From the Departments of Anesthesiology and
¶ Physiology, UCLA School of Medicine,
Los Angeles, California 90095
Received for publication, October 26, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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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 Vasopressin (1) 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 (1). Furthermore, in the case of the -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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
adrenergic receptor (
2-AR), the endothelin receptor, and
the cholecystokinin receptor, an additional internalization mechanism
has been described involving caveolin (2-4). Once internalized in the
different types of vesicles, different receptors can remain separate or
join in endosomal compartments (see Refs. 5 and 6 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 (7),
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" (8).
View larger version (37K):
[in a new window]
Fig. 1.
Intracellular pathways recycling GPCRs.
The schematic represents a model of the hypothetical
pathways recycling different GPCRs. Diverse types of vesicles collect
endocytosed receptors, in some case converging in common endosomes.
From the endosomal compartment, at least three diverse pathways diverge
to different destinations: lysosomes (degradative pathway), perinuclear
recycling compartment (long cycle), or directly to the plasma membrane
(short cycle).
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 (9). 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
(10).2 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.
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The fate of the intracellularly retained V2R is not immediate lysosomal
degradation, since the internalized receptor remains intact for several
hours (11), 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
2-adrenergic receptors (12, 13) with the purpose to
identify possible differences in their intracellular localization and
to determine at which point the recycling and nonrecycling pathways
physically diverged.
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EXPERIMENTAL PROCEDURES |
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Materials--
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 -arrestin 2 polyclonal developed against amino
acids 333-410 of rat
-arrestin 2 fused to glutathione
S-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).
Construction of Mutant V2 Receptors-- 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.
Cell Culture and Transfection-- 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 (12, 14). The clone expressing the V2R-S363A
was obtained as previously described (10). The clone expressing
2-AR was a generous gift from Dr. M. von Zastrow (University of California at San Francisco).
Kinetic Analysis of Receptor Recycling-- To examine receptor recycling, cells were plated 24 h after transfection in polylysine-coated 24-well plates (1.5-2.5 × 105 cells/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 mM MgCl2. 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.
Confocal Laser-scanning Microscopy--
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.
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RESULTS |
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The "Retention Domain" Is Entirely Contained in the C Terminus of the V2R-- 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 (15). 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 (10). 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.
The Recycling Pathway of the V1aR Diverges from the Intracellular Path Followed by the V2R-- 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 (12, 14).
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
2-AR (6). 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.
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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."
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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.
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The Endocytosed V2R Accumulates in the Pericentriolar Recycling
Compartment--
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.
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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 (16). Rab11, a small GTP-binding protein, has
been found mainly associated with the PNRC by Trischler et
al. (8) and others. As illustrated in Fig.
7, staining with a polyclonal antibody
specific for Rab11 (17) 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
2-adrenergic receptors was compared with that of Rab11.
For the
2-AR, only marginal colocalization was observed
in vesicles appearing as smaller endosomes surrounding the PNRC. The
V1aR was practically absent from the PNRC.
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Retention in the PNRC Is Not Mediated by -Arrestin
Binding--
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 (10). 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
2-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.
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It has been suggested that a strong and long term interaction with
-arrestin protects the phosphorylated sites from phosphatases and is
responsible for the nonrecycling of the V2R (13). 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
-arrestin was used to examine its location
after ligand-mediated internalization. An immunoblot against an HEK
cell lysate, shown in Fig. 9A,
indicated that the antibody detected both isoforms of the endogenous
protein. Surprisingly, as depicted in Fig. 9B, after 60 min
of hormone treatment,
-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
2-adrenergic
receptors failed to colocalize with arrestin that was found dispersed
throughout the cytosol, as previously described for many receptors
(7).
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DISCUSSION |
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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 2-AR (18). 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 (11). 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. (6) reported receptor-mediated Tfn internalization in HEK 293 cells at 16 °C, while agonist-bound 2-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 (13). The pits collecting
the AVP receptors are therefore likely to be the same as those
gathering the
2-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. (19), who failed to
detect colocalization of the receptor with caveolin, and according to
Chini et al.,3 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 (20). 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 (21), 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 (10) and allow
receptor recycling. Accordingly, dephosphorylation has recently been
shown to be a requirement for the exocytosis of internalized
2-AR in human epidermoid carcinoma cells A431 (22) and
opioid receptor in human neuroblastoma SK-N-BE cells (23). By
colocalizing the internalized human V2R expressed in HEK cells with
TfnR, our experiments confirmed recent data reported by Fahrenholz and
co-workers (19). Additionally, by utilizing the small GTPase Rab11 as a
marker (24), 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 (25), and the "long cycle," where the receptors appeared
in large vesicles and tubules forming the PNRC in close proximity to
the microtubule organizing center (16, 26-28).
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
(29-31). 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
2-AR is initiated in early endosomes (32) or perhaps
even earlier at the plasma membrane (18, 32, 33, 34), 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 (35, 36). 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 -arrestins to GPCRs, and it
has been suggested that a high affinity interaction between V2R and
-arrestin protected the C terminus from phosphatases, offering a
biochemical explanation for the trapping of the receptor (13). 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. (13) 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
2-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
-arrestin from the intracellular retention of the WT V2R, these results dispute the hypothesis that stable binding to
-arrestin directs internalized receptors to the lysosomes as proposed by Bremnes et al. (31). The significance of the
translocation of
-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 (37).
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.
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ACKNOWLEDGEMENTS |
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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--arrestin antibody, and Dr. Mark von Zastrow for the
2-AR-stable HEK clone.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK-41-244 (to M. B.).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.
§ Present address: DIBIT, Scientific Institute San Raffaele, via Olgettina 58,I-20132 Milan, Italy.
To whom correspondence should be addressed. Tel.:
310-794-6695; Fax: 310-825-6711; E-mail: marielb@ucla.edu.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M009780200
2 C. Le Gouill, G. Innamorati, and M. Birnbaumer, manuscript in preparation.
3 B. Chini, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G-protein-coupled receptor;
AVP, [Arg8]vasopressin;
V2R, vasopressin type 2 receptor;
V1a, vasopressin type 1a receptor;
V1b, vasopressin type 1b
receptor;
HEK, human embryonic kidney;
PCR, polymerase chain reaction;
2-AR,
2-adrenergic receptor;
HA, hemagglutinin;
D-PBS, Dulbecco's phosphate-buffered saline;
Tfn, transferrin;
TfnR, transferrin receptor;
PNRC, pericentriolar or
perinuclear recycling compartment;
WT, wild type.
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