarchical dephosphorylation could be mirroring the stepwise incorporation of phosphates that has been proposed for the reverse process driven by GRKs (35Prossnitz E.R. Kim C.M. Benovic J.L. Ye R.D. J. Biol. Chem. 1995; 270: 1130-1137Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 36Ohguro H. Palczewski K. Ericsson L.H. Walsh K.A. Johnson R.S. Biochemistry. 1993; 32: 5718-5724Crossref PubMed Scopus (152) Google Scholar). The wild type receptor would thus reach the PNRC after incomplete dephosphorylation. Vice versa, the mutants (already lacking phosphates at selected sites) would be better substrates for the phosphatases and, therefore, reaching the PNRC with few or no phosphates would pass through it and finally recycle to the cell surface. Either way, the retention signal acting as an anchor in the PNRC functions as an additional regulatory step at which phosphorylation controls the abundance of receptor available to the hormone and therefore the intensity of the tissue response to AVP. Phosphorylation enhanced the binding of β-arrestins to GPCRs, and it has been suggested that a high affinity interaction between V2R and β-arrestin protected the C terminus from phosphatases, offering a biochemical explanation for the trapping of the receptor (13Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). However, two lines of experimental evidence support a distinction between the binding of the V2R to arrestin and recycling. The first is that the two domains, although partially overlapping, do not coincide; the domain sufficient to determine the lasting interaction with arrestin described by Oakley et al. (13Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar) did not include amino acids like Ser357 that are part of the retention domain (this report).2 The second is that only the wild type V2R should have manifested a lasting association with arrestin; instead, the recycling mutant V2R also concentrated arrestin in the PNRC, a phenomenon that was not detected with the V1a or the β2-AR. Thus, the translocation of arrestin from the plasma membrane to the organelle, rather than being the determining factor distinguishing recycling from nonrecycling receptors, was dependent on the identity of the receptor protein. In addition to dissociating the long lasting interaction with β-arrestin from the intracellular retention of the WT V2R, these results dispute the hypothesis that stable binding to β-arrestin directs internalized receptors to the lysosomes as proposed by Bremnes et al. (31Bremnes T. Paasche J.D. Mehlum A. Sandberg C. Bremnes B. Attramadal H. J. Biol. Chem. 2000; 275: 17596-17604Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The significance of the translocation of β-arrestin to the PNRC remains elusive and suggests that a better definition of the role of this protein is required, especially in view of its ubiquitous presence inside the cell, nucleus included (37Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (146) Google Scholar). In summary, the data demonstrate that the V1a and V2 receptors, although internalized by the same early endosomes, are routed via two distinct intracellular pathways, namely the short and the long cycle. The long term interaction of the V2R with arrestin was shown to be compatible with receptor recycling via a pathway that includes transit through the PNRC, a step apparently regulated by dephosphorylation of a specific C-terminal domain in the human V2R. We thank Dr. David Scott for valuable discussion and helpful advice with laser-scanning microscopy and Dr. George Sachs for generous access to the microscope, Dr. David Sabatini for the anti-Rab11 antibody, Dr. Robert Lefkowitz for the anti-β-arrestin antibody, and Dr. Mark von Zastrow for the β2-AR-stable HEK clone. G-protein-coupled receptor [Arg8]vasopressin vasopressin type 2 receptor vasopressin type 1a receptor vasopressin type 1b receptor human embryonic kidney polymerase chain reaction β2-adrenergic receptor hemagglutinin Dulbecco's phosphate-buffered saline transferrin transferrin receptor pericentriolar or perinuclear recycling compartment wild type" @default.
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