Regulation of Anterograde Transport of α2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments*

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 α2-adrenergic receptors (α2-ARs) in the anterograde transport from the endoplasmic reticulum (ER) through the Golgi to the cell surface. The N-terminal-truncated α2B-AR mutant is completely unable to target to the cell surface. A single Met-6 residue is essential for the export of α2B-AR from the ER, likely through modulating correct α2B-AR folding in the ER. The Tyr-Ser motif, highly conserved in the membrane-proximal N termini of all α2-AR subtypes, is required for the exit of α2A-AR and α2B-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.

geted 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 cellsurface 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)(22)(23)(24). We have demonstrated that Rab1, a Ras-like small GTPase that coordi-nates protein transport specifically from the ER to the Golgi, selectively regulates the transport of AT1R and ␤ 2 -AR. In contrast, the transport from the ER to the cell surface of ␣ 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 ␣ 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 (FX 6 LL), which is required for the export of AT1R and ␣ 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 ␣ 2 -ARs in the transport from the ER to the cell surface. We demonstrated that the single Met-6 residue modulates ␣ 2B -AR export from the ER, and the Tyr-Ser motif, which is highly conserved in the membraneproximal N termini of all ␣ 2 -AR subtypes, regulates exit of ␣ 2A -AR and ␣ 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.
Plasmid Constructions-␣ 2B -AR tagged with GFP at its C terminus (␣ 2B -AR-GFP) was generated as described previously (21). For generation of GFP-tagged ␣ 2B -AR-12 construct in which the N-terminal 12 amino acid residues (Ser-2-Ser-13) were deleted from ␣ 2B -AR, the full-length ␣ 2B -AR-GFP was amplified by PCR (forward primer, 5Ј-GATCAAGCTT-ATGGTGCAGGCCACCGCCGCCATCGCGTCG-3Ј; reverse primer, 5Ј-GATCGTCGACGCCCAGCCAGTCTGGGTC-3Ј) in which the truncated ␣ 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 ␣ 2A -AR and the ␣ 2A -AR mutant lacking the N-terminal 28 residues (Gly-2-Ser-29). For generation of the C-terminal YFP-tagged ␣ 2B -AR, ␣ 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 g/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 g of GFP-tagged receptors. To determine the influence of the endocytotic pathway on ␣ 2B -AR expression at the cell surface, HEK293T cells were transfected with 0.25 g of GFP-tagged ␣ 2B -AR or its mutants together with 0.75 g 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 g of pDsRed2-ER and equal amounts of receptors. The transfection efficiency was estimated to be about 80% based on the GFP fluorescence.
Measurement of ␣ 2 -AR Expression at the Cell Surface-Cellsurface ␣ 2 -AR expression was measured by [ 3 H]RX821002 binding to intact cells. HEK293T cells were cultured on 6-well plates and transfected with ␣ 2 -AR constructs for 6 h. The cells were then split onto poly-L-lysine-coated 12-well plates at a density of 4 ϫ 10 5 cells/well and grown for 24 h. To determine whether the reduced temperature could rescue the transport of mutated ␣ 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 Me 2 SO on ␣ 2B -AR export, HEK293T cells were cultured at 37°C for 24 h and then incubated with Me 2 SO at a concentration of 2% for another 24 h. The cells were incubated with Dulbecco's modified Eagle's medium containing 20 nM [ 3 H]RX821002 for 90 min at room temperature with constant shaking (40 rpm). The nonspecific binding was determined in the presence of nonradioactive rauwolscine (10 M). The cells were washed twice with 1 ml of ice-cold phosphate-buffered saline (PBS), and the cell surface-bound [ 3 H]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 ϫ 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 CO 2 -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 M for 5 min. Stimulation was terminated by the addition of 1 ϫ SDS gel loading buffer. After solubilizing the cells, 20 l 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 ␣ 2B -AR-HEK293T cells cultured on 100-mm dishes were transfected with 4 g of HA-tagged ␣ 2B -AR together with 4 g of the pEGFP-N1 vector or GFPtagged wild-type or mutated ␣ 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 l 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 ϫ g, and the supernatant was incubated with 50 l of protein G-Sepharose for 1 h at 4°C to remove nonspecific bound proteins. Samples were then incubated with 5 g of anti-GFP antibodies overnight at 4°C with gentle rotation followed by an incubation with 50 l of protein G-Sepharose beads for 5 h. Resin was collected by centrifugation and washed 3 ϫ 500 l of lysis buffer. Immunoprecipitated receptors were eluted with 100 l of 1 ϫ 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 Ͻ 0.05 was considered as statistically significant. Data are expressed as the mean Ϯ S.E.

RESULTS
A Requirement of the N terminus of ␣ 2B -AR for the Transport to the Cell Surface-We have recently identified the FX 6 LL motif in the membrane-proximal C termini of ␣ 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 ␣ 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 ␣ 2B -AR to the cell surface. ␣ 2B -AR and its mutant lacking N-terminal 12 amino acid residues (Ser-2-Ser-13) (␣ 2B -AR-12) were conjugated with the GFP at their C termini and transiently expressed in HEK293T cells. The cell-surface expression of ␣ 2B -AR-12 was markedly reduced by 95% as compared with wild-type (WT) ␣ 2B -AR as measured by intact cell ligand binding using [ 3 H]RX821002 (Fig. 1A). Consistent with the lack of ␣ 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 ␣ 2B -AR-12 ( Fig.  1B). In contrast, ERK1/2 activation was stimulated dose-dependently by UK14304 in cells transiently transfected with ␣ 2B -AR (Fig. 1B). Subcellular localization of the receptors indicated that ␣ 2B -AR-12 was trapped in the perinuclear region, whereas ␣ 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 ␣ 2B -AR transport to the cell surface.
Identification of Amino Acid Residues in the N Terminus Required for the Cell-surface Expression of ␣ 2B -AR-To identify amino acid residues important for ␣ 2B -AR cell-surface expression, each amino acid residue (except Gly-3) in the N terminus ( 2 SGPTMDHQEPYS 13 ) ( 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 ␣ 2B -AR. In contrast, single substitution of Met-6 with Ala abolished ␣ 2B -AR transport to the cell surface, and mutation of Tyr-12 and Ser-13 significantly attenuated the cell-surface expression of ␣ 2B -AR by 65 and 55%, respectively, as compared with WT ␣ 2B -AR (Fig. 2B). Simultaneous mutation of Tyr-12 and Ser-13 (Y12A/S13A) to Ala more profoundly inhibited the cell-surface expression of ␣ 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 Export Trafficking of ␣ 2 -Adrenergic Receptor DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50 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 ␣ 2B -AR (Fig. 2D).
The subcellular localization of each ␣ 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 expres-sion 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 ␣ 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 ␣ 2B -AR export to the cell surface.
Residues Met-6, Tyr-12, and Ser-13 Modulate ␣ 2B -AR Export at Distinct Organelles-The preceding data have demonstrated that residues Met-6, Tyr-12, and Ser-13 in the ␣ 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 ␣ 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 ␣ 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 ␣ 2B -AR, ␣ 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 ␣ 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 ␣ 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 ␣ 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 ␣ 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 ␣ 2B -AR endocytotic trafficking, and Rab5 is involved in the transport from the plasma membrane to the endosome of many GPCRs (3,(25)(26)(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 ␣ 2B -AR and its mutants Y12A, S13A, and Y12A/S13A.  Site-directed mutagenesis identifies key residues at the N terminus required for the cell-surface expression of ␣ 2B -AR. A, the sequence of the ␣ 2B -AR N terminus, in which each amino acid residue (except Gly-3) was mutated to Ala individually or in combination. B, specific [ 3 H]RX821002 binding to intact HEK293T cells transfected with ␣ 2B -AR and its mutants. HEK293T cells were transiently transfected with ␣ 2B -AR and its mutants, and their expression at the cell surface was determined by intact cell ligand binding with [ 3 H]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 ␣ 2B -AR and are presented as the mean Ϯ S.E. of three separate experiments. *, p Ͻ 0.05 versus cells transfected with WT ␣ 2B -AR. C, Western blot analysis of ␣ 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 g of GFP-tagged ␣ 2B -AR or its mutants. The cells were then solubilized in 300 l of 1ϫ SDS gel loading buffer. Five l 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 ␤-actin antibodies (lower panel). D, ERK1/2 activation by UK14304 in HEK293T cells transiently transfected with ␣ 2B -AR or its mutants M6A, Y12A, S13A, and Y12A/S13A. HEK293T cells were transfected with ␣ 2B -AR (squares) and its mutants (triangles), and the cells were then treated with UK14304 (0.01-10 M) 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 ␣ 2B -AR and stimulated with 10 ⌴ 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 ␣ 2B -AR and its mutants. GFP-conjugated WT and mutated ␣ 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 m. DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50

JOURNAL OF BIOLOGICAL CHEMISTRY 38547
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 ␣ 2B -AR and almost abolished the cell-surface expression of Y12A, S13A, and Y12A/S13A mutants (Fig. 5B). BFA treatment arrested ␣ 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.
Hydrophobic Properties of Met-6 and Tyr-12 Are Important for Their Function in ␣ 2B -AR Export-To characterize physiochemical properties required for the function of Met-6, Tyr-12, and Ser-13 in ␣ 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 A, co-localization of ␣ 2B -AR, ␣ 2B -AR-12, and M6A with the ER marker pDsRed2-ER in fixed cells. HEK293T cells were transfected with GFP-tagged ␣ 2B -AR, ␣ 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 ␣ 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 ␣ 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, ␣ 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 m. 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 ␣ 2B -AR export.
We next determined if the function of Tyr-12 and Ser-13 residues in ␣ 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 ␣ 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 ␣ 2B -AR export.
Rescue of M6A and Y12A/S13A Transport by Low Temperature and Me 2 SO Treatment-To determine if Met-6 and the Tyr-Ser motif are involved in the proper ␣ 2B -AR folding, we determined the effect of low temperature culture and treatment with Me 2 SO, 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 ␣ 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. Me 2 SO treatment facilitated the transport to the cell surface of ␣ 2B -AR and M6A mutant as determined by intact cell ligand binding (Fig. 7B) and subcellular localization (Fig. 7C). Furthermore, the Me 2 SO-enhanced cell-surface expression was much greater for M6A mutant compared with WT ␣ 2B -AR (Fig. 7B). In contrast, Me 2 SO 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 Me 2 SO 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 ␣ 2B -AR folding in the ER.
Effect of Mutation of Met-6 and the Tyr-Ser Motif on Dimerization of ␣ 2B -AR-We previously demonstrated that ␣ 2B -AR constitutively forms dimers in the ER, and dimerization of ␣ 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 ␣ 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 ␣ 2B -AR when co-expressed in HEK293T cells. HA-␣ 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 ␣ 2B -AR and its mutants, HEK293T cells were separately transfected with ␣ 2B -AR or its mutants, mixed, and immunoprecipitated with anti-GFP antibodies. Anti-GFP antibodies did not immunoprecipitate HAtagged ␣ 2B -AR from the mixture (data not shown). These data indicate that ␣ 2B -AR forms heterodimers with M6A and Y12A/ S13A when co-expressed in same cell populations.  DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50

JOURNAL OF BIOLOGICAL CHEMISTRY 38549
The Tyr-Ser Motif Modulates ␣ 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 ␣ 2A -, ␣ 2B -, and ␣ 2C -AR subtypes in different species (Fig. 9A). To test if the Tyr-Ser motif is also important for the export of other ␣ 2 -ARs, we investigated the effect of mutating Tyr-28 and Ser-29 residues on the cell-surface expression and subcellular localization of ␣ 2A -AR, as the majority of ␣ 2C -AR is normally expressed intracellularly (28,29). Similar to ␣ 2B -AR, mutation of Tyr-28 and Ser-29 significantly inhibited ␣ 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 ␣ 2A -AR expression at the cell surface by 75%. Consistent with the attenuated cell-surface expression, the ␣ 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 ␣ 2A -AR transport from the Golgi to the cell surface. These results together with the data obtained from ␣ 2B -AR suggest that the conserved Tyr-Ser motif may function as a common signal specifically modulating Golgi export of the ␣ 2 -AR subfamily.

DISCUSSION
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 EX 3 LL motif in vasopressin V2 receptor, the FX 3 FX 3 F motif in dopamine D1 receptor, and the FNX 2 LLX 3 L motif in vasopressin V1b/V3 receptor (15)(16)(17). We have recently identified the FX 6 LL motif, which is necessary for export of ␣ 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 ␣ 1D -AR and cannabinoid receptor 1 (CB1), and deletion of the N terminus does not influence ␣ 1B -AR transport to the cell surface (30 -32). In the present study we determined the role of N termini in the export of ␣ 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 ␣ 2B -AR. First, the ␣ 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 cellsurface expression, ␣ 2B -AR-12 was unable to activate ERK1/2. Third, subcellular localization analysis revealed that ␣ 2B -AR-12 was extensively trapped in the ER. These results together with 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 ␣ 2B -AR. B, subcellular localization of ␣ 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 ␣ 2B -AR. D, subcellular localization of ␣ 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 cellsurface expression of ␣ 2B -AR. HEK293T cells were transfected with ␣ 2B -AR or its mutants, their expression at the cell surface was determined by intact cell ligand binding with [ 3 H]RX821002, and subcellular localization was revealed by fluorescence microscopy detecting GFP signal as described in the legend of Fig. 1 our previous data (14) strongly indicate that both extracellular and intracellular terminal tails of ␣ 2B -AR contain structural determinants for its targeting to the cell surface.
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 ␣ 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 ␣ 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  HEK293T cells were transfected with HA-tagged ␣ 2B -AR plus the pEGFP-N1 vector (control), GFP-tagged ␣ 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of ␣ 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA-␣ 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged ␣ 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. DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50 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 ␣ 2B -AR possesses multiple signals that are required for the export from the ER and the Golgi.

Export Trafficking of ␣ 2 -Adrenergic Receptor
There are at least two possibilities regarding the accumulation of ␣ 2B -AR in the Golgi induced by mutation of the Tyr-Ser motif. It is possible that the Tyr-Ser motif regulates ␣ 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 ␣ 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 ␣ 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 Me 2 SO. These data suggest that the function of Met-6 in modulating ␣ 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 ␣ 2B -AR constitutively forms dimers in the ER, and dimerization of ␣ 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 ␣ 2B -AR, indicating that Met-6 is not involved in the dimerization of ␣ 2B -AR in the ER. Furthermore, we demonstrated that the function of Met-6 in modulating ␣ 2B -AR export depends on its hydrophobic property. It is possible that the hydrophobic characteristic of Met-6 is important for proper ␣ 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 traf- ficking 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 ␣ 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 ␣ 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 ␣ 2A -, ␣ 2B -, and ␣ 2C -AR subtypes in different species. Mutation of Tyr-28 and/or Ser-29 residues attenuated ␣ 2A -AR transport to the cell surface and induced ␣ 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 ␣ 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)(38)(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⌽ motif (where X is any amino acid, and ⌽ has a bulky hydrophobic side chain) modulates protein transport from the TGN to the lysosome or basolateral membranes in polarized cells (42)(43)(44)(45). Therefore, the Tyr-Ser motif may represent a novel Tyrbased motif that is required for the transport of ␣ 2 -ARs from the Golgi to the cell surface.
The molecular mechanism underlying the function of the Tyr-Ser motif in regulating ␣ 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 ␣ 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 ␣ 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 ␣ 2 -AR exit from the Golgi. Therefore, the molecular mechanism underlying the function of the Tyr-Ser motif in mediating ␣ 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.