ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of α2B-adrenergic receptor (AR), β2-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that α2B-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the α2B-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.
ADP-ribosylation factors (ARFs) belong to the superfamily of Ras-related small GTPases and modulate vesicle-mediated transport (D'Souza-Schorey and Chavrier, 2006). Six ARF members (ARF1–6) have been identified in mammalian cells, but ARF2 is not expressed in humans. Based on their amino acid sequence homology and gene organization, ARFs are divided into three classes: class I (ARF1–3), class II (ARF4–5), and class III (ARF6). Among these ARF GTPases, ARF1 and ARF6 are the best studied and well understood members. ARF1 plays a crucial role in both anterograde and retrograde trafficking, whereas ARF6 is mainly involved in regulation of endocytosis and actin cytoskeleton remodeling (Stearns et al., 1990; Palacios et al., 2001; Spang, 2002). Although ARF3 has not been well studied, it is generally considered that the functions of ARF1 and ARF3 are interchangeable. In contrast, the physiological roles for the class II ARFs remain poorly characterized.
Like other Ras-related GTPases, the function of ARFs is highly regulated by their recycling between active GTP-bound and inactive GDP-bound conformations (Gsandtner et al., 2005; Lee et al., 2005). Inactive GDP-bound ARFs may be recruited from cytosol onto the membrane by interacting with receptor proteins, and their association with the membrane is mediated through the N-terminal myristoylated amphipathic helix domain. On the membrane, ARFs undergo the exchange of GDP for GTP, which is catalyzed by guanine nucleotide exchange factors (GEFs). Active GTP-bound ARFs subsequently interact with downstream effectors. It has been shown that the GTP-bound ARF1 recruits distinct protein complexes onto different intracellular compartments, resulting in the formation of different transport vesicles. In the early secretory pathway, activation of ARF1 results in the recruitment of a complex of cytosolic proteins, collectively known as coatomers, leading to the formation of COPI-coated vesicles, which mediate cargo transport from the Golgi to the endoplasmic reticulum (ER), from the ER-Golgi intermediate complex (ERGIC) to the Golgi, and between Golgi cisternae (Spang, 2002). In the post-Golgi transport, the activated ARF1 recruits the adaptor protein complex and Golgi-localized γ-ear-containing ARF1-binding proteins (Bonifacino, 2004) to initiate the formation of the clathrin-coated vesicles, which mediate protein transport between the trans-Golgi network (TGN) and the endosomal compartment. The release of the cargo-carrying vesicles from the membrane is controlled by the hydrolysis of GTP to GDP of ARFs, a process facilitated by GTPase-activating proteins (GAPs).
G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors and regulate a variety of cell functions (Pierce et al., 2002). The magnitude of receptor-elicited cellular response to a given stimulus is dictated by elaborately regulated intracellular trafficking processes that control the cell surface expression and subcellular compartment targeting of the receptors. Similar to many plasma membrane proteins, the life of GPCRs begins at the ER, where they are synthesized, folded, and assembled. Properly folded receptors are transported from the ER through the ERGIC, the Golgi apparatus, and the TGN to the plasma membrane (Dong et al., 2007). At the plasma membrane, GPCRs may undergo internalization on stimulation by their ligands. The internalized receptors in the early endosome may be sorted to the lysosome for degradation or to the recycling endosome for return to the plasma membrane. In contrast to the extensive studies on the endocytic, recycling, and degradation pathways (Goodman et al., 1996; Puthenveedu and von Zastrow, 2006), the molecular mechanism underlying the export of newly synthesized GPCRs from the ER to the cell surface remains less well understood.
As an initial approach to elucidate the mechanism of nascent GPCR targeting to the cell surface, we have determined the role of Sar1 and several Rab GTPases in regulating cell surface transport of representative GPCRs, including the angiotensin II and adrenergic receptor (AR) subfamilies (Wu et al., 2003; Filipeanu et al., 2004; Filipeanu et al., 2006; Dong and Wu, 2007; Dong et al., 2008; Zhang et al., 2009). Our studies indicate that the cell surface transport of different GPCRs may be mediated through distinct pathways and suggest a novel pathway mediating α2B-AR traffic from the ER to the Golgi apparatus (Wu et al., 2003). In this study we investigated the role of ARF GTPases in the anterograde transport from the ER to the cell surface of four family A GPCRs, including α2B-AR, β2-AR, angiotensin II type 1 receptor (AT1R), and chemokine (CXC motif) receptor 4 (CXCR4).
Materials and Methods
Antibodies against ARF1, ARF5, and ARF6 were purchased from Assay Designs, Inc. (Ann Arbor, MI). Antibodies against ARF3, GM130, and p230 and PE-conjugated monoclonal anti-CXCR4 and IgG2a antibodies were from BD Biosciences (San Jose, CA). ERGIC-53 antibodies were from Alexis Laboratories (San Diego, CA). Antibodies against phospho-extracellular signal-regulated kinase 1 and 2 (ERK1/2) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ERK1/2 antibodies were from Cell Signaling Technology Inc. (Danvers, MA). High affinity fluorescein-conjugated anti-hemagglutinin (HA) antibody 3F10 and anti-HA mouse monoclonal antibodies 12CA5 were from Roche Diagnostics (Mannheim, Germany). Isoproterenol (ISO), UK-14,034, brefeldin A (BFA), and anti-His antibodies were from Sigma-Aldrich (St. Louis, MO). Human angiotensin II (Ang II) was purchased from EMD Chemicals (Gibbstown, NJ). Human stromal cell-derived factor 1α (SDF-1α) was purchased from PeproTech (Rocky Hill, NJ). Penicillin/streptomycin, l-glutamine, and trypsin/EDTA were from Invitrogen (Carlsbad, CA). [3H]CGP-12177 (specific activity = 51 Ci/mmol), [3H]RX 821002 (41 Ci/mmol), l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS, 80 Ci/mmol), and (3-(125I)-iodotyrosyl 4) Ang II (125I-Ang II) (2000 Ci/mmol) were purchased from GE Healthcare (Piscataway, NJ). Alexa Fluor 594-labeled secondary antibodies and 4,6-diamidino-2-phenylindole were from Invitrogen. All the other materials were obtained as described elsewhere (Wu et al., 2003; Duvernay et al., 2009a).
α2B-AR, β2-AR, and AT1R tagged with green fluorescent protein (GFP) at their C termini were generated as described previously (Wu et al., 2003). α2B-AR, AT1R, and ARF4 tagged with three HA at their N termini were generated as described previously (Duvernay et al., 2009a). CXCR4 tagged with HA at its N terminus was obtained from Missouri S&T cDNA Resource Center (Rolla, MO). GFP and HA have been widely used to tag GPCRs without altering their trafficking and function (Wu et al., 2003; Duvernay et al., 2009a). Glutathione transferase (GST) fusion proteins encoding the C terminus of α2B-AR were generated into the pGEX-4T-1 vector using a strategy as described previously (Wu et al., 1997, 1998). ARF mutants were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA). The sequence of each construct used in this study was verified by restriction mapping and nucleotide sequence analysis.
Cell Culture and Transient Transfection.
Human embryonic kidney (HEK) 293 cells and ghost cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Wu et al., 2003). NG108-15 neuroblastoma-glioma cells expressing endogenous α2B-AR subtype were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. Transient transfection of the cells was carried out using Lipofectamine 2000 reagent (Invitrogen) as described previously (Wu et al., 2003). The transfection efficiency was estimated to be greater than 75% based on the GFP fluorescence. To determine the effect of BFA treatment on the cell surface expression and subcellular distribution of receptors, the cells transfected with the receptors were incubated with BFA at a concentration of 5 μg/ml culture medium for 8 h.
Cell surface expression of α2B-AR, β2-AR, M3-muscarinic receptor (M3-MR), and AT1R in HEK293 cells was measured by ligand binding of intact live cells as previously described using [3H]RX 821002, [3H]CGP-12177, [3H]NMS, and 125I-Ang II (Filipeanu et al., 2004; Duvernay et al., 2009a), respectively. In brief, HEK293 cells were cultured on six-well dishes and transfected with 200 ng of the α2B-AR, β2-AR, or M3-MR plasmid together with 800 ng of pcDNA3 or ARFs. After 6 h, the cells were split into 12-well plates at a density of 4 × 105 cells/well and cultured for an additional 24 h. For measurement of β2-AR expression at the cell surface, the cells were incubated with DMEM containing the ligand [3H]CGP-12177 at a concentration of 20 nM for 90 min at room temperature. Nonspecific binding was determined by preincubation with alprenolol at a concentration of 20 μM for 30 min followed by incubation with [3H]CGP-12177 (20 nM) in the presence of alprenolol (20 μM). For measurement of the cell surface expression of the exogenously expressed α2B-AR in HEK293 cells and endogenous α2B-AR in NG108-15 cells, the cells were incubated with DMEM plus [3H]RX 821002 at a concentration of 20 nM for 90 min at room temperature. The nonspecific binding was determined in the presence of rauwalscine (10 μM). For measurement of M3-MR expression at the cell surface, the cells were incubated with the ligand [3H]NMS at a concentration of 20 nM for 2 h at 4°C. Nonspecific binding was determined in the presence of atropine (20 μM). The cells were washed twice with 1 ml of phosphate-buffered saline (PBS), and the cell surface-bound ligands were extracted by 1 M NaOH treatment for 2 h. The radioactivity was counted by liquid scintillation spectrometry in 3.5 ml of Ecoscint A scintillation solution (National Diagnostics, Atlanta, GA).
For measurement of AT1R expression at the cell surface, HEK293 cells were cultured on six-well plates and transfected with 0.5 μg of AT1R together with 1.5 μg of pcDNA3 or individual ARF1 mutants. The cells then were split into 12-well plates and starved for 24 h followed by an incubation with 400 μl of DMEM containing 125I-Ang II at a concentration of 20 pM for 5 h at 4°C to avoid AT1R internalization. The nonspecific binding was determined in the presence of nonradioactive Ang II (3 μM). After washing the cells twice with 1 ml of DMEM, the bound ligand was extracted by mild acid treatment (2 × 5 min with 0.5 ml of buffer containing 50 mM glycine and 125 mM NaCl, pH 3). The radioactivity was counted in a gamma counter. All the radioligand binding assays were performed in triplicate.
Flow Cytometric Analysis of the Cell Surface and Total Expression of Receptors.
The cell surface expression of α2B-AR, AT1R, and CXCR4 was measured by flow cytometry as described previously (Zhang et al., 2009). In brief, the receptors were tagged with three HA at their N termini and transiently expressed into HEK293 cells. The cells were suspended at a density of 1 × 107 cells/ml in PBS containing 1% FBS and incubated with high affinity anti-HA-fluorescein (3F10) at a final concentration of 2 μg/ml for 30 min at 4°C. For measurement of the cell surface expression of endogenous CXCR4 in HEK293 and ghost cells, the cells were incubated with anti-CXCR4 antibodies or the control antibodies IgG2a (20 μl of antibodies/100 μl of cells) for 30 min at room temperature. After washing three times with 1.0 ml of PBS plus 1% FBS, the cells were resuspended and the fluorescence was analyzed on a flow cytometer (FACSCalibur; BD Biosciences). Because the staining with anti-HA antibodies was carried out in the unpermeabilized cells and only those receptors expressed at the cell surface were accessible to the anti-HA antibodies, the measured fluorescence reflected the amount of receptor expressed at the cell surface.
Fluorescence microscopic analysis of receptor subcellular localization was carried out as described previously (Wu et al., 2003). HEK293 cells were grown on coverslips precoated with poly-l-lysine in six-well plates and transfected with 40 ng of GFP-tagged receptors with or without cotransfection with 400 ng of the pcDNA3 vector or the ARF1 mutants. For colocalization of the receptors with the ER marker DsRed2-ER, HEK293 cells were transfected with 100 ng of GFP-tagged receptors and 100 ng of pDsRed2-ER with 400 ng of the pcDNA3 vector or ARF1 mutants. The cells were fixed with 4% paraformaldehyde/4% sucrose mixture in PBS for 15 min and stained with 4,6-diamidino-2-phenylindole for 5 min. For colocalization of the receptors with the ERGIC, Golgi, and TGN markers, HEK293 cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 min and blocked with 5% normal donkey serum for 1 h. The cells were then incubated with antibodies against ERGIC-53, GM130, or p230 for another 2 h. After washing with PBS (3 × 5 min), the cells were incubated with Alexa Fluor 594-labeled secondary antibody (1:2000 dilution) for 1 h at room temperature. The coverslips were mounted, and fluorescence was detected with a Leica DMRA2 epifluorescent microscope (Leica Microsystems, Inc., Bannockburn, IL). Images were deconvolved using SlideBook software and the nearest neighbor deconvolution algorithm (Intelligent Imaging Innovations, Denver, CO).
Measurement of ERK1/2 Activation.
HEK293 or ghost cells were cultured on six-well plates and transfected with 1.5 μg of the pcDNA3 vector or ARF1T31N with or without cotransfection with 0.5 μg of the receptor plasmids. At 6 to 8 h after transfection, the cells were split and cultured for 24 to 30 h. The cells were starved for at least 2 h and then stimulated with agonists as indicated in the figure legends. The stimulation time and drug concentrations used in these experiments will produce the maximal ERK1/2 in response to each agonist (Wu et al., 2003; Duvernay et al., 2004). Stimulation was terminated by addition of 1× SDS gel loading buffer as described previously (Wu et al., 2003). After solubilizing the cells, 20 μl of total cell lysates was separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes. ERK1/2 activation was determined by Western blotting measuring the levels of phosphorylation of ERK1/2 with phosphospecific ERK1/2 antibodies. The membranes were then stripped and reprobed with anti-ERK1/2 or anti-ARF1 antibodies to determine the total amount of ERK1/2 and ARF1, respectively. The signal was detected using Enhanced Chemiluminescence Plus (PerkinElmer Life and Analytical Sciences, Waltham, MA) and a Fujifilm (Tokyo, Japan) luminescent image analyzer (LAS-1000 Plus) and quantitated using the Image Gauge program (version 3.4; Fujifilm).
For coimmunoprecipitation of α2B-AR and ARF1, HEK293 cells cultured on 100-mm dishes were transfected with 2 μg of HA- or GFP-tagged α2B-AR or the pEGFP-N1 vector together with ARF1 for 36 h. The cells were washed twice with PBS, harvested, and lysed with 500 μl of lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, and Complete Mini protease inhibitor mixture (Roche Diagnostics). After gentle rotation for 1 h, samples were centrifuged for 15 min at 14,000g, and the supernatant was incubated with 50 μl of protein G-Sepharose for 1 h at 4°C to remove nonspecific bound proteins. Samples were then incubated with 1 μg of anti-HA or anti-GFP antibodies overnight at 4°C with gentle rotation followed by incubation with 50 μl of protein G Sepharose 4B beads for 5 h. Resin was collected by centrifugation and washed three times with 500 μl of lysis buffer without SDS. Immunoprecipitated receptors were eluted with SDS gel loading buffer, separated by 10% SDS-PAGE, and visualized by immunoblotting using anti-HA or anti-GFP antibodies (Dong and Wu, 2006). ARF1 in the immunoprecipitates was detected by using anti-ARF1 antibodies.
GST Fusion Protein Pull-Down Assay.
The GST fusion proteins containing the C terminus of α2B-AR were expressed in bacteria and purified using a glutathione affinity matrix as described previously (Wu et al., 1997, 1998). The fusion proteins immobilized on the glutathione resin were either used immediately or stored at 4°C for no longer than 3 days. Each batch of fusion protein used in experiments was first analyzed by Coomassie Blue staining after SDS-PAGE. GST fusion proteins tethered to the glutathione resin were incubated with cell lysate prepared from HEK293 cells transfected with GFP-tagged ARF1 in 500 μl of binding buffer (20 mM Tris-HCl, pH 7.5, 2% Nonidet P40, and 70 mM NaCl) at 4°C overnight. To determine whether the α2B-AR C terminus could directly interact with ARF1, ARF1 was tagged with the epitope His at its N terminus, and His-ARF1 was purified and eluted by using the His SpinTrap kit from GE Healthcare. One microgram of purified His-ARF1 was incubated with GST-α2B-AR C-terminal fusion protein at 4°C for 2 h. The resin was washed four times with 1 ml of binding buffer, and the retained proteins were solubilized in 1× SDS gel loading buffer and separated by SDS-PAGE. Bound ARF1-GFP and His-ARF1 were detected by immunoblotting using anti-GFP and anti-His antibodies, respectively.
Differences were evaluated using Student's t test, and P < 0.05 was considered statistically significant. Data are expressed as the mean ± S.E.
Inhibition of the Cell Surface Expression of α2B-AR, β2-AR, AT1R, and CXCR4 by BFA Treatment.
To investigate the role of ARF GTPases in the anterograde transport of GPCRs, we first determined the effect of BFA treatment on the cell surface expression of four family A GPCRs, including α2B-AR, β2-AR, AT1R, and CXCR4. BFA treatment is the well characterized tool for studying the function of ARF GTPases. BFA is a fungal metabolite that inserts at the interface between GDP-ARF and the catalytic Sec7 domain of GEFs, thus preventing the function of GEFs in facilitating the displacement of GDP with GTP. α2B-AR-GFP, β2-AR-GFP, AT1R-GFP, or HA-CXCR4 was transiently expressed into HEK293 cells, and their cell surface expression was measured by radioligand binding in intact live cells (GFP-tagged receptors) or flow cytometry after staining with anti-HA antibodies in unpermeabilized cells (HA-tagged receptors). BFA treatment at a concentration of 5 μg/ml for 8 h dramatically inhibited the cell surface expression of all four receptors by approximately 80% (Fig. 1A).
We then determined the effect of the BFA treatment on the subcellular distribution of the receptors. GFP-tagged α2B-AR, β2-AR, and AT1R HA-tagged CXCR4 were transiently expressed in HEK293 cells, and their subcellular localization at steady state was revealed by fluorescence microscopy analysis. As anticipated, each receptor robustly expressed at the cell surface in control cells. BFA treatment arrested the receptors in the perinuclear regions of the cell (Fig. 1B), and the intracellularly accumulated receptors were extensively colocalized with the ER marker DsRed2-ER (data not shown). These data suggest that ARF GTPases may play a crucial role in the anterograde transport from the ER to the cell surface of a group of GPCRs.
Five Human ARF GTPases Differentially Modulate the Cell Surface Transport of α2B-AR.
We next asked the question of which ARF GTPases regulate GPCR transport to the cell surface. To address this question, we defined the role of each of the five identified human ARF GTPases in the cell surface transport of α2B-AR. The loss of function of each ARF GTPase was achieved by expressing its GTP-bound (Q mutated to L), GDP-bound (T mutated to N), and guanidine nucleotide-deficient (N mutated to I) mutants, which have been shown to function as the dominant negative mutants. Expression of ARF GTPases was determined by immunoblotting (Fig. 2A). Transient expression of each of the three ARF1 mutants markedly attenuated the cell surface expression of α2B-AR as measured by intact cell ligand binding. Expression of ARF3Q71L and ARF3N126I also strongly inhibited α2B-AR transport to the cell surface. In contrast, ARF3T31N, ARF5T31N, and ARF6Q67L mutants only moderately attenuated α2B-AR transport by approximately 25%, whereas all three ARF4 mutants, ARF5Q71L, ARF5T31N, ARF6T27N, and ARF6N122I did not clearly alter the cell surface expression of α2B-AR (Fig. 2B). These data show that five ARF GTPases differentially modulate the cell surface targeting of nascent α2B-AR.
Attenuation of ARF1 Function Inhibits the Cell Surface Expression of α2B-AR, β2-AR, AT1R, CXCR4, and M3-MR.
Our preceding data show that expression of the ARF1 mutants produced the most profound inhibitory effect on α2B-AR expression at the cell surface as measured by intact cell ligand binding. To eliminate the possible effect of the ARF1 mutants on the ligand binding ability of α2B-AR, HA-α2B-AR was expressed together with the ARF1 mutants, and its cell surface expression was measured by flow cytometry. Consistent with the intact cell ligand binding data, the cell surface expression of α2B-AR was dramatically attenuated by the ARF1 mutants (Fig. 3A). In contrast, expression of the ARF1 mutants had no noticeable effects on the total α2B-AR expression as quantified by measuring the GFP signal using flow cytometry (Fig. 3A).
We next determined the effect of expression of the ARF1 mutants on the cell surface expression of β2-AR, AT1R, and CXCR4. Similar to α2B-AR, the cell surface expression of β2-AR, AT1R, and CXCR4 was markedly inhibited by each of the three ARF1 mutants by 60 to 80% (Fig. 3B). These data strongly show that ARF1 is essential for the cell surface transport of α2B-AR, β2-AR, AT1R, and CXCR4.
It was reported that transient expression of ARF1T31N did not significantly influence the plasma membrane expression of M3-MR in 1321N1 human astrocytoma cells (Mitchell et al., 2003). To determine whether ARF1 could selectively modulate the transport of distinct GPCRs, we measured the effect of the three ARF1 mutants on the cell surface expression of M3-MR in HEK293 cells using the same system as for α2B-AR, β2-AR, AT1R, and CXCR4. Our data show that transient expression of each of the three ARF1 mutants markedly inhibited the cell surface expression of M3-MR as measured by intact cell ligand binding (Fig. 3B).
We then determined the effect of transient expression of cytohesin-2 and ARF GTPase-activating protein 1 (ARFGAP1), which facilitates the exchange of GDP for GTP and the GTP hydrolysis of ARF1, respectively, on the cell surface transport of α2B-AR. Expression of cytohesin-2 did not alter α2B-AR cell surface expression, whereas expression of ARFGAP1 significantly inhibited by 22% (Fig. 3C). These data suggest that the GTP hydrolysis of ARF1 may play an important role in α2B-AR transport.
ARF1T31N and ARF1Q71L Arrest the Receptors in Different Intracellular Compartments.
To further characterize how ARF1 regulates GPCR anterograde transport, we visualized the subcellular distribution of the receptors in cells expressing the GDP-bound ARF1T31N or GTP-bound ARF1Q71L mutants. Consistent with the marked reduction in the cell surface expression as measured by radioligand binding and flow cytometry, GFP-tagged α2B-AR, β2-AR, and AT1R were unable to transport to the cell surface in cells transfected with ARF1T31N or ARF1Q71L. Interestingly, these two ARF1 mutants clearly arrested the receptors in distinct intracellular compartments (Fig. 4). Similar effects were obtained for CXCR4 (data not shown). These data suggest that inhibition of the ARF1 function by expressing its GDP- and GTP-bound mutants may selectively block receptor transport at different intracellular compartments along the biosynthetic pathway.
Subcellular colocalization of the intracellularly accumulated α2B-AR with different organelle markers revealed that α2B-AR was strongly colocalized with the ER marker DsRed2-ER in cells expressing ARF1T31N (Fig. 5A), whereas the receptor was partially colocalized with the ERGIC marker ERGIC-53, the Golgi marker GM130, and the TGN marker p230 in cells expressing ARF1Q71L (Fig. 5B). Similar colocalization patterns were obtained for β2-AR, AT1R, and CXCR4 (data not shown). These data suggest that, similar to the BFA treatment, expression of the GDP-bound ARF1T31N mutant blocks receptor transport from the ER, whereas the GTP-bound ARF1Q71L mutant inhibits receptor export at the ERGIC, the Golgi, and the TGN (Fig. 5C).
Expression of ARF1T31N Blocks ERK1/2 Activation by α2B-AR, β2-AR, AT1R, and CXCR4.
To evaluate whether the attenuated receptor cell surface expression caused by expression of the ARF1 mutants could result in a concomitant defective signaling, we measured the activation of the mitogen-activated protein kinase pathway in response to stimulation with receptor agonists in cells expressing ARF1T31N. Stimulation with the agonists UK-14,304 (Fig. 6A), ISO (Fig. 6B), Ang II (Fig. 6C), and SDF-1α (Fig. 6D and Supplemental Fig. 1) strongly activated ERK1/2 in cells expressing α2B-AR, β2-AR, AT1R, and CXCR4, respectively. ERK1/2 activation by each agonist was significantly compromised in cells coexpressing ARF1T31N and individual receptor compared with cells expressing receptor alone (Fig. 6, A–E). These data are consistent with the inhibitory effect of ARF1T31N on the cell surface expression of the receptors.
Expression of ARF1T31N Attenuates the Cell Surface Expression and Signaling of Endogenous α2B-AR and CXCR4.
To further explore the physiological significance of ARF1 regulation of GPCR trafficking, we determined the effect of ARF1T31N on the cell surface expression of endogenous α2B-AR in NG108-15 cells and endogenous CXCR4 in HEK293 and ghost cells. Expression of ARF1T31N moderately but consistently reduced the cell surface expression of endogenous α2B-AR by 25% in NG108-15 cells as measured by intact cell ligand binding, whereas it markedly reduced the cell surface transport of endogenous CXCR4 by 70 and 44% in HEK293 and ghost cells, respectively (Fig. 7A) as measured by flow cytometry after staining with anti-CXCR4 antibodies. Consistent with the reduction in the CXCR4 cell surface expression, ERK1/2 activation by SDF-1α was clearly reduced in both HEK293 and ghost cells expressing ARF1T31N (Fig. 7B). These data indicate that ARF1 modulates the cell surface expression of endogenous α2B-AR and CXCR4.
Interaction of α2B-AR with ARF1.
To elucidate the possible molecular mechanism underlying the function of ARF1 in regulating GPCR transport, we determined whether α2B-AR is able to physically associate with ARF1 in vivo. α2B-AR tagged with HA or GFP was transiently expressed in HEK293 cells, and the interaction between α2B-AR and ARF1 was measured by coimmunoprecipitation with anti-HA or anti-GFP antibodies. A significant amount of ARF1 was found in the anti-HA and anti-GFP immunoprecipitates (Fig. 8), suggesting that α2B-AR may associate with ARF1.
We then sought to identify the ARF1-binding domain in α2B-AR by focusing on the C terminus as the C termini have been shown to modulate the ER-to-cell surface transport for many GPCRs, including α2B-AR (Duvernay et al., 2004, 2009b). The C terminus was generated as a GST fusion protein (Fig. 9, A and B), and its interaction with ARF1 was determined in the GST fusion protein pull-down assay. The GST fusion protein encoding the C terminus of α2B-AR, but not GST alone, was able to interact with ARF1 (Fig. 9C).
To determine whether the interaction between the α2B-AR C terminus and ARF1 is direct or indirect, ARF1 was generated as His-tagged fusion proteins, and the purified His-ARF1 was also able to interact with GST-α2B-AR C terminus fusion protein (Fig. 9D). These data show that the small GTPase ARF1 directly interacts with the α2B-AR C terminus.
As a continuous effort to elucidate the molecular mechanism underlying the export trafficking of GPCRs, we determined the function of the small GTPase ARFs in regulating cell surface expression, subcellular distribution, and signaling of GPCRs. The results presented here have shown for the first time that, of the five ARFs identified in humans, ARF1 plays a crucial role in the anterograde transport of a group of the family A GPCRs. Transient expression of three different ARF1 mutants dramatically inhibited the cell surface transport of α2B-AR, β2-AR, AT1R, and CXCR4. Consistent with the remarked reduction in the cell surface expression, the receptors were expressed intracellularly, and receptor-mediated signaling measured as ERK1/2 activation was significantly attenuated in cells transfected with the ARF1 mutants. An important role for ARF1 in receptor export trafficking was further supported by a reduction of α2B-AR cell surface expression in cells expressing ARFGAP1, which facilitates the GTP hydrolysis of ARF1 (Lee et al., 2005). These data strongly indicate that the cell surface transport of α2B-AR, β2-AR, AT1R, and CXCR4 uses an ARF1-dependent pathway.
Interestingly, expression of the GDP-bound mutant ARF1T31N strongly arrested the receptors in the ER, whereas the GTP-bound mutant ARF1Q71L inhibited receptor transport from the ERGIC, the Golgi, and the TGN. These data are consistent with other studies showing that expression of different ARF1 mutants blocked protein transport at distinct intracellular compartments (Dascher and Balch, 1994; Ward et al., 2001). ARF1 GTPase has been shown to modulate the assembly and budding of the COPI-coated transport vesicles that are involved in anterograde transport from the ERGIC to the Golgi and retrograde transport from the Golgi to the ER (Stearns et al., 1990; Balch et al., 1992; D'Souza-Schorey and Chavrier, 2006). Expression of the GDP-bound mutant ARF1T31N would induce malfunction of the COPI-mediated retrograde transport system and disrupt the recycling of components of transport machinery required for anterograde transport from the ER. Therefore, expression of ARF1T31N results in the receptor accumulation in the ER. Similar to the expression of ARF1T31N, the treatment with BFA, which blocks the function of GEFs and arrests ARFs in the GDP-bound state, induced an ER accumulation of the receptors. On the contrary, expression of the GTP-bound ARF1Q71L mutant would disrupt the release of the COPI-coated vesicles from the ERGIC and the Golgi, leading to receptor accumulation in these compartments.
The molecular mechanism underlying the transport of GPCRs from the TGN to the plasma membrane remains poorly defined, and the vesicles that mediate the post-Golgi transport of GPCRs have not been identified. The fact that the expression of ARF1Q71L arrested receptors in the TGN suggests that, in addition to modulating receptor traffic at the ERGIC and the Golgi, ARF1 is also involved in the regulation of the post-Golgi transport of the receptors. It has been shown that ARF1 plays an important role in the formation of the TGN-derived clathrin-coated transport vesicles, which are mainly responsible for the transport of lysosomal enzymes between the TGN and the endosomal compartment (Puertollano et al., 2001; Misra et al., 2002; Bonifacino, 2004). Furthermore, a recent study has shown that ARF1 modulates the cytoplasmic protein complex termed exomer, which is important for the TGN-to-plasma membrane transport in yeast (Wang et al., 2006). In addition, ARF1 has been shown to regulate the Golgi-to-plasma membrane transport of internalized protease-activated receptor-2 (Luo et al., 2007). It is also interesting to note that ARF1 has been shown to directly interact with GPCRs (Mitchell et al., 1998), which may contribute to the mechanism of receptor transport regulation by ARF1. Our current study shows that ARF1 modulates multiple transport steps involved in the biosynthetic pathway, including export from the TGN, of a group of the family A GPCRs. We have also shown that ARF1 is able to physically associate with α2B-AR, and the interaction is mediated through the C terminus of the receptor. The interaction between α2B-AR and ARF1 appears to be direct as two purified fusion proteins containing the α2B-AR C terminus and ARF1 (i.e., GST-C terminus and His-ARF1) are able to interact, forming a complex. These data imply that the function of ARF1 in coordinating GPCR transport may be mediated through its direct interaction with the intracellular domains of the receptors.
The current studies, together with previous reports, have shown that the regulation of cell surface targeting of nascent GPCRs by ARF GTPases appears complicated. ARF1 is involved in the cell surface transport of all the GPCRs, including α2B-AR, β2-AR, AT1R, CXCR4, and M3-MR, as examined in this study. Inhibition of M3-MR cell surface expression by three ARF1 mutants is in contrast to a previous study showing that ARF1T31N did not alter the plasma membrane numbers of M3-MR (Mitchell et al., 2003). The differential effects of the ARF1 mutants on M3-MR cell surface transport could be a result of the different systems (e.g., different cell types) used in these two studies. Similar to ARF1, ARF3 also clearly modulates α2B-AR cell surface expression, as expression of each of the three ARF3 mutants significantly attenuated the α2B-AR numbers at the cell surface. Strong inhibition of α2B-AR transport by the ARF1 and ARF3 mutants supports the notion that ARF1 and ARF3 may have overlapping functions (Volpicelli-Daley et al., 2005). It has been shown that ARF4 modulates the post-Golgi traffic of rhodopsin, which is mediated via a direct interaction between ARF4 and the C terminus of the receptor (Deretic et al., 2005). However, expression of any of the three ARF4 mutants did not influence α2B-AR expression at the cell surface, whereas expression of ARF5N126I moderately inhibited. These data show that ARF5, but not ARF4, may play a role in the α2B-AR biosynthesis. These data also implicate that the class II ARF GTPases, ARF4 and ARF5, may selectively modulate the cell surface transport of distinct GPCRs. Furthermore, ARF6 has been showed to modulate the cell surface expression of vasopressin V2 receptor (Madziva and Birnbaumer, 2006). Moderate inhibition of α2B-AR transport by ARF6Q67L suggests that ARF6 may also regulate α2B-AR cell surface transport. However, the reason for differential effects of the GTP-bound, the GDP-bound, and the guanidine nucleotide-deficient mutants of ARF3, ARF5, and ARF6 on the α2B-AR transport remains unknown. Nevertheless, these studies show that different ARF GTPases may differentially modulate the transport of distinct GPCRs. As our previous studies have shown that Rab1 GTPase selectively modulates the cell surface transport of GPCRs (Wu et al., 2003; Filipeanu et al., 2006), it will be interesting to study the effect of ARF-Rab1 combination on the export trafficking of distinct GPCRs.
α2B-AR, β2-AR, AT1R, and CXCR4 couple to different heterotrimeric G proteins and regulate distinct signaling pathways. Expression of the ARF1T31N mutant significantly attenuated ERK1/2 activation by the receptor agonists, paralleling its inhibitory effects on the cell surface transport of the receptors. Therefore, the attenuation of receptor-mediated ERK1/2 activation is caused, at least in part, by less receptor transport to the cell surface in the ARF1T31N-transfected cells. However, we cannot exclude the possibility that ARF1T31N expression may also influence the intracellular trafficking and function of other signaling molecules that are involved in regulation of receptor-mediated ERK1/2 activation. It is also possible that, in addition to its well established function in regulating vesicle-mediated transport, ARF1 may function as a signaling entity directly involved in the activation of the ERK1/2 pathway. Consistent with this possibility, ARF1 has been shown to be involved in receptor-mediated phospholipase D activation (Brown et al., 1993; Cockcroft et al., 1994; Massenburg et al., 1994).
In contrast to extensive investigation on the endocytic process of GPCRs, the studies on the export trafficking of GPCRs from the ER through the Golgi to the cell surface have just begun (Dong et al., 2007; Achour et al., 2008; Ge et al., 2009). Recent studies on the functional roles of Ras-like small GTPases, particularly the Rab subfamily, in regulating GPCR expression at the cell surface and identification of highly conserved export motifs, which dictate receptor exit at distinct intracellular organelles, have greatly advanced our understanding of the cell surface targeting of newly synthesized GPCRs and have also shown that receptor export trafficking plays a crucial role in regulating receptor function and in the development of a number of diseases (Duvernay et al., 2004, 2009a; Dong et al., 2007). To further explore the regulatory mechanisms of the ER-to-cell surface transport of GPCRs may provide an important foundation for developing new therapeutic strategies in treating diseases involving abnormal trafficking and signaling of the receptors.
We thank Drs. Stephen M. Lanier, John D. Hildebrandt, Kenneth E. Bernstein, Jürgen Zezula, and Victor W. Hsu for sharing reagents.
- Received September 10, 2009.
- Accepted January 20, 2010.
C.D. and X.Z. contributed equally to this work.
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM076167].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- ADP-ribosylation factor
- guanine nucleotide exchange factor
- endoplasmic reticulum
- endoplasmic reticulum-Golgi intermediate complex
- trans-Golgi network
- GTPase-activating protein
- G protein-coupled receptor
- adrenergic receptor
- angiotensin II type 1 receptor
- chemokine (CXC motif) receptor 4
- extracellular signal-regulated kinase 1 and 2
- brefeldin A
- Ang II
- angiotensin II
- stromal cell-derived factor 1α
- RX 821002
- green fluorescent protein
- glutathione transferase
- human embryonic kidney
- Dulbecco's modified Eagle's medium
- fetal bovine serum
- M3-muscarinic receptor
- phosphate-buffered saline
- polyacrylamide gel electrophoresis
- ADP-ribosylation factor GTPase-activating protein 1.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics