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Originally published In Press as doi:10.1074/jbc.M605734200 on October 12, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38543-38554, December 15, 2006
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Regulation of Anterograde Transport of {alpha}2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments*

Chunmin Dong and Guangyu Wu1

From the Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

Received for publication, June 15, 2006 , and in revised form, September 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The studies on the intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly focused on the C termini of the receptors. In this report we determined the role of the extracellular N termini of {alpha}2-adrenergic receptors ({alpha}2-ARs) in the anterograde transport from the endoplasmic reticulum (ER) through the Golgi to the cell surface. The N-terminal-truncated {alpha}2B-AR mutant is completely unable to target to the cell surface. A single Met-6 residue is essential for the export of {alpha}2B-AR from the ER, likely through modulating correct {alpha}2B-AR folding in the ER. The Tyr-Ser motif, highly conserved in the membrane-proximal N termini of all {alpha}2-AR subtypes, is required for the exit of {alpha}2A-AR and {alpha}2B-AR from the Golgi apparatus, thus representing a novel Tyr-based motif modulating GPCR transport at the Golgi level. These data provide the first evidence indicating an essential role of the N termini of GPCRs in the export from distinct intracellular compartments along the secretory pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs)2 constitute a superfamily of membrane proteins that respond to a vast array of sensory and chemical stimuli and regulate downstream effectors such as adenylyl cyclases, phospholipases, protein kinases, and ion channels through coupling to heterotrimeric G proteins (1, 2). GPCRs are synthesized in the ER. After being correctly folded in the ER, newly synthesized receptors are packaged into the ER-derived COPII transport vesicles and move to the ER-Golgi intermediate complex and the Golgi apparatus through which they are post-translationally modified. The receptors then move from the Golgi to the trans-Golgi network (TGN) to attain fully matured statuses (3) and are further targeted to their functional destination at the plasma membrane. GPCRs at the plasma membrane may undergo internalization upon stimulation with their agonists. The internalized receptors in the endosome may be sorted to target to the lysosome for degradation or to recycle back to the plasma membrane. Therefore, expression of an individual GPCR at the plasma membrane is determined by the overall balance of export from the ER to the cell surface, internalization, recycling, and degradation. However, compared with the extensive studies performed on the events of the endocytotic pathway (4-6), molecular mechanisms governing the export trafficking of GPCRs from the ER through the Golgi to the cell surface and their role in regulating receptor expression at the cell surface and function is relatively less well understood (3).

The progress achieved over the past few years indicates that export from the ER, the first step in intracellular trafficking of GPCRs, is a highly regulated process and influences the cell-surface expression level of GPCRs (3, 7). GPCR export from the ER is modulated by direct interactions with multiple regulatory proteins such as the ER chaperones and receptor activity modifying proteins (8, 9). GPCR dimerization also plays an important role in receptor folding and export from the ER. Several studies have indicated that some GPCR dimers are constitutively formed in the ER and that dimerization is required for their transport from the ER to the cell surface (10-13). Furthermore, the identification of conserved sequences in the membrane-proximal C termini essential for ER export indicates that GPCR export from the ER may be directed by specific motifs (14-17).

Protein transport from the Golgi may be mediated through constitutive or regulatory pathways (18). Recently, several studies have demonstrated that protein export from the Golgi is mediated through highly specified motifs. For example, vesicular stomatitis virus glycoprotein uses the Tyr-based di-acidic motif found in its cytoplasm tail to export from the TGN through recruiting adaptor protein complex 3 (19). The cytoplasmic N-terminal positively charged residues are necessary for the efficient export of inward rectifier potassium channels from the Golgi complex (20). However, the specific sequences for exit from the Golgi of the GPCR superfamily have not been identified.

As an initial approach to understanding the export pathways for different GPCRs, we focused on the adrenergic (AR) and angiotensin II type 1 receptor (AT1R) (14, 21-24). We have demonstrated that Rab1, a Ras-like small GTPase that coordinates protein transport specifically from the ER to the Golgi, selectively regulates the transport of AT1R and beta2-AR. In contrast, the transport from the ER to the cell surface of {alpha}2B-AR is independent of Rab1 (21). These data demonstrated that different GPCRs may use distinct pathways for their transport from the ER to the cell surface. Most importantly, the transport of {alpha}2B-AR from the ER to the cell surface is mediated through a non-conventional Rab1-independent pathway. We then identified a motif consisting of a Phe and double Leu spaced by six residues (FX6LL), which is required for the export of AT1R and {alpha}2B-AR from the ER (14). This motif is highly conserved in the membrane-proximal C termini of GPCRs and, therefore, may provide a common signal in mediating export of the receptors from the ER. To further define the intrinsic structural determinants for GPCR export trafficking, in this manuscript we determined the role of the N termini of {alpha}2-ARs in the transport from the ER to the cell surface. We demonstrated that the single Met-6 residue modulates {alpha}2B-AR export from the ER, and the Tyr-Ser motif, which is highly conserved in the membrane-proximal N termini of all {alpha}2-AR subtypes, regulates exit of {alpha}2A-AR and {alpha}2B-AR from the Golgi. These data provide strong evidence for the first time indicating that the N termini of GPCRs contain multiple signals modulating the export of the receptors from distinct intracellular compartments.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Rat {alpha}2B-AR in vector pcDNA3 was kindly provided by Dr. Stephen M. Lanier (Dept. of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center). Human {alpha}2A-AR and {alpha}2B-AR tagged with three HAs were purchased from UMR cDNA Resource Center (Rolla, MO). The dominant negative arrestin-3 mutant Arr3-(201-409) and the dominant negative dynamin mutant DynK44A were kindly provided by Dr. Jeffery L. Benovic (Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University). Antibodies against green fluorescent protein (GFP) and phospho-ERK1/2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ERK antibodies detecting total ERK1/2 expression were from Cell Signaling Technology, Inc. (Beverly, MA). Anti-HA monoclonal antibody 12CA5 was from Roche Applied Science. Antibodies against GM130 and p230 were from Transduction Laboratories (San Diego, CA). Brefeldin A (BFA), UK14304, rauwolscine, and dimethyl sulfoxide (Me2SO) were obtained from Sigma-Aldrich. Alexa Fluor 594-labeled secondary antibodies and 4,6-diamidino-2-phenylindole were from Molecular Probes, Inc. (Eugene, OR). The ER markers pDsRed2-ER and pECFP-ER were from BD Biosciences. Normal donkey serum was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). [3H]RX821002 (45.0 Ci/mmol) was purchased from PerkinElmer Life Sciences. Penicillin/streptomycin, L-glutamine, trypsin/EDTA, and Lipofectamine 2000 reagent were from Invitrogen. Polyvinylidene difluoride membranes were obtained from Gelman Sciences (Ann Arbor, MI). All other materials were obtained as described elsewhere (14, 21).

Plasmid Constructions—{alpha}2B-AR tagged with GFP at its C terminus ({alpha}2B-AR-GFP) was generated as described previously (21). For generation of GFP-tagged {alpha}2B-AR-12 construct in which the N-terminal 12 amino acid residues (Ser-2—Ser-13) were deleted from {alpha}2B-AR, the full-length {alpha}2B-AR-GFP was amplified by PCR (forward primer, 5'-GATCAAGCTTATGGTGCAGGCCACCGCCGCCATCGCGTCG-3'; reverse primer, 5'-GATCGTCGACGCCCAGCCAGTCTGGGTC-3') in which the truncated {alpha}2B-AR was in-frame with GFP, restricted with HindIII and SalI, and ligated into the pEGFP-N1 vector (Invitrogen). Similar strategies were used to generate C-terminal GFP-tagged {alpha}2A-AR and the {alpha}2A-AR mutant lacking the N-terminal 28 residues (Gly-2—Ser-29). For generation of the C-terminal YFP-tagged {alpha}2B-AR, {alpha}2B-AR was released from the pEGFP-N1 vector by digestion with HindIII and SalI and ligated into the pEYFP-N1 vectors (Invitrogen), which was cleaved with the same restriction enzymes. Receptor mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using GFP-tagged receptors as templates. The sequence of each construct used in this study was verified by restriction mapping and nucleotide sequence analysis (Louisiana State University Health Sciences Center DNA Sequence Core).

Cell Culture and Transient Transfection—HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transient transfection of the HEK293T cells was carried out using Lipofectamine 2000 reagent as described previously (21). For measurement of cell-surface receptor expression and ERK1/2 activation, HEK293T cells were cultured on 6-well plates and transfected with 0.5 µg of GFP-tagged receptors. To determine the influence of the endocytotic pathway on {alpha}2B-AR expression at the cell surface, HEK293T cells were transfected with 0.25 µg of GFP-tagged {alpha}2B-AR or its mutants together with 0.75 µg of dominant negative mutants Arr3-(201-409), DynK44A, or Rab5S34N. For co-localization with pDsRed2-ER, an ER marker, HEK293T cells were co-transfected with 0.5 µg of pDsRed2-ER and equal amounts of receptors. The transfection efficiency was estimated to be about 80% based on the GFP fluorescence.

Measurement of {alpha}2-AR Expression at the Cell Surface—Cell-surface {alpha}2-AR expression was measured by [3H]RX821002 binding to intact cells. HEK293T cells were cultured on 6-well plates and transfected with {alpha}2-AR constructs for 6 h. The cells were then split onto poly-L-lysine-coated 12-well plates at a density of 4 x 105 cells/well and grown for 24 h. To determine whether the reduced temperature could rescue the transport of mutated {alpha}2B-AR, HEK293T cells were grown at 30 °C for 40 h after being split onto 12-well plates. To determine the effect of the chemical chaperone Me2SO on {alpha}2B-AR export, HEK293T cells were cultured at 37 °C for 24 h and then incubated with Me2SO at a concentration of 2% for another 24 h. The cells were incubated with Dulbecco's modified Eagle's medium containing 20 nM [3H]RX821002 for 90 min at room temperature with constant shaking (40 rpm). The nonspecific binding was determined in the presence of nonradioactive rauwolscine (10 µM). The cells were washed twice with 1 ml of ice-cold phosphate-buffered saline (PBS), and the cell surface-bound [3H]RX821002 was extracted by 1 M NaOH treatment for 2 h at 37 °C. The radioactivity was counted by liquid scintillation spectrometry in 5 ml of Ecoscint A scintillation solution (National Diagnostics, Inc., Atlanta, GA).

Immunofluorescence Microscopy—HEK293T cells were grown on coverslips and fixed with a 4% paraformaldehyde, 4% sucrose mixture in PBS for 15 min. The cells were stained with 4,6-diamidino-2-phenylindole for 5 min, and the coverslips were mounted. For co-localization of the receptor with intracellular markers, HEK293T 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 GM130 or p230 (1:50) for 1 h. After washing with PBS (3 x 5 min), the cells were incubated with Alexa Fluor 594-labeled secondary antibody (1:2000 dilution) for 1 h at room temperature. The fluorescence was detected with a Leica DMRA2 epifluorescent microscope.

For co-localization of YFP-tagged receptors with the ER marker pECFP-ER in live cells, HEK293T cells were plated on poly-L-lysine-precoated 35-mm glass bottom dishes and transiently transfected with 100 ng of YFP-tagged receptor and 100 ng of the ER marker pECFP-ER with Lipofectamine 2000 reagent for 24 h. One hour before imaging, culture medium was replaced with CO2-independent medium (Invitrogen). Fluorescence was detected with a Zeiss Axiovert microscope (200M). Images were deconvolved using SlideBook software and the nearest neighbors deconvolution algorithm (Intelligent Imaging Innovations, Denver, CO).

Measurement of ERK1/2 Activation—HEK293T cells were cultured on 6-well plates and transfected as described above. At 36 h after transient transfection, HEK293T cells were starved for at least 3 h and then stimulated with UK14304 at concentrations from 0.01 to 10 µM for 5 min. Stimulation was terminated by the addition of 1 x SDS gel loading buffer. After solubilizing the cells, 20 µl of total cell lysates was separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The ERK1/2 activation was determined by measuring the levels of phosphorylation of ERK1/2 with phosphospecific ERK1/2 antibodies (21). The signal was detected using ECL Plus (PerkinElmer Life Sciences) and a Fuji Film luminescent image analyzer (LAS-1000 Plus) and quantitated using the Image Gauge program (Version 3.4). The membranes were stripped and reprobed with anti-ERK1/2 antibodies to confirm equal protein loading.

Immunoprecipitation of {alpha}2B-AR—HEK293T cells cultured on 100-mm dishes were transfected with 4 µg of HA-tagged {alpha}2B-AR together with 4 µg of the pEGFP-N1 vector or GFP-tagged wild-type or mutated {alpha}2B-AR in the pEGFP-N1 vector for 28 h. The cells were washed twice with PBS and harvested. The cells were then lysed with 500 µl of lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and Complete Mini protease inhibitor mixture. After gentle rotation for 1 h, samples were centrifuged for 15 min at 14,000 x g, 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 5 µg of anti-GFP antibodies overnight at 4 °C with gentle rotation followed by an incubation with 50 µl of protein G-Sepharose beads for 5 h. Resin was collected by centrifugation and washed 3 x 500 µl of lysis buffer. Immunoprecipitated receptors were eluted with 100 µl of 1 x SDS-PAGE loading buffer, separated by 8% SDS-PAGE, and visualized by immunoblotting using anti-HA antibodies.

Statistical Analysis—Differences were evaluated using Student's t test, and p < 0.05 was considered as statistically significant. Data are expressed as the mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Requirement of the N terminus of {alpha}2B-AR for the Transport to the Cell Surface—We have recently identified the FX6LL motif in the membrane-proximal C termini of {alpha}2B-AR and AT1R, which is required for their export from the ER (14). To further define the structural determinants for export trafficking of GPCRs, we determined the role of the N terminus of {alpha}2B-AR in its anterograde trafficking from the ER to the cell surface. We first determined the effect of deleting the entire N terminus on the transport of {alpha}2B-AR to the cell surface. {alpha}2B-AR and its mutant lacking N-terminal 12 amino acid residues (Ser-2—Ser-13) ({alpha}2B-AR-12) were conjugated with the GFP at their C termini and transiently expressed in HEK293T cells. The cell-surface expression of {alpha}2B-AR-12 was markedly reduced by 95% as compared with wild-type (WT) {alpha}2B-AR as measured by intact cell ligand binding using [3H]RX821002 (Fig. 1A). Consistent with the lack of {alpha}2B-AR-12 cell-surface expression as measured by ligand binding, ERK1/2 activation by the agonist UK14304 was completely lost in cells transfected with {alpha}2B-AR-12 (Fig. 1B). In contrast, ERK1/2 activation was stimulated dose-dependently by UK14304 in cells transiently transfected with {alpha}2B-AR (Fig. 1B). Subcellular localization of the receptors indicated that {alpha}2B-AR-12 was trapped in the perinuclear region, whereas {alpha}2B-AR was mainly localized at the cell surface (Fig. 1C), which was confirmed by co-localization with tetramethylrhodamine-conjugated concanavalin A, a plasma membrane marker (data not shown). These data indicate that the extracellular N-terminal portion is required for {alpha}2B-AR transport to the cell surface.

Identification of Amino Acid Residues in the N Terminus Required for the Cell-surface Expression of {alpha}2B-AR—To identify amino acid residues important for {alpha}2B-AR cell-surface expression, each amino acid residue (except Gly-3) in the N terminus (2SGPTMDHQEPYS13) (Fig. 2A) was substituted with Ala individually or in combination, and the cell-surface expression of each receptor mutant was determined by intact cell ligand binding. Mutation of Ser-2, Pro-4, Thr-5, Asp-7, His-8, Gln-9, Glu-10, and Pro-11 to Ala did not significantly influence the cell-surface expression of {alpha}2B-AR. In contrast, single substitution of Met-6 with Ala abolished {alpha}2B-AR transport to the cell surface, and mutation of Tyr-12 and Ser-13 significantly attenuated the cell-surface expression of {alpha}2B-AR by 65 and 55%, respectively, as compared with WT {alpha}2B-AR (Fig. 2B). Simultaneous mutation of Tyr-12 and Ser-13 (Y12A/S13A) to Ala more profoundly inhibited the cell-surface expression of {alpha}2B-AR than either mutant (Fig. 2B). Western blot analysis of total cell lysate using anti-GFP antibodies demonstrated that the reduction in the cell-surface expression of the mutant receptors was not due to the differences in total receptor expression as expression levels of these mutants were comparable with their WT (Fig. 2C). Consistent with the attenuated cell-surface expression, ERK1/2 activation in response to stimulation with UK14304 was also significantly inhibited in cells transfected with M6A, Y12A, S13A, or Y12A/S13A when compared with cells transfected with WT {alpha}2B-AR (Fig. 2D).


Figure 1
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  		FIGURE 1.
Effect of the deletion of extracellular N terminus on {alpha}2B-AR transport to the cell surface. A, quantitation of the cell-surface expression of {alpha}2B-AR and {alpha}2B-AR-12 lacking the N-terminal 12 residues (Ser-2—Ser-13). HEK293T cells cultured on 6-well plates were transfected with 0.5 µg of GFP-conjugated {alpha}2B-AR or {alpha}2B-AR-12 and then split onto 12-well plates. The cell-surface expression of the receptors were measured by intact cell binding assays using [3H]RX821002 at a concentration of 20 nM in duplicate. The non-specific binding was determined in the presence of nonradioactive rauwolscine (10 µM) as described under "Experimental Procedures." The mean values of specific [3H]RX821002 binding were 16981 ± 956 and 849 ± 339 cpm (n = 3) from cells transfected with {alpha}2B-AR and {alpha}2B-AR-12, respectively. The data shown are percentages of the mean value obtained from cells transfected with {alpha}2B-AR and are presented as the mean ± S.E. of three experiments. *, p < 0.05 versus cells transfected with {alpha}2B-AR. B, effect of the deletion of the N terminus of {alpha}2B-AR on ERK1/2 activation. HEK293T cells cultured on 6-well plates were transfected with {alpha}2B-AR (squares) or {alpha}2B-AR-12 (triangles). At 8-12 h after transfection, the cells were split and cultured for an additional 24 h. The cells were then stimulated with increasing concentrations of UK14304 (0.01-10 µM) for 5 min. ERK1/2 activation was determined by Western blot analysis using phosphospecific ERK1/2 antibodies. Left upper panel,a representative blot of ERK1/2 activation; left lower panel, total ERK1/2 expression; right panel, quantitative data expressed as percent of the ERK1/2 activation obtained in the cells transfected with {alpha}2B-AR and stimulated with 10 µM UK14304 (control). Similar results were obtained in at least three separate experiments. C, subcellular distribution of {alpha}2B-AR and {alpha}2B-AR-12. HEK293T cells cultured on coverslips were transfected with GFP-conjugated {alpha}2B-AR or {alpha}2B-AR-12, and subcellular distribution of the receptors was revealed by detecting GFP fluorescence as described under "Experimental Procedures." The data are representative images of at least five independent experiments. Scale bar, 10 µm.

 
The subcellular localization of each {alpha}2B-AR mutant was then visualized. Consistent with quantitative measurement of receptor expression at the cell surface by intact cell ligand binding, M6A mutant was completely unable to transport to the cell surface, and Y12A and S13A mutants were partially trapped inside the cells. In contrast, S2A, P4A, T5A, D7A, H8A, Q9A, E10A, and P11A mutants exhibited clear cell-surface expression patterns (Fig. 2E). Ala substitution of Val-14, Gln-15, and Thr-17 at the beginning of the first transmembrane domain also did not significantly influence the cell-surface expression and subcellular localization of {alpha}2B-AR (data not shown). These data strongly indicate that three residues, Met-6, Tyr-12, and Ser-13, at the N terminus are critical for {alpha}2B-AR export to the cell surface.

Residues Met-6, Tyr-12, and Ser-13 Modulate {alpha}2B-AR Export at Distinct Organelles—The preceding data have demonstrated that residues Met-6, Tyr-12, and Ser-13 in the {alpha}2B-AR N terminus are required for cell-surface targeting. Interestingly, the subcellular localization pattern of M6A mutant is apparently different from those of Y12A, S13A, and Y12A/S13A mutants. To define the intracellular compartments in which the {alpha}2B-AR mutants M6A, Y12A, S13A, and Y12A/S13A were retained, each mutant was co-localized with markers of the ER, the Golgi, and the TGN. M6A mutant as well as {alpha}2B-AR-12 was extensively co-localized with the ER marker pDsRed2-ER (Fig. 3A) but not with the Golgi marker GM130 and the TGN marker p230 (data not shown) in fixed cells. To eliminate the possible nonspecific influence of cell fixation on the subcellular localization of {alpha}2B-AR, {alpha}2B-AR and its mutant M6A were tagged with YFP at their C termini, and their subcellular co-localization with the ER marker pECFP-ER was visualized by microscopic analysis in live cells. Similar to the results obtained from the fixed cells, M6A mutant was strongly co-localized with pECFP-ER in live HEK293T cells (Fig. 3B). These data demonstrate that M6A mutant was unable to export from the ER and indicate that Met-6 modulates {alpha}2B-AR export at the level of the ER.

In contrast to M6A retained in the ER, Y12A, S13A, and Y12A/S13A, mutants were strongly co-localized with GM130 (Fig. 4A) but not with pDsRed2-ER (data not shown) and p230 (Fig. 4B). Similar results were obtained from COS-7 cells (data not shown), suggesting the role of the Tyr-12—Ser-13 motif in modulating {alpha}2B-AR export is not cell-type specific. These data demonstrate that these mutants were able to exit from the ER and transport to the Golgi, but their abilities to export from the Golgi to the TGN were impaired. These data indicate that the Tyr-12—Ser-13 motif modulates {alpha}2B-AR export at the level of the Golgi.

Effect of the Dominant Negative Mutants of Arrestin-3, Dynamin, and Rab5 and Treatment with BFA on {alpha}2B-AR Export—To eliminate the possibility that the accumulation of the Y12A, S13A, and Y12A/S13A mutants in the Golgi is caused by their constitutive internalization induced by the mutation, we determined the effect of transient expression of the dominant negative mutants Arr3-(201-409), DynK44A, and Rab5S34N on the cell-surface expression and subcellular localization of the mutated receptors. Arrestin-3 and dynamin modulate {alpha}2B-AR endocytotic trafficking, and Rab5 is involved in the transport from the plasma membrane to the endosome of many GPCRs (3, 25-27). Expression of Arr3-(201-409), DynK44A, and Rab5S34N did not have clear influence on the cell-surface expression (Fig. 5A) or subcellular localization (data not shown) of {alpha}2B-AR and its mutants Y12A, S13A, and Y12A/S13A.


Figure 2
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  		FIGURE 2.
Site-directed mutagenesis identifies key residues at the N terminus required for the cell-surface expression of {alpha}2B-AR. A, the sequence of the {alpha}2B-AR N terminus, in which each amino acid residue (except Gly-3) was mutated to Ala individually or in combination. B, specific [3H]RX821002 binding to intact HEK293T cells transfected with {alpha}2B-AR and its mutants. HEK293T cells were transiently transfected with {alpha}2B-AR and its mutants, and their expression at the cell surface was determined by intact cell ligand binding with [3H]RX821002 as described in the legend of Fig. 1. The data shown are the percentages of the mean value obtained from cells transfected with WT {alpha}2B-AR and are presented as the mean ± S.E. of three separate experiments. *, p < 0.05 versus cells transfected with WT {alpha}2B-AR. C, Western blot analysis of {alpha}2B-AR and its mutants M6A, Y12A, S13A, and Y12A/S13A (YS-AA). HEK293T cells were cultured on 6-well plates and transfected with 0.5 µg of GFP-tagged {alpha}2B-AR or its mutants. The cells were then solubilized in 300 µl of 1x SDS gel loading buffer. Five µl of total cell lysate were separated by 10% SDS-PGAE, receptor expression was visualized by immunoblotting using GFP antibodies (upper panel), and the blots were stripped and then probed with beta-actin antibodies (lower panel). D, ERK1/2 activation by UK14304 in HEK293T cells transiently transfected with {alpha}2B-AR or its mutants M6A, Y12A, S13A, and Y12A/S13A. HEK293T cells were transfected with {alpha}2B-AR (squares) and its mutants (triangles), and the cells were then treated with UK14304 (0.01-10 µM) for 5 min. P-, phosphorylated. Left panel, representative blots of ERK1/2 activation. Right panel, quantitative data expressed as percent of the ERK1/2 activation obtained in the cells transfected with {alpha}2B-AR and stimulated with 10 µM UK14304 (control). There is no significant difference in total ERK1/2 expression between samples (data not shown). Similar results were obtained in at least three separate experiments. E, subcellular localization of {alpha}2B-AR and its mutants. GFP-conjugated WT and mutated {alpha}2B-AR were transiently expressed in HEK293T cells, and their subcellular distribution was revealed by fluorescence microscopy detecting GFP as described under "Experimental Procedures." The data shown are representative images of three independent experiments. Scale bar, 10 µm.

 


Figure 3
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  		FIGURE 3.
Effect of mutation of Met-6 on the subcellular localization of {alpha}2B-AR. A, co-localization of {alpha}2B-AR, {alpha}2B-AR-12, and M6A with the ER marker pDsRed2-ER in fixed cells. HEK293T cells were transfected with GFP-tagged {alpha}2B-AR, {alpha}2B-AR-12, or M6A mutant together with pDsRed2-ER, and the subcellular distribution and co-localization of the receptors with pDsRed2-ER were revealed by fluorescence microscopy as described under "Experimental Procedures." B, co-localization of {alpha}2B-AR and M6A mutant with the ER marker pECFP-ER in live cells. HEK293T cells plated on poly-L-lysine-precoated 35-mm glass-bottom dishes were transfected with YFP-tagged {alpha}2B-AR or M6A together with pECFP-ER, and the subcellular localization and co-localization of the receptors with pECFP-ER were obtained in living cells with a Zeiss Axiovert microscope. Green, {alpha}2B-AR tagged with GFP (A) or YFP (B); red, the ER markers pDsRed2-ER (A) and pECFP-ER (B); yellow, co-localization of the receptors with the ER; blue, DNA staining by 4,6-diamidino-2-phenylindole (nuclei). The data shown in A and B are representative images of at least three independent experiments. Scale bars, 10 µm.

 
We then determined the effect of blocking anterograde protein transport by BFA treatment on the cell-surface expression and subcellular localization of the Tyr-Ser motif mutants. BFA is a fungal metabolite that disrupts the structures of the Golgi and blocks protein transport from the ER to the Golgi. Treatment with BFA dramatically inhibited the cell-surface expression of {alpha}2B-AR and almost abolished the cell-surface expression of Y12A, S13A, and Y12A/S13A mutants (Fig. 5B). BFA treatment arrested {alpha}2B-AR in the perinuclear regions of the transfected cells, presumably in the ER. Y12A, S13A, and Y12A/S13A mutants were redistributed from the Golgi to the ER in the presence of BFA (Fig. 5C). These data suggest that the Golgi accumulation of the Tyr-Ser motif mutants is likely caused by defective export from the Golgi rather than the constitutive endocytotic transport from the plasma membrane.


Figure 4
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  		FIGURE 4.
Effect of mutation of Tyr-12 and Ser-13 on the subcellular localization of {alpha}2B-AR. HEK293T cells were transfected with {alpha}2B-AR or its mutants Y12A, S13A, and Y12A/S13A and their co-localization with the Golgi (A) and the TGN (B) markers were revealed by fluorescence microscopy after staining with antibodies against GM130 (A) and p230 (B) (1:50 dilution), respectively, as described under "Experimental Procedures." Green, {alpha}2B-AR tagged with GFP; red, the Golgi marker GM130 (A) or the TGN marker p230 (B); yellow, co-localization of the receptors with the Golgi (A) and the TGN markers (B); blue, DNA staining by 4,6-diamidino-2-phenylindole (nuclei). The data shown in A and B are representative images of at least five independent experiments. Scale bars, 10 µm. YS-AA, Y12A/S13A.

 
Hydrophobic Properties of Met-6 and Tyr-12 Are Important for Their Function in {alpha}2B-AR Export—To characterize physiochemical properties required for the function of Met-6, Tyr-12, and Ser-13 in {alpha}2B-AR transport to the cell surface, we first determined if hydrophobicity of Met-6 and Tyr-12 played a role in their function by mutating them to hydrophobic residues (M6L and Y12F) and non-hydrophobic residue (M6Q and Y12Q). The cell-surface expression of M6L and Y12F mutants was markedly enhanced as compared with M6A and Y12A mutants, respectively. In contrast, the cell-surface expression of M6Q and Y12Q mutants was the same as their respective Ala mutants (Fig. 6, A and C). Consistently, subcellular localization analysis showed that M6L and Y12F mutants were able to transport to the cell surface, whereas M6Q and Y12Q mutants were trapped inside the cell (Fig. 6, B and D). These data indicate that the hydrophobic characteristics of the residues Met-6 and Tyr-12 are crucial for their function in {alpha}2B-AR export.


Figure 5
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  		FIGURE 5.
Effect of blocking endocytotic and anterograde transport on the transport of the Tyr-Ser motif mutants. A, specific [3H]RX821002 binding to intact HEK293T cells co-transfected with {alpha}2B-AR and internalization blockers. HEK293T cells were transfected with {alpha}2B-AR, Y12A, S13A, or Y12A/S13A (YS-AA) together with pcDNA3 vector (control) or the dominant negative mutants Arr3-(201-409), DynK44A, or Rab5S34N. Receptor expression at the cell surface was measured by intact cell ligand binding as described in the legend of Fig. 1. The data shown are percentages of the mean value obtained from cells transfected with WT {alpha}2B-AR and pcDNA3 and are presented as the mean ± S.E. of three separate experiments. Receptor cell-surface expression has no significant difference in cells transfected with Arr3-(201-409), DynK44A, or Rab5S34N as compared with their respective controls. B, specific [3H]RX821002 binding to intact HEK293T cells transfected with {alpha}2B-AR and treated with BFA. HEK293 cells were transfected with{alpha}2B-AR or its mutants and then incubated with ethanol (control) or BFA at a concentration of 5 µg/ml for 8 h. The data shown are percentages of the mean value obtained from cells transfected with WT{alpha}2B-AR and treated with ethanol and are presented as the mean ± S.E. of three separate experiments. *, p < 0.05 versus respective control. C, effect of BFA treatment on the subcellular distribution of {alpha}2B-AR and its mutants. HEK293T cells were transfected with GFP-conjugated {alpha}2B-AR or its mutants, and subcellular distribution of the receptors was revealed by detecting GFP fluorescence as described under "Experimental Procedures." The data are representative images of at least three independent experiments. Scale bar,10 µm.

 
We next determined if the function of Tyr-12 and Ser-13 residues in {alpha}2B-AR export was regulated by their potential phosphorylation. Similar to the mutation to Ala, substitution of Tyr-12 and Ser-13 with Asp, which will mimic the status of phosphorylation, significantly attenuated {alpha}2B-AR export to the cell surface (Fig. 6E). Mutation of Ser-13 to Thr also inhibited receptor expression at the cell surface (Fig. 6E). These data suggest that phosphorylation of Tyr-12 and Ser-13 residues is unlikely involved in regulating {alpha}2B-AR export.

Rescue of M6A and Y12A/S13A Transport by Low Temperature and Me2SO Treatment—To determine if Met-6 and the Tyr-Ser motif are involved in the proper {alpha}2B-AR folding, we determined the effect of low temperature culture and treatment with Me2SO, a chemical chaperone, on the cell-surface expression of the M6A and Y12A/S13A mutants. HEK293T cells cultured at a reduced temperature (30 °C) significantly enhanced the cell-surface expression of M6A mutant without influencing WT {alpha}2B-AR transport as measured by intact cell ligand binding (Fig. 7A) and subcellular distribution (Fig. 7C), suggesting that 30 °C is a permissive temperature for proper M6A folding to achieve a status competent for export from the ER. Similar to M6A, the transport of the Y12A/S13A mutant was also rescued by reducing culture temperature. However, the magnitude of rescue by low temperature was greater for M6A than Y12A/S13A.

Me2SO treatment facilitated the transport to the cell surface of {alpha}2B-AR and M6A mutant as determined by intact cell ligand binding (Fig. 7B) and subcellular localization (Fig. 7C). Furthermore, the Me2SO-enhanced cell-surface expression was much greater for M6A mutant compared with WT {alpha}2B-AR (Fig. 7B). In contrast, Me2SO treatment did not produce a significant effect on the transport of Y12A/S13A mutant (Fig. 7, B and C). These data indicate that both low temperature culture and the chemical chaperone Me2SO could rescue at least in part the cell-surface delivery of the ER-retained M6A mutant and suggest that Met-6 residue may be involved in the regulation of correct {alpha}2B-AR folding in the ER.

Effect of Mutation of Met-6 and the Tyr-Ser Motif on Dimerization of {alpha}2B-AR—We previously demonstrated that {alpha}2B-AR constitutively forms dimers in the ER, and dimerization of {alpha}2B-AR plays a crucial role in modulating its transport from the ER to the cell surface (23). To test if the influence of Met-6 and the Tyr-Ser motif on export trafficking of {alpha}2B-AR was caused by disrupting its dimerization in the ER, we determined whether GFP-tagged M6A or Y12A/S13A could form heterodimers with HA-tagged WT {alpha}2B-AR when co-expressed in HEK293T cells. HA-{alpha}2B-AR was found in the anti-GFP immunoprecipitate from cells transfected with either GFP-tagged M6A (Fig. 8A)or Y12A/S13A (Fig, 8B), but not from cells expressing GFP. To determine specific interaction of {alpha}2B-AR and its mutants, HEK293T cells were separately transfected with {alpha}2B-AR or its mutants, mixed, and immunoprecipitated with anti-GFP antibodies. Anti-GFP antibodies did not immunoprecipitate HA-tagged {alpha}2B-AR from the mixture (data not shown). These data indicate that {alpha}2B-AR forms heterodimers with M6A and Y12A/S13A when co-expressed in same cell populations.


Figure 6
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  		FIGURE 6.
Characterization of Met-6, Tyr-12, and Ser-13. A, effect of mutating Met-6 to hydrophobic Leu (M6L) and non-hydrophobic Gln (M6Q) residues on the cell-surface expression of {alpha}2B-AR. B, subcellular localization of {alpha}2B-AR and its mutants M6L and M6Q. C, effect of mutating Tyr-12 to Leu (Y12L) and Gln (Y12Q) on the cell-surface expression of {alpha}2B-AR. D, subcellular localization of {alpha}2B-AR and its mutants Y12L and Y12Q. E, effect of mutating Tyr-12 to Asp (Y12D) and Ser-13 to Asp (S13D) and Thr (S13T) on the cell-surface expression of {alpha}2B-AR. HEK293T cells were transfected with {alpha}2B-AR or its mutants, their expression at the cell surface was determined by intact cell ligand binding with [3H]RX821002, and subcellular localization was revealed by fluorescence microscopy detecting GFP signal as described in the legend of Fig. 1. The data shown in A, C, and E are percentages of the mean value obtained from cells transfected with WT {alpha}2B-AR and are presented as the mean ± S.E. of three separate experiments. *, p < 0.05 versus cells transfected with WT {alpha}2B-AR; **, p < 0.05 versus cells transfected with M6A (A) and Y12A (C). The data shown in B and D are representative images of three independent experiments. Scale bars, 10 µm.

 
The Tyr-Ser Motif Modulates {alpha}2A-AR Export from the Golgi—We then searched the GPCR data base to see if the Tyr-Ser motif is conserved in the superfamily of GPCRs. The Tyr-Ser motif is highly conserved in the membrane-proximal N termini of {alpha}2A-, {alpha}2B-, and {alpha}2C-AR subtypes in different species (Fig. 9A). To test if the Tyr-Ser motif is also important for the export of other {alpha}2-ARs, we investigated the effect of mutating Tyr-28 and Ser-29 residues on the cell-surface expression and subcellular localization of {alpha}2A-AR, as the majority of {alpha}2C-AR is normally expressed intracellularly (28, 29). Similar to {alpha}2B-AR, mutation of Tyr-28 and Ser-29 significantly inhibited {alpha}2A-AR transport to the cell surface by 57 and 46%, respectively (Fig. 9B). Simultaneous mutation of Tyr-28 and Ser-29 (Y28A/S29A) blocked {alpha}2A-AR expression at the cell surface by 75%. Consistent with the attenuated cell-surface expression, the {alpha}2A-AR mutants Y28A, S29A (data not shown) and Y28A/S29A were accumulated in the perinuclear regions of the transfected cells (Fig. 9C) and were extensively co-localized with the Golgi marker GM130 (data not shown). These data indicate that Tyr-28 and Ser-29 are important for {alpha}2A-AR transport from the Golgi to the cell surface. These results together with the data obtained from {alpha}2B-AR suggest that the conserved Tyr-Ser motif may function as a common signal specifically modulating Golgi export of the {alpha}2-AR subfamily.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The studies on the intrinsic structural determinants for GPCR export trafficking have been mainly focused on the C termini of the receptors. A number of sequences essential for exit from the ER have been identified in the membrane-proximal C termini of GPCRs including the EX3LL motif in vasopressin V2 receptor, the FX3FX3F motif in dopamine D1 receptor, and the FNX2LLX3L motif in vasopressin V1b/V3 receptor (15-17). We have recently identified the FX6LL motif, which is necessary for export of {alpha}2B-AR and AT1R from the ER (14). In contrast to the C termini, the roles of the N termini in regulating GPCR export trafficking have been much less investigated and remain controversial. For instance, whereas proteolytic cleavage of the N-terminal 64 amino acid residues reduces the expression of the endothelin B receptor at the cell surface, removal of the N termini facilitates the cell-surface transport of {alpha}1D-AR and cannabinoid receptor 1 (CB1), and deletion of the N terminus does not influence {alpha}1B-AR transport to the cell surface (30-32). In the present study we determined the role of N termini in the export of {alpha}2-ARs from the ER through the Golgi to the cell surface. Our data demonstrated that, similar to the intracellular C terminus, the extracellular N terminus is essential for cell-surface targeting of {alpha}2B-AR. First, the {alpha}2B-AR-12 mutant lacking the N-terminal 12 residues was unable to transport to the cell surface, as quantified by intact cell radioligand binding. Second, consistent with the lack of receptor cell-surface expression, {alpha}2B-AR-12 was unable to activate ERK1/2. Third, subcellular localization analysis revealed that {alpha}2B-AR-12 was extensively trapped in the ER. These results together with our previous data (14) strongly indicate that both extracellular and intracellular terminal tails of {alpha}2B-AR contain structural determinants for its targeting to the cell surface.


Figure 7
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  		FIGURE 7.
Effect of reduced temperature and Me2SO treatment on the cell-surface expression of M6A and Y12A/S13A mutants. A, specific [3H]RX821002 binding to intact HEK293T cells transfected with {alpha}2B-AR, M6A, or Y12A/S13A (YS-AA) and cultured at 37 and 30 °C for 40 h. Left panel, data shown are percentages of the mean value obtained from cells transfected with WT {alpha}2B-AR and cultured at 37 °C and are presented as the mean ± S.E. of three separate experiments. Right panel, -fold increase in receptor cell-surface expression at 30 °C over 37 °C. *, p < 0.05 versus cells cultured at 37 °C (left panel) and WT (right panel). B, specific [3H]RX821002 binding to intact HEK293T cells transfected with {alpha}2B-AR, M6A or Y12A/S13A and treated with Me2SO (DMSO). HEK293T cells transfected with {alpha}2B-AR, M6A, or Y12A/S13A were incubated with 2% Me2SO for 24 h. Left panel, the data shown are percentages of the mean value obtained from cells transfected with WT {alpha}2B-AR (control) and are presented as the mean ± S.E. of three separate experiments. Right panel, -fold increase in receptor cell-surface expression after Me2SO treatment. *, p < 0.05 versus control (left panel) and WT (right panel). Con, control. C, the effect of low temperature culture and Me2SO treatment on the subcellular localization of {alpha}2B-AR, M6A, and Y12A/S13A. The data shown are representative images of three independent experiments. Arrows indicate {alpha}2B-AR expressed at the plasma membrane. Scale bar, 10 µm.

 
The most important finding in this manuscript is the identification of three specific amino acid residues, Met-6, Tyr-12, and Ser-13, within the N terminus by an alanine-scanning mutagenesis approach, which are essential for proper {alpha}2B-AR export at discrete intracellular compartments. M6A mutant was completely unable to transport to the cell surface, whereas Y12A and S13A transport to the cell surface was markedly attenuated. Double mutation of Tyr-12 and Ser-13 (Y12A/S13A) more profoundly inhibited {alpha}2B-AR transport to the cell surface. Consistently, ERK1/2 activation by M6A, Y12A, S13A, and Y12A/S13A mutants was significantly blocked. More importantly, subcellular co-localization of the mutated receptors with intracellular organelle markers demonstrated that M6A, Y12A, S13A, and Y12A/S13A mutants were arrested in distinct subcellular compartments. M6A mutant was extensively co-localized with the ER marker pDsRed2-ER and pECFP-ER, indicating M6A mutant was unable to export from the ER. In contrast to M6A mutant, Y12A, S13A, and Y12A/S13A mutants were markedly co-localized with the Golgi marker GM130 but not the ER marker pDsRed2-ER and the TGN marker p230, indicating that they were able to exit from the ER and were further transported to the Golgi compartment but could not export from the Golgi to reach the TGN. These data indicate that the N terminus of {alpha}2B-AR possesses multiple signals that are required for the export from the ER and the Golgi.


Figure 8
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  		FIGURE 8.
Western blot (IB) analysis of heterodimerization of {alpha}2B-AR and M6A or Y12A/S13A mutants. A, dimerization of {alpha}2B-AR and M6A. HEK293T cells were transfected with HA-tagged {alpha}2B-AR plus the pEGFP-N1 vector (control), GFP-tagged {alpha}2B-AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of {alpha}2B-AR and Y12A/S13A. HEK293T cells were transfected with HA-{alpha}2B-AR together with the pEGFP-N1 vector (control), GFP-tagged {alpha}2B-AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immunoprecipitated with anti-GFP antibodies. The immunoprecipitate was separated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HA antibodies (upper panels). The blots were then striped and reprobed with anti-GFP antibodies (lower panels). The data shown in A and B are representative of three independent experiments.

 


Figure 9
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  		FIGURE 9.
Effect of mutation of the Tyr-Ser motif on the cell-surface expression and subcellular localization of {alpha}2A-AR. A, the conserved Tyr-Ser motif in the N termini of {alpha}2A-, {alpha}2B-, and {alpha}2C-AR subtypes. The entire N-terminal sequences of {alpha}2A-, {alpha}2B-, and {alpha}2C-ARs from human, rat, and mouse are shown. The Tyr-Ser motifs are underlined. B, specific [3H]RX821002 binding to intact HEK293T cells transfected with {alpha}2A-AR and its mutants Y28A, S29A, and Y28A/S29A (YS-AA) as described in the legend of Fig. 1. The mean values of specific [3H]RX821002 binding were 18873 ± 1432, 8115 ± 2264, 10568 ± 1321, and 4718 ± 755 cpm (n = 3) from cells transfected with {alpha}2A-AR, Y28A, S29A, and Y28A/S29A, respectively. The data shown are percentages of the mean value obtained from cells transfected with WT {alpha}2A-AR and are presented as the mean ± S.E. of three experiments. *, p < 0.05 versus cells transfected with {alpha}2A-AR. C, subcellular localization of {alpha}2A-AR and Y28A/S29A. GFP-conjugated WT and mutated {alpha}2A-AR were transiently expressed in HEK293T cells, and their subcellular distribution was revealed by fluorescence microscopy as described under "Experimental Procedures." The data shown are representative images of at lease three independent experiments. Scale bar, 10 µm.

 
There are at least two possibilities regarding the accumulation of {alpha}2B-AR in the Golgi induced by mutation of the Tyr-Ser motif. It is possible that the Tyr-Ser motif regulates {alpha}2B-AR export from the Golgi, and mutation of this motif results in defective export of the receptor from the Golgi. It is also possible that mutation of the Tyr-Ser motif does not alter {alpha}2B-AR export trafficking, but the mutated receptors are constitutively internalized from the plasma membrane to the intracellular compartments in the absence of agonists. To dissect these possibilities, we determined the effect of blocking endocytotic and anterograde transport pathways on the cell-surface expression of the Tyr-Ser motif mutants. Our data demonstrated that the internalization blockers Arr3-(201-409), DynK44A, and Rab5S34N did not influence the cell-surface expression and subcellular localization of the Tyr-Ser motif mutants. In contrast, inhibition of the anterograde ER-to-Golgi transport by BFA treatment abolished cell-surface expression of the motif mutants and arrested them in the ER. These data indicate that the accumulation of the Tyr-Ser motif mutants in the Golgi is due to defective export rather than constitutive internalization.

The absolute requirement of the single Met-6 residue for the exit of {alpha}2B-AR from the ER is quite surprising. The role of the Met residue in GPCR trafficking has been suggested in opioid receptors as mutation of a Met residue in the third intracellular loop altered internalization and lysosomal targeting of the receptors (33). We demonstrated that the cell-surface expression of M6A mutant was significantly restored by reduced temperature during culture and the chemical chaperone Me2SO. These data suggest that the function of Met-6 in modulating {alpha}2B-AR export from the ER is likely mediated through regulating proper receptor folding and that Met-6 is not involved in the insertion of the receptor into the ER membrane. We previously demonstrated that {alpha}2B-AR constitutively forms dimers in the ER, and dimerization of {alpha}2B-AR plays an important role in modulating its export from the ER. Our data showed that M6A was able to form dimers with WT {alpha}2B-AR, indicating that Met-6 is not involved in the dimerization of {alpha}2B-AR in the ER. Furthermore, we demonstrated that the function of Met-6 in modulating {alpha}2B-AR export depends on its hydrophobic property. It is possible that the hydrophobic characteristic of Met-6 is important for proper {alpha}2B-AR folding to achieve a conformation competent for passing the ER quality control mechanism.

Structural determinants for exit from the Golgi of the GPCR superfamily have not been identified. Olfactory and chemokine receptors have been reported to be released from the ER but accumulated in the Golgi (34, 35), suggesting that multiple trafficking steps are involved in the regulation of GPCR export. Our studies identified the first motif consisting of a Tyr and a Ser in the GPCR superfamily, which is crucial for export from the Golgi. Similar to Met-6, the function of Tyr-12 in {alpha}2-AR export is dependent on its hydrophobic property. However, the function of the Tyr-Ser motif is unlikely modulated by their phosphorylation, as mutation of Tyr-12 and Ser-13 to Asp and Ser-13 to Thr produced an inhibitory effect on the {alpha}2B-AR export similar to their mutation to Ala. By searching the GPCR data base, we found that the Tyr-Ser motif is highly conserved in the membrane-proximal N termini of {alpha}2A-, {alpha}2B-, and {alpha}2C-AR subtypes in different species. Mutation of Tyr-28 and/or Ser-29 residues attenuated {alpha}2A-AR transport to the cell surface and induced {alpha}2A-AR accumulation in the Golgi. These data suggest that the Tyr-Ser motif in the N-terminal portion may provide a common mechanism for the Golgi export of this subgroup of adrenergic receptors.

The Tyr-Ser motif identified in the membrane-proximal N termini of {alpha}2-ARs is apparently different from other Tyr-based signals, which have been demonstrated to modulate intracellular trafficking at distinct steps for a variety of proteins including GPCRs (36). For example, the NPXY motif functions as an internalization signal for non-GPCRs (37-39), and the NPXXY motif, which is highly conserved in many GPCRs within the putative seventh transmembrane domain near the cytoplasmic face of the plasma membrane, is involved in the endocytotic trafficking of the receptors (40, 41). In contrast, the YXX{Phi} motif (where X is any amino acid, and {Phi} has a bulky hydrophobic side chain) modulates protein transport from the TGN to the lysosome or basolateral membranes in polarized cells (42-45). Therefore, the Tyr-Ser motif may represent a novel Tyr-based motif that is required for the transport of {alpha}2-ARs from the Golgi to the cell surface.

The molecular mechanism underlying the function of the Tyr-Ser motif in regulating {alpha}2-AR trafficking remains largely unknown. Our data show that the Tyr-Ser motif mutants were exported from the ER and transported to the Golgi. These data suggest that these mutants were correctly inserted into the ER membrane for export and properly folded to pass through the ER quality control mechanism. In addition, mutation of the Tyr-Ser motif did not alter {alpha}2B-AR ability to dimerize in the ER. It has been well demonstrated that ER export motifs such as DXE and FF motifs are decoded by physical interaction with components of the ER-derived COPII-coated vesicles, which exclusively transport cargo proteins from the ER to the ER-Golgi intermediate complex (46-48). Interestingly, the function of the FF motif can be functionally substituted by other dihydrophobic motifs such as YY, LL, II, and VV. The DXE motif also interacts with adaptor protein 3 to coordinate vesicular stomatitis virus glycoprotein export from the TGN (24). The function of the C-terminal motifs in the ER export of GPCRs, as discussed above, may be mediated through direct interaction with the transport machinery or by regulating correct receptor folding in the ER. However, the Tyr-Ser motif within the N terminus of {alpha}2-AR are positioned toward the lumen of the ER or the Golgi during the export process and are unable to directly interact with components of the transport machinery or other proteins in the cytoplasm. It is unlikely that the Tyr-Ser motif would function as linear independent export motifs directing {alpha}2-AR exit from the Golgi. Therefore, the molecular mechanism underlying the function of the Tyr-Ser motif in mediating {alpha}2-AR export is different from those proposed for the export motifs identified in the C termini of receptors and other membrane proteins.

The physiological functions of GPCRs are dependent on their precise localization in the cell, and defective GPCR transport from the ER through the Golgi to the cell surface is associated with the pathogenesis of a variety of human diseases. These diseases may result from defects in ER export (resulting in ER retention) and/or in the machinery for transport after ER exit. For example, numerous naturally occurring mutations in the GPCRs themselves prevent proper folding and lead to ER retention. Such mutations have been implicated in the inherited diseases such as nephrogenic diabetes insipidus, retinitis pigmentosa, and male pseudohermaphroditism (49-51). Further characterization of the molecular mechanisms of the motif-facilitated traffic of GPCRs may be used as an important foundation for developing new therapeutic strategies in treating diseases by targeting GPCR export trafficking.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01GM076167 (to G. W.) and P20RR018766 (to G. W. and Stephen M. Lanier) and by the American Heart Association, Southeast affiliate (to C. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology and Experimental Therapeutics, LA State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-2236; Fax: 504-568-2361; E-mail: gwu{at}lsuhsc.edu.

2 The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; AT1R, angiotensin II type 1 receptor; ER, endoplasmic reticulum; TGN, trans-Golgi network; GFP, green fluorescent protein; YFP, yellow fluorescent protein; ERK1/2, extracellular signal-regulated kinase 1/2; WT, wild type; BFA, brefeldin A; PBS, phosphate-buffered saline; HA, hemagglutinin. Back


   ACKNOWLEDGMENTS
 
We are grateful to Stephen M. Lanier and Jeffery L. Benovic for sharing reagents. We also thank Fuguo Zhou and Erin Fugetta for superb technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Premont, R. T., Inglese, J., and Lefkowitz, R. J. (1995) FASEB J. 9, 175-182[Abstract]
  2. Wess, J. (1998) Pharmacol. Ther. 80, 231-264[CrossRef][Medline] [Order article via Infotrieve]
  3. Duvernay, M. T., Filipeanu, C. M., and Wu, G. (2005) Cell. Signal. 17, 1457-1465[CrossRef][Medline] [Order article via Infotrieve]
  4. von Zastrow, M. (2003) Life Sci. 74, 217-224[CrossRef][Medline] [Order article via Infotrieve]
  5. Marchese, A., Chen, C., Kim, Y. M., and Benovic, J. L. (2003) Trends Biochem. Sci. 28, 369-376[CrossRef][Medline] [Order article via Infotrieve]
  6. Tan, C. M., Brady, A. E., Nickols, H. H., Wang, Q., and Limbird, L. E. (2004) Annu. Rev. Pharmacol. Toxicol. 44, 559-609
  7. Petaja-Repo, U. E., Hogue, M., Laperriere, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727-13736[Abstract/Free Full Text]
  8. Tai, A.W., Chuang, J. Z., Bode, C., Wolfrum, U., and Sung, C. H. (1999) Cell 97, 877-887[CrossRef][Medline] [Order article via Infotrieve]
  9. McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333-339[CrossRef][Medline] [Order article via Infotrieve]
  10. Jones, K. A., Borowsky, B., Tamm, J. A., Craig, D. A., Durkin, M. M., Dai, M., Yao, W. J., Johnson, M., Gunwaldsen, C., Huang, L. Y., Tang, C., Shen, Q., Salon, J. A., Morse, K., Laz, T., Smith, K. E., Nagarathnam, D., Noble, S. A., Branchek, T. A., and Gerald, C. (1998) Nature 396, 674-679[CrossRef][Medline] [Order article via Infotrieve]
  11. Hague, C., Uberti, M. A., Chen, Z., Hall, R. A., and Minneman, K. P. (2004) J. Biol. Chem. 279, 15541-15549[Abstract/Free Full Text]
  12. Salahpour, A., Angers, S., Mercier, J. F., Lagace, M., Marullo, S., and Bouvier, M. (2004) J. Biol. Chem. 279, 33390-33397[Abstract/Free Full Text]
  13. Overton, M. C., Chinault, S. L., and Blumer, K. J. (2003) J. Biol. Chem. 278, 49369-49377[Abstract/Free Full Text]
  14. Duvernay, M. T., Zhou, F., and Wu, G. (2004) J. Biol. Chem. 279, 30741-30750[Abstract/Free Full Text]
  15. Schulein, R., Hermosilla, R., Oksche, A., Dehe, M., Wiesner, B., Krause, G., and Rosenthal, W. (1998) Mol. Pharmacol. 54, 525-535[Abstract/Free Full Text]
  16. Bermak, J. C., Li, M., Bullock, C., and Zhou, Q. Y. (2001) Nat. Cell Biol. 3, 492-498[CrossRef][Medline] [Order article via Infotrieve]
  17. Robert, J., Clauser, E., Petit, P. X., and Ventura, M. A. (2005) J. Biol. Chem. 280, 2300-2308[Abstract/Free Full Text]
  18. Traub, L. M., and Kornfeld, S. (1997) Curr. Opin. Cell Biol. 9, 527-533[CrossRef][Medline] [Order article via Infotrieve]
  19. Nishimura, N., Plutner, H., Hahn, K., and Balch, W. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6755-6760[Abstract/Free Full Text]
  20. Stockklausner, C., and Klocker, N. (2003) J. Biol. Chem. 278, 17000-17005[Abstract/Free Full Text]
  21. Wu, G., Zhao, G., and He, Y. (2003) J. Biol. Chem. 278, 47062-47069[Abstract/Free Full Text]
  22. Filipeanu, C. M., Zhou, F., Claycomb, W. C., and Wu, G. (2004) J. Biol. Chem. 279, 41077-41084[Abstract/Free Full Text]
  23. Zhou, F., Filipeanu, C. M., Duvernay, M. T., and Wu, G. (2006) Cell. Signal. 18, 318-327[CrossRef][Medline] [Order article via Infotrieve]
  24. Filipeanu, C. M., Zhou, F., Fugetta, E. K., and Wu, G. (2006) Mol. Pharmacol. 69, 1571-1578[Abstract/Free Full Text]
  25. DeGraff, J. L., Gagnon, A. W., Benovic, J. L., and Orsini, M. J. (1999) J. Biol. Chem. 274, 11253-11259[Abstract/Free Full Text]
  26. Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842[Abstract/Free Full Text]
  27. DeGraff, J. L., Gurevich, V. V., and Benovic, J. L. (2002) J. Biol. Chem. 277, 43247-43252[Abstract/Free Full Text]
  28. Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017-5024[Abstract/Free Full Text]
  29. von Zastrow, M., Link, R., Daunt, D., Barsh, G., and Kobilka, B. (1999) J. Biol. Chem. 268, 763-766
  30. Grantcharova, E., Furkert, J., Reusch, H. P., Krell, H. W., Papsdorf, G., Beyermann, M., Schulein, R., Rosenthal, W., and Oksche, A. (2002) J. Biol. Chem. 277, 43933-43941[Abstract/Free Full Text]
  31. Andersson, H., D'Antona, A. M., Kendall, D. A., von Heijne, G., and Chin, C. N. (2003) Mol. Pharmacol. 64, 570-577[Abstract/Free Full Text]
  32. Hague, C., Chen, Z., Pupo, A. S., Schulte, N. A., Toews, M. L., and Minneman, K. P. (2004) J. Pharmacol. Exp. Ther. 309, 388-397[Abstract/Free Full Text]
  33. Wang, W., Loh, H. H., and Law, P. Y. (2003) J. Biol. Chem. 278, 36848-58368[Abstract/Free Full Text]
  34. Gimelbrant, A. A., Haley, S. L., and McClintock, T. S. (2001) J. Biol. Chem. 276, 7285-7290[Abstract/Free Full Text]
  35. Venkatesan, S., Petrovic, A., Van Ryk, D. I., Locati, M., Weissman, D., and Murphy, P. M. (2002) J. Biol. Chem. 277, 2287-2301[Abstract/Free Full Text]
  36. Bonifacino, J. S., and Traub, L. M. (2003) Annu. Rev. Biochem. 72, 395-447
  37. Chen, W. J., Goldstein, J. L., and Brown, M. S. (1990) J. Biol. Chem. 265, 3116-3123[Abstract/Free Full Text]
  38. Collawn, J. F., Kuhn, L. A., Liu, L. F., Tainer, J. A., and Trowbridge, I. S. (1991) EMBO J. 10, 3247-3253[Medline] [Order article via Infotrieve]
  39. Bouley, R., Sun, T. X., Chenard, M., McLaughlin, M., McKee, M., Lin, H. Y., Brown, D., and Ausiello, D. (2003) Am. J. Physiol. Cell Physiol. 285, 750-762
  40. Kalatskaya, I., Schussler, S., Blaukat, A., Muller-Esterl, W., Jochum, M., Proud, D., and Faussner, A. (2004) J. Biol. Chem. 279, 31268-31276[Abstract/Free Full Text]
  41. Lee, T. H., and Linstedt, A. D. (2000) Mol. Biol. Cell 11, 2577-2590[Abstract/Free Full Text]
  42. Williams, M. A., and Fukuda, M. (1990) J. Cell Biol. 111, 955-966[Abstract/Free Full Text]
  43. Harter, C., and Mellman, I. (1992) J. Cell Biol. 117, 311-325[Abstract/Free Full Text]
  44. Hunziker, W., Harter, C., Matter, K., and Mellman, I. (1991) Cell 66, 907-920[CrossRef][Medline] [Order article via Infotrieve]
  45. Rajasekaran, A. K., Humphrey, J. S., Wagner, M., Miesenbock, G., Le Bivic, A., Bonifacino, J. S., and Rodriguez-Boulan, E. (1994) Mol. Biol. Cell 5, 1093-1103[Abstract]
  46. Nishimura, N., and Balch, W. E. (1997) Science 277, 556-558[Abstract/Free Full Text]
  47. Fiedler, K., Veit, M., Stamnes, M. A., and Rothman, J. E. (1996) Science 273, 1396-1399[Abstract]
  48. Nufer, O., Guldbrandsen, S., Degen, M., Kappeler, F., Paccaud, J. P., Tani, K., and Hauri, H. P. (2002) J. Cell Sci. 115, 619-628[Abstract/Free Full Text]
  49. Stojanovic, A., and Hwa, J. (2002) Receptors Channels 8, 33-50[CrossRef][Medline] [Order article via Infotrieve]
  50. Morello, J. P., and Bichet, D. G. (2001) Annu. Rev. Physiol. 63, 607-630[CrossRef][Medline] [Order article via Infotrieve]
  51. Themmen, A. P., and Brunner, H. G. (1996) Eur. J. Endocrinol. 134, 533-540[Abstract/Free Full Text]

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