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• W2036820171 abstract "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. 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. G protein-coupled receptors (GPCRs) 2The 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. 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 (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (472) Google Scholar, 2Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (369) Google Scholar). 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 (3Duvernay M.T. Filipeanu C.M. Wu G. Cell. Signal. 2005; 17: 1457-1465Crossref PubMed Scopus (128) Google Scholar) 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 (4von Zastrow M. Life Sci. 2003; 74: 217-224Crossref PubMed Scopus (153) Google Scholar, 6Tan C.M. Brady A.E. Nickols H.H. Wang Q. Limbird L.E. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 559-609Crossref PubMed Scopus (174) Google Scholar), 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 (3Duvernay M.T. Filipeanu C.M. Wu G. Cell. Signal. 2005; 17: 1457-1465Crossref PubMed Scopus (128) Google Scholar). 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 (3Duvernay M.T. Filipeanu C.M. Wu G. Cell. Signal. 2005; 17: 1457-1465Crossref PubMed Scopus (128) Google Scholar, 7Petaja-Repo U.E. Hogue M. Laperriere A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). GPCR export from the ER is modulated by direct interactions with multiple regulatory proteins such as the ER chaperones and receptor activity modifying proteins (8Tai A.W. Chuang J.Z. Bode C. Wolfrum U. Sung C.H. Cell. 1999; 97: 877-887Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 9McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1856) Google Scholar). 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 (10Jones 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. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (925) Google Scholar, 13Overton M.C. Chinault S.L. Blumer K.J. J. Biol. Chem. 2003; 278: 49369-49377Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). 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 (14Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 17Robert J. Clauser E. Petit P.X. Ventura M.A. J. Biol. Chem. 2005; 280: 2300-2308Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Protein transport from the Golgi may be mediated through constitutive or regulatory pathways (18Traub L.M. Kornfeld S. Curr. Opin. Cell Biol. 1997; 9: 527-533Crossref PubMed Scopus (194) Google Scholar). 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 (19Nishimura N. Plutner H. Hahn K. Balch W.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6755-6760Crossref PubMed Scopus (80) Google Scholar). The cytoplasmic N-terminal positively charged residues are necessary for the efficient export of inward rectifier potassium channels from the Golgi complex (20Stockklausner C. Klocker N. J. Biol. Chem. 2003; 278: 17000-17005Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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) (14Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Filipeanu C.M. Zhou F. Fugetta E.K. Wu G. Mol. Pharmacol. 2006; 69: 1571-1578Crossref PubMed Scopus (48) Google Scholar). 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 β2-AR. In contrast, the transport from the ER to the cell surface of α2B-AR is independent of Rab1 (21Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). 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 α2B-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 α2B-AR from the ER (14Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). 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 α2-ARs in the transport from the ER to the cell surface. We demonstrated that the single Met-6 residue modulates α2B-AR export from the ER, and the Tyr-Ser motif, which is highly conserved in the membrane-proximal N termini of all α2-AR subtypes, regulates exit of α2A-AR and α2B-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. Materials—Rat α2B-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 α2A-AR and α2B-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 (14Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Plasmid Constructions—α2B-AR tagged with GFP at its C terminus (α2B-AR-GFP) was generated as described previously (21Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). For generation of GFP-tagged α2B-AR-12 construct in which the N-terminal 12 amino acid residues (Ser-2—Ser-13) were deleted from α2B-AR, the full-length α2B-AR-GFP was amplified by PCR (forward primer, 5′-GATCAAGCTTATGGTGCAGGCCACCGCCGCCATCGCGTCG-3′; reverse primer, 5′-GATCGTCGACGCCCAGCCAGTCTGGGTC-3′) in which the truncated α2B-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 α2A-AR and the α2A-AR mutant lacking the N-terminal 28 residues (Gly-2—Ser-29). For generation of the C-terminal YFP-tagged α2B-AR, α2B-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 (21Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). 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 α2B-AR expression at the cell surface, HEK293T cells were transfected with 0.25 μg of GFP-tagged α2B-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 α2-AR Expression at the Cell Surface—Cell-surface α2-AR expression was measured by [3H]RX821002 binding to intact cells. HEK293T cells were cultured on 6-well plates and transfected with α2-AR constructs for 6 h. The cells were then split onto poly-l-lysine-coated 12-well plates at a density of 4 × 105 cells/well and grown for 24 h. To determine whether the reduced temperature could rescue the transport of mutated α2B-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 α2B-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 × 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 × 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 (21Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). 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 α2B-AR—HEK293T cells cultured on 100-mm dishes were transfected with 4 μg of HA-tagged α2B-AR together with 4 μg of the pEGFP-N1 vector or GFP-tagged wild-type or mutated α2B-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 × 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 × 500 μl of lysis buffer. Immunoprecipitated receptors were eluted with 100 μl of 1 × 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. A Requirement of the N terminus of α2B-AR for the Transport to the Cell Surface—We have recently identified the FX6LL motif in the membrane-proximal C termini of α2B-AR and AT1R, which is required for their export from the ER (14Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). To further define the structural determinants for export trafficking of GPCRs, we determined the role of the N terminus of α2B-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 α2B-AR to the cell surface. α2B-AR and its mutant lacking N-terminal 12 amino acid residues (Ser-2—Ser-13) (α2B-AR-12) were conjugated with the GFP at their C termini and transiently expressed in HEK293T cells. The cell-surface expression of α2B-AR-12 was markedly reduced by 95% as compared with wild-type (WT) α2B-AR as measured by intact cell ligand binding using [3H]RX821002 (Fig. 1A). Consistent with the lack of α2B-AR-12 cell-surface expression as measured by ligand binding, ERK1/2 activation by the agonist UK14304 was completely lost in cells transfected with α2B-AR-12 (Fig. 1B). In contrast, ERK1/2 activation was stimulated dose-dependently by UK14304 in cells transiently transfected with α2B-AR (Fig. 1B). Subcellular localization of the receptors indicated that α2B-AR-12 was trapped in the perinuclear region, whereas α2B-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 α2B-AR transport to the cell surface. Identification of Amino Acid Residues in the N Terminus Required for the Cell-surface Expression of α2B-AR—To identify amino acid residues important for α2B-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 α2B-AR. In contrast, single substitution of Met-6 with Ala abolished α2B-AR transport to the cell surface, and mutation of Tyr-12 and Ser-13 significantly attenuated the cell-surface expression of α2B-AR by 65 and 55%, respectively, as compared with WT α2B-AR (Fig. 2B). Simultaneous mutation of Tyr-12 and Ser-13 (Y12A/S13A) to Ala more profoundly inhibited the cell-surface expression of α2B-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 α2B-AR (Fig. 2D). The subcellular localization of each α2B-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 α2B-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 α2B-AR export to the cell surface. Residues Met-6, Tyr-12, and Ser-13 Modulate α2B-AR Export at Distinct Organelles—The preceding data have demonstrated that residues Met-6, Tyr-12, and Ser-13 in the α2B-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 α2B-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 α2B-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 α2B-AR, α2B-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 α2B-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 α2B-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 α2B-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 α2B-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 α2B-AR endocytotic trafficking, and Rab5 is involved in the transport from the plasma membrane to the endosome of many GPCRs (3Duvernay M.T. Filipeanu C.M. Wu G. Cell. Signal. 2005; 17: 1457-1465Crossref PubMed Scopus (128) Google Scholar, 25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 27DeGraff J.L. Gurevich V.V. Benovic J.L. J. Biol. Chem. 2002; 277: 43247-43252Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). 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 α2B-AR and its mutants Y12A, S13A, and Y12A/S13A. 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 α2B-AR and almost abolished the cell-surface expression of Y12A, S13A, and Y12A/S13A mutants (Fig. 5B). BFA treatment arrested α2B-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 α2B-AR Export—To characterize physiochemical properties required for the function of Met-6, Tyr-12, and Ser-13 in α2B-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 α2B-AR export. We next determined if the function of Tyr-12 and Ser-13 residues in α2B-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 α2B-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 α2B-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 α2B-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 α2B-AR transport as measured by intact cell" @default.
• W2036820171 created "2016-06-24" @default.
• W2036820171 creator A5008175920 @default.
• W2036820171 creator A5034206493 @default.
• W2036820171 date "2006-12-01" @default.
• W2036820171 modified "2023-10-12" @default.
• W2036820171 title "Regulation of Anterograde Transport of α2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments" @default.
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