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• W1963988769 abstract "After agonist-induced internalization, the vasopressin V2 receptor (V2R) does not recycle to the plasma membrane. The ADP-ribosylation factor (ARF) proteins initiate vesicular intracellular traffic by promoting the recruitment of adaptor proteins; thus, we sought to determine whether ARF6 could promote V2 R recycling. Neither the agonist-induced internalization nor the recycling of the V2 Rwas regulated by ARF6, but a constitutively active mutant of ARF6 reduced cell-surface V2Rs 10-fold in the absence of agonist treatment. Visualization of the ARF6 mutant-expressing cells revealed a vacuolar-staining pattern of the V2R instead of the normal plasmamembrane expression. Analysis of V2R maturation revealed that reduced cell-surface expression was due to the diminished ability of the newly synthesized receptor to migrate from the endoplasmic reticulum to the Golgi network. The same mechanism affected processing of the V1aR and acetylcholine M2 receptors. Therefore, ARF6 controls the exit of the V2 and other receptors from the endoplasmic reticulum in addition to its established role in the trafficking of plasma-membrane-derived vesicles. After agonist-induced internalization, the vasopressin V2 receptor (V2R) does not recycle to the plasma membrane. The ADP-ribosylation factor (ARF) proteins initiate vesicular intracellular traffic by promoting the recruitment of adaptor proteins; thus, we sought to determine whether ARF6 could promote V2 R recycling. Neither the agonist-induced internalization nor the recycling of the V2 Rwas regulated by ARF6, but a constitutively active mutant of ARF6 reduced cell-surface V2Rs 10-fold in the absence of agonist treatment. Visualization of the ARF6 mutant-expressing cells revealed a vacuolar-staining pattern of the V2R instead of the normal plasmamembrane expression. Analysis of V2R maturation revealed that reduced cell-surface expression was due to the diminished ability of the newly synthesized receptor to migrate from the endoplasmic reticulum to the Golgi network. The same mechanism affected processing of the V1aR and acetylcholine M2 receptors. Therefore, ARF6 controls the exit of the V2 and other receptors from the endoplasmic reticulum in addition to its established role in the trafficking of plasma-membrane-derived vesicles. The vasopressin V2 receptor (V2R) 2The abbreviations used are: V2R, vasopressin V2 receptor; ARF, ADP-ribosylation factor; ARNO, ARF nucleotide binding site opener; AVP, arginine vasopressin; GPCR, G-protein-coupled receptor; LAMP1, lysosomal-associated membrane protein 1; Tfn, transferrin; TfnR Tfn receptor; β-gal, β-galactosidase; HA, hemagglutinin; GFP, green fluorescent protein; HEK cells, human embryonic kidney cells; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation assay buffer. is a member of the G-protein-coupled receptor (GPCR) superfamily of receptors that are characterized by seven membrane-spanning domains (1Birnbaumer M. Seibold A. Gilbert S. Ishido M. Barberis C. Antaramian A. Brabet P. Rosenthal W. Nature. 1992; 357: 333-335Crossref PubMed Scopus (489) Google Scholar). GPCR plasma membrane levels are maintained at a steady state by a combination of new receptor synthesis, proteolytic receptor degradation, physical sequestration from the plasma membrane, and recycling back to the plasma membrane (2Koenig J.A. Edwardson J.M. Trends Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar). Receptor removal from the cell surface to an intracellular compartment is typically induced by the application of agonist. V2Rs have been shown to internalize in clathrin-coated pits via a process that requires both arrestin and dynamin (3Bowen-Pidgeon D. Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 2001; 59: 1395-1401Crossref PubMed Scopus (34) Google Scholar). Strikingly, upon agonist removal, the V2R does not recycle back to the plasma membrane even 6 h after agonist removal (4Innamorati G. Sadeghi H. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). Regardless of the V2R expression levels in stable or transient lines or the cell line in which the receptor is expressed, the V2R still does not recycle. The receptor remains localized in an intracellular compartment, tentatively identified as the perinuclear recycling compartment, as determined by co-localization with established intracellular markers (5Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Post-translational modifications also affect the expression and distribution of V2Rs. The palmitoylation of cysteines 341 and 342 in the carboxyl terminus of the V2R, although not essential for plasma membrane delivery, results in increased receptor abundance at the plasma membrane (6Sadeghi H. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar). Agonist-induced phosphorylation of the V2R, exclusively by G-protein-coupled receptor kinases, resulted in receptor sequestration from the plasma membrane and trapping inside the cell (7Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 5Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). After removal of the agonist, persistent phosphorylation accounted for the failure of the V2Rs to recycle to the plasma membrane (8Innamorati G. Sadeghi H. Birnbaumer M. J. Recept. Signal Transduct. Res. 1999; 19: 315-326Crossref PubMed Scopus (30) Google Scholar). Elimination of the phosphate acceptor sites resident in the V2R carboxyl terminus imparted recycling properties to these mutant receptor species (4Innamorati G. Sadeghi H. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). Visualization of the phosphorylation-deficient V2Rs revealed their sorting to the same perinuclear compartment as the wild type receptor, but unlike the wild type V2R, their lower level of phosphorylation allowed them to recycle (4Innamorati G. Sadeghi H. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). The ADP-ribosylation factor (ARF) family of proteins mediates various intracellular trafficking processes that involve membrane-bound organelles (9Nie Z. Hirsch D.S. Randazzo P.A. Curr. Opin. Cell Biol. 2003; 15: 396-404Crossref PubMed Scopus (188) Google Scholar). ARFs are small GTPases of ∼20 kDa in size that act as molecular switches. In the GDP-bound “off” state, ARFs are inactive, whereas when bound to GTP they are in the “on” state. Active, GTP-bound ARFs associate with membranes where they have effects on local lipid generation and actin rearrangement (10Donaldson J.G. J. Biol. Chem. 2003; 278: 41573-41576Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). In general, ARFs exert their effects on cellular trafficking by recruiting coat proteins to membranes, activating lipid-modifying enzymes and modulating the actin cytoskeletal arrangement (11Donaldson J.G. Jackson C.L. Curr. Opin. Cell Biol. 2000; 12: 475-482Crossref PubMed Scopus (319) Google Scholar). Of the six members in the ARF family, ARF1 has been the most extensively characterized to date. Whereas ARF1 is localized to the Golgi network, ARF6 was described to exclusively affect trafficking between the plasma membrane and endosomes (12Peters P.J. Hsu V.W. Eng-Ooi C. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). Specifically, ARF6 was shown to control a clathrin-independent recycling pathway that was dependent on the full ARF6 activation/inactivation cycle as well as phospholipid hydrolysis (13Radhakrishna H. Klausner R.D. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar, 14Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar). Overexpression of a GTPase-activating protein for ARF6, which catalyzed the hydrolysis of GTP to GDP thereby inactivating ARF6, reduced β2-adrenergic receptor internalization (15Premont R.T. Claing A. Vitale N. Freeman J.L.R. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Crossref PubMed Scopus (255) Google Scholar), highlighting the importance of ARF6 in the internalization of a GPCR. Point mutations of ARF6 have been generated to elucidate the localization and function of ARF6 in vivo. Using ARF1 as a model, substitution of glutamine with leucine at position 67 (Q67L ARF6) resulted in an ARF6 isoform that could not hydrolyze GTP and was, thus, constitutively active (16D'souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Furthermore, substitution of threonine with asparagine at position 27 resulted in an ARF6 that could not exchange GDP for GTP and was, thus, inactive. Even though much of the T27N ARF6 protein aggregates intracellularly, a sufficient level of T27N ARF6 localizes to the plasma membrane where it acts as a dominant-negative inhibitor of ARF6 function by sequestering nucleotide exchange factors that activate ARF6 (17Macia E. Luton F. Partisani M. Cherfils J. Chardin P. Franco M. J. Cell Sci. 2004; 117: 2389-2398Crossref PubMed Scopus (74) Google Scholar). These tools have been used to examine the ARF6 dependence of cell-surface protein trafficking. It has been shown that Q67L ARF6 enhanced the rate and extent of recycling of the transferrin (Tfn) receptor as well as diminishing TfnR endocytosis (16D'souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). TfnRs have been used as models for vesicular and GPCR trafficking, and so it was interesting to determine whether ARF6 would play an analogous role in the trafficking of the V2R given the failure of this particular GPCR to recycle. In the absence of agonist application, Q67L ARF6 reduced cell-surface V2R number by more than 10-fold, although neither the agonist-induced internalization nor the recycling of the V2R was altered by the ARF6 constructs. This is contrary to what happens with the TfnR, whereby TfnR levels at the plasma membrane were enhanced 2-fold (16D'souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar), but similar to the effect of Q67L ARF6 on acetylcholine M2 receptor expression (18Delaney K.A. Murph M.M. Brown L.M. Radhakrishna H. J. Biol. Chem. 2002; 277: 33439-33446Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Thus, while attempting to delineate the route(s) of V2R trafficking affected by ARF6, we found a role for ARF6 in GPCR maturation. Reagents—Cell culture supplies and media were obtained from Invitrogen. Tritiated AVP and 35S-Express protein labeling mix were from PerkinElmer Life Sciences. CALPHOS™ mammalian transfection kit and anti-LAMP1 monoclonal antibody were from BD Biosciences. Complete™ protease inhibitor mixture was from Roche Applied Science. The following reagents were obtained from Molecular Probes (Eugene, OR): Alexa Fluor 488-conjugated anti-HA monoclonal, Alexa Fluor 488-conjugated goat anti-mouse, Alexa Fluor 568-conjugated goat anti-rabbit, and Texas-red conjugated Tfn. Anti-endosomal autoantigen 1 polyclonal was from Upstate Biotechnology, Inc. (Lake Placid, NY). Vectorshield mounting medium was from Vector Laboratories (Burlingame, CA). Wild-type, Q67L, T27N AFR6 constructs and the ARF6-specific rabbit polyclonal antibody were generous gifts from Dr. J. G. Donaldson (National Institutes of Health, Bethesda, MD). Plasmids encoding fragment 319-418 arrestin 2 GFP and K44A dynamin were generous gifts from Dr L. Slice (University of California, Los Angeles, CA). Plasmids encoding ARNO were a generous gift from Dr. J. E. Casanova (University of Virginia at Charlottesville, VA). The plasmid encoding the phospholipase D1 pleckstrin homology domain fused to the green fluorescent protein (PLCδ1-PH-GFP) was a generous gift of Dr. T. Balla (NICHD, National Institutes of Health, Bethesda, MD). All other reagents were obtained from Sigma. Cell Culture and Transfection—HEK 293-T cells were transiently transfected with 6 μg of plasmid DNA using the CALPHOS™ mammalian transfection kit according to the manufacturer's instructions. After 24 h the cells were plated onto 24-well plates or 100-mm dishes and analyzed 24 h later. Immunoblot Analysis of V2R and ARF6 Expression—HEK 293-T cells were lysed by incubation in hypotonic buffer (50 mm Tris.HCl, pH 7.5, 0.5 mm MgCl2, 150 mm potassium acetate, 1% Nonidet P-40, 1.5 mm dithiothreitol) containing the Complete™ protease inhibitor mixture). Lysis was achieved by drawing the cells through needles of decreasing gauge (20-25 gauge) fitted to a 1-ml syringe. A post-nuclear supernatant was prepared by centrifugation at 3000 rpm for 5 min. After protein concentration determination, the supernatant was incubated with 2× sample buffer for 20 min. Samples were resolved by SDS-PAGE. The V2R was detected using a peptide-purified rabbit polyclonal antibodies raised against a peptide corresponding to the carboxyl terminus (antibody 3) of the human V2R (7Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). ARF6 was detected using an ARF6-specific rabbit polyclonal antibody raised against the carboxyl region of ARF6 (19Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (417) Google Scholar). Proteins were transferred to nitrocellulose using a Trans-Blot semidry blotter (Bio-Rad). The nitrocellulose was blocked for 30 min in blotto (20 mm Tris.HCl, pH 7.5, 150 mm NaCl, 5% powdered milk, 0.1% Tween 20) followed by incubation with primary antibody (1:1000) overnight at 4 °C. The blot was washed 3 times for 10 min in blotto and incubated with horseradish peroxidase-coupled secondary antibody) for 1 h at room temperature. After 3 more 10-min washes in blotto, the proteins were visualized by ECL. Measurement of Receptor Binding—Transfected cells were plated onto 24-well plates that had been previously coated with poly-l-lysine (100 μg/well). The cells were washed twice with ice-cold PBS and incubated with 20 nm [3H]AVP in 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 PBS, and 0.5 ml of 0.1 m NaOH was added to extract radioactivity. After 30 min at 37 °C, the fluid from each well was transferred to a scintillation vial containing 3.5 ml of scintillation fluid. Nonspecific binding was determined under the same conditions in the presence of 20 μm unlabeled AVP. Each experimental point was assayed in triplicate. Data are presented as the mean ± S.E. of 3-5 experiments. Flow Cytometry—V2R-expressing HEK 293-T cells stained with an Alexa 488-conjugated anti-HA antibody (1:100) were examined on a FACSort flow cytometer (BD Biosciences) in the presence of propidium iodide to exclude cells that had lost their membrane integrity. Cells were excited at 488 nm, and the Alexa 488 and propidium iodide fluorescence were detected at 530 and >650 nm, respectively. Ten thousand viable cells were examined per sample and analyzed using CellQuest software. An analysis gate was set on the control cells to determine the number of cells that exhibited a decrease in V2R expression. Immunofluorescence and Confocal Laser-scanning Microscopy—Transfected cells were seeded on glass coverslips, and if necessary, the requisite intracellular markers were added 24 h later. The cells were then incubated at 37 °C followed by 2 washes with 37 °C PBS. Cells were fixed with 4% paraformaldehyde at 4 °C for 1 h followed by 3 washes for 10 min with PBS. The cells were then permeabilized with a solution of PBS containing 0.1% saponin and 0.2% gelatin. After incubation of the cells with the primary antibody for 2 h at room temperature, the cells were washed 3 times for 10 min with PBS. The cells were then incubated with the secondary antibodies for 90 min at room temperature followed by three 10-min washes with PBS. The coverslips were mounted on microscope slides in VectorShield™ mounting medium. Cells were then visualized at 22 °C using a Zeiss LSM 510UV confocal laser-scanning microscope using an oil 100× objective, numerical aperture of 1 (Carl Zeiss, Inc.). The 488- and 543-nm line from the included argon ion laser were the excitation sources, and a 505-550-nm band-pass filter or 560-nm long-pass filter, respectively, were used for the emission. The software used for acquisition was Zeiss LSM510 Version 3.2 for Windows 2000, and for analysis, LSM Image Examiner (licensed) Version 3.2 for Windows 2000. Metabolic Labeling with [35S]Methionine/Cysteine and Immunoprecipitation of GPCRs—Proteins were labeled in 100-mm dishes using a modification of the method published by Keefer and Limbird (20Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar) as follows. 48 h after transfection the cells were incubated for 1 h at 37°C in methionine/cysteine-free Dulbecco's modified Eagle's medium. 100 μCi of 35S-Express protein-labeling mix was then added to each plate for 30 min at 37 °C (pulse). The medium was aspirated, and the cells were washed with 37 °C PBS and returned to the incubator for the stated intervals (chase). The cells were rinsed and harvested in PBS, and the cell pellet from each plate was homogenized in 500 μl of RIPA buffer (150 mm NaCl, 50 mm Tris.HCl pH 8.0, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS containing the Complete™ protease inhibitor mixture) by drawing the cells through needles of decreasing gauge (20-25 gauge) fitted to a 1-ml syringe. Cell extracts were clarified by mixing with 50 μl of a 50% slurry of pre-washed protein A-Sepharose. Pre-washed protein A-Sepharose was prepared by incubating the resin with 100 mg/ml bovine serum albumin in RIPA buffer for 1 h followed by 2 washes with RIPA buffer alone. The clarified extracts were then incubated overnight at 4 °C with a monoclonal 12CA5 anti-HA antibody (21Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar). Antigen-antibody complexes were then separated by incubating the mixture with pre-washed protein A-Sepharose for 2 h at 4°C. The beads were centrifuged, incubated 4 times for 4 min at room temperature with RIPA buffer, and recovered each time by centrifugation. Proteins were eluted with 80 μl of 2 × Laemmli buffer containing 10% β-mercaptoethanol. The samples were electrophoresed in 10% polyacrylamide gels and visualized by exposing the dried gels to Eastman Kodak Co. BioMax film at -70 °C. Densitometric analysis of the labeled bands was carried out using a ChemiDoc XRS gel-imaging system (Bio-Rad). Constitutively Active ARF6 Inhibits Cell-surface V2R Expression—We have previously shown that the mature wild-type V2R migrates as a diffuse band of 45-55 kDa on SDS-PAGE gels (21Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar). This migratory pattern arises from both N- and O-linked glycosylation, which takes place in the endoplasmic reticulum and Golgi apparatus (22Sadeghi H. Birnbaumer M. Glycobiology. 1999; 9: 731-737Crossref PubMed Scopus (50) Google Scholar). A V2R mutant in which asparagine at position 22 was changed to glutamine (N22Q V2R) lacks N-linked glycosylation and migrates as a focused band of 40 kDa. As previously reported, this receptor form is unaltered in its ligand binding affinity, second-messenger generation, and trafficking patterns (21Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar). As a result, the N22Q V2R was used as the wild-type V2R for this study to obtain sharper, better-resolved bands during protein electrophoresis. Published reports describing a role for ARF6 in the recycling of internalized endosomes to the plasma membrane led us to examine whether this small GTPase could promote the recycling of the V2R. For this purpose, cDNAs encoding wild-type, constitutively active (Q67L), or dominant-negative (T27N) forms of ARF6 were co-transfected with V2Rs in HEK 293-T cells. In the absence of any agonist treatment, coexpression of the V2R with Q67L ARF6 reduced the cell-surface levels of receptor by greater than 90% (Fig. 1A, left y axis), as detected by radioligand binding. After a 20-min incubation with a saturating concentration of agonist (100 nm AVP), neither the extent of endocytosis nor lack of recycling of the V2R was affected by expression of either T27N or Q67L ARF6 (data not shown). More modest reductions in V2R surface expression were observed when the V2R was co-expressed with either wild-type or T27N ARF6 (Fig. 1A). In the absence of any agonist treatment, co-expression of the V2R with either the wild-type or T27N (GDP-bound and, thus, inactive) ARF6 reduced surface receptors in whole cells by 42 ± 2 and 22 ± 8%, respectively. Both levels were significantly different (n = 3, p < 0.01, one-way analysis of variance, Dunnett's post-test) from control (that is, those transfected with the V2R and β-galactosidase (β-gal). This Q67L-mediated effect was not restricted to the Gs-coupled V2R, as the recycling-competent Gq-coupled V1aR was subject to similar regulation (Fig. 1A, left y axis). A similar ARF6 effect on the acetylcholine M2 muscarinic receptor cell-surface expression has been reported (18Delaney K.A. Murph M.M. Brown L.M. Radhakrishna H. J. Biol. Chem. 2002; 277: 33439-33446Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Support for these results was obtained using fluorescence-assisted cell sorting analysis (Fig. 1A, right y axis). Here too, the proportion of viable cells showing fluorescence emanating from Alexa 488-labeled V2Rs was indistinguishable between the wild-type and T27N-transfected HEK 293-T cells. However, a clear difference was observed in the fluorescence intensity of the Q67L-expressing cells, which showed 33% of the intensity of control cells (Fig. 1A, right y axis). To examine whether ARF6 modified V2R synthesis, HEK 293-T cells transfected with the V2R plus the ARF6 constructs were lysed by hypotonic buffer, as described under “Materials and Methods,” and the aqueous extract obtained from this process was immunoblotted for the presence of the V2R (Fig. 1B) or ARF6 (Fig. 1C). Overexpression of the ARF6 constructs resulted in an ∼10-fold increase in ARF6 expression over control, untransfected cells. For the V2R, the mature 40-kDa form (arrow) and the 33-kDa precursor form (arrowhead) were detected in the cells co-transfected with the ARF6 constructs. As can be seen, there was a decrease in mature V2R expression in the presence of Q67L ARF6. The mature V2R form is the one responsible for [3H]AVP binding, and so reduced expression would have accounted for diminished ligand binding. Thus, the immunoblotting data were consistent with those obtained via radioligand binding and fluorescence-assisted cell sorting analysis. In the presence of Q67L ARF6, it was clear that there was a prevalence of the precursor form of the V2R and concomitant depletion of the mature form. This was in contrast to the other lanes whereby there were approximately equal levels of V2R expression in either form. Therefore, it appeared that the constitutively active Q67L ARF6 reduced the amount of mature V2R. These results were contrary to what happened with the prototypical model of recycling membrane proteins, the TfnR. The net effect observed was an increase of TfnR present at the plasma membrane unlike what we have observed (16D'souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Thus, we attempted to delineate the route(s) for this trafficking phenomenon. Intracellular Localization of the V2R in the Presence of Q67L ARF6—To determine whether the overexpressed V2Rs and ARFs interacted in vivo, co-immunoprecipitation experiments were carried out as described under “Materials and Methods.” As shown in Fig. 2A, immunoprecipitation of the V2R gave the previously observed pattern of V2R expression whereby the Q67L ARF6 drastically inhibited V2R expression. When parallel samples were immunoblotted for the presence ARF6 (Fig. 2B), only the Q67L ARF6 mutant was found to be associated with the V2R. The association was stable for the duration of the immunoprecipitation as it was unnecessary to add cross-linkers to recover ARF6 Q67L from the V2R precipitate. As expected, visualization of the V2R when co-transfected with either wild-type or T27N ARF6 showed a predominantly plasma-membrane distribution (Fig. 3, A and G, respectively), as there was very little co-localization of cell-surface V2R and overexpressed wild-type or T27N ARF6 (Fig. 3, B and F, respectively). Endogenous ARF6 as well as overexpressed wild-type and Q67L ARF6 have been reported to have both an intracellular and plasma membrane distribution (12Peters P.J. Hsu V.W. Eng-Ooi C. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). In the presence of the Q67L ARF6 (Fig. 3E), the V2R was observed in “vacuolar” structures (Fig. 3D) that were distributed in distinct cytoplasmic areas. These vacuolar bodies also contained the overexpressed Q67L ARF6, as evidenced by the yellow regions on the merged image (Fig. 3F). This vacuolar pattern induced by Q67L ARF6 has been observed as early as 18 h after transfection in Cos and HeLa cells (14Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar) and persisted for at least 48 h after transfection in HEK 293-T cells. By virtue of residing in the same compartment, this result lent support to the co-immunoprecipitation studies that had indicated an interaction between the V2R and Q67L ARF6.FIGURE 3Localization of overexpressed V2Rs and ARF6 constructs. Transfected HEK 293-T cells growing on glass coverslips were fixed and stained for overexpressed V2Rs and ARF6. V2Rs (A, D, and G) were visualized using an Alexa 488-conjugated anti-HA antibody (1:100). Overexpressed wild-type (WT) (B), Q67L (E), and T27N ARF6 (H) were visualized using a polyclonal antibody raised against ARF6 (1:500) and an Alexa 568-conjugated goat anti-rabbit secondary antibody (1:100). The corresponding overlays (C, F, and I) are shown on the right-hand side. Scale bar,10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the nature of these vacuoles, co-localization studies using established intracellular markers were carried out. Intracellular Tfn accumulation (Fig. 4, B and E) was used to identify early and recycling endosomes (23Maxfield F.R. McGraw T.E. Nat. Rev. Mol. Cell Biol. 2004; 5: 121-132Crossref PubMed Scopus (1494) Google Scholar). Tfn uptake was efficient in untransfected cells as well as in those only expressing the V2R and β-gal (Fig. 4C). Cells coexpressing the V2R and Q67L ARF6 did not show any Tfn endocytosis (Fig. 4F). Neighboring untransfected cells internalized the Tfn, indicating that the Q67L ARF6 construct functioned as previously reported (16D'souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Other intracellular markers were used to identify localization of the V2R (Fig. 5, A, D, G, and J); that is, early endosomal autoantigen 1 (EEA1) and lysosomal-associated membrane protein (LAMP1) to identify endosomes and lysosomes, respectively (23Maxfield F.R. McGraw T.E. Nat. Rev. Mol. Cell Biol. 2004; 5: 121-132Crossref PubMed Scopus (1494) Google Scholar). To test if origin of the vacuoles was the plasma membrane, the localization of PLCδ1-PH-GFP fusion protein in HEK cells was examined. When expressed alone, the PLCδ1-PH-GFP protein was not only restricted to the plasma membrane as it has been shown in other cell lines but was also found in intracellular membranes (supplemental Images 1 and 2). In addition, V2R-containing “vacuoles” did not co-localize with either endosomal autoantigen 1 (EEA1) (Fig. 5, B and E) or LAMP1 (Fig. 5, H and K), ruling out their identity as early/recycling endosomes or late endosomes/lysosomes. The lack of co-localization with recycling endosomes indicated that the V2R did not reside in a compartment from where it could be recycled to the cell surface.FIGURE 5Visualization of Q67L ARF6 vacuoles and intracellular organelle markers. HEK 293-T cells co-expressing either the V2R and β-gal or the V2R and Q67L ARF6 were fixed and stained for endosomal autoantigen 1 (EEA1) (B and E at 1:100 dilution) or LAMP1 (H and K at 1:100 dilution). Alexa 568 anti-rabbit polyclonal (1:100) was used as the secondary antibody. The V2Rs (A, D, G, and J) were visualized using an Alexa 488-conjugated anti-HA antibody (1:100). The respective overlays (C, F, I, and L) are shown. Scale bar,10 μm.View Large Image Figure" @default.
• W1963988769 created "2016-06-24" @default.
• W1963988769 creator A5019687222 @default.
• W1963988769 creator A5056461765 @default.
• W1963988769 date "2006-04-01" @default.
• W1963988769 modified "2023-09-30" @default.
• W1963988769 title "A Role for ADP-ribosylation Factor 6 in the Processing of G-protein-coupled Receptors" @default.
• W1963988769 cites W1532176495 @default.
• W1963988769 cites W1978475018 @default.
• W1963988769 cites W1990398484 @default.
• W1963988769 cites W2009468127 @default.
• W1963988769 cites W2009776121 @default.
• W1963988769 cites W2012642649 @default.
• W1963988769 cites W2023397454 @default.
• W1963988769 cites W2039060112 @default.
• W1963988769 cites W2041097342 @default.
• W1963988769 cites W2049170665 @default.
• W1963988769 cites W2050020385 @default.
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