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• W2019247237 abstract "It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α2B-adrenergic receptor (α2B-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α2B-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α2B-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α2B-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α2B-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules. It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α2B-adrenergic receptor (α2B-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α2B-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α2B-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α2B-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α2B-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules. The precise function of G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; AT1R, angiotensin II type 1 receptor; CT, C terminus; ER, endoplasmic reticulum. relies on highly regulated intracellular trafficking processes, including export of nascent receptors from the endoplasmic reticulum (ER) to the cell surface, agonist-evoked internalization of the receptors from the cell surface to endosomes, recycling of the internalized receptors from the endosomes back to the cell surface, and targeting to the lysosome for degradation, which dictate proper expression and correct targeting to the functional destination of the receptors. Over the past decades, substantial studies have been focused on the events of endocytosis, recycling, and degradation of GPCRs (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2097) Google Scholar, 2von Zastrow M. Life Sci. 2003; 74: 217-224Crossref PubMed Scopus (153) Google Scholar, 3Tan 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, 4Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 5Wu G. Bogatkevich G.S. Mukhin Y.V. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 2000; 275: 9026-9034Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In contrast, the molecular mechanisms underlying the export trafficking of newly synthesized GPCRs from the ER to the cell surface remain largely unknown. It has been well documented that GPCR export to the cell surface involve direct interactions with multiple regulatory proteins such as ER chaperones, accessory proteins, and receptor activity modifying proteins, which may stabilize receptor conformation, facilitate receptor maturation, and promote receptor delivery to the plasma membrane (6Dwyer N.D. Troemel E.R. Sengupta P. Bargmann C.I. Cell. 1998; 93: 455-466Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 7Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 8Ferreira P.A. Nakayama T.A. Pak W.L. Travis G.H. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) 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 (1853) Google Scholar, 10Achour L. Scott M.G. Shirvani H. Thuret A. Bismuth G. Labbé-Jullié C. Marullo S. Blood. 2009; 113: 1938-1947Crossref PubMed Scopus (44) Google Scholar, 11Chen Y. Chen C. Kotsikorou E. Lynch D.L. Reggio P.H. Liu-Chen L.Y. J. Biol. Chem. 2009; 284: 1673-1685Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In addition, GPCR dimerization may regulate proper receptor folding and assembly, which will influence the receptors ability to pass through the ER quality control mechanism (12Salahpour A. Angers S. Mercier J.F. Lagacé M. Marullo S. Bouvier M. J. Biol. Chem. 2004; 279: 33390-33397Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 13Zhou F. Filipeanu C.M. Duvernay M.T. Wu G. Cell Signal. 2006; 18: 318-327Crossref PubMed Scopus (48) Google Scholar). As an initial approach to study GPCR cell surface transport, we have determined the role of the Ras-like small GTPases in the ER-to-cell surface movement of nascent GPCRs. Our studies have shown that the Rab and Sar1/ARF subfamilies play an important role in regulation of GPCR transport from the ER to the cell surface along the secretory pathway. More importantly, the small GTPases Rab1, Rab6, Rab8, and Sar1 are able to selectively or differentially modulate the cell surface transport of GPCRs, suggesting that distinct GPCRs with similar structural features may use different pathways to move to the cell surface en route from the ER and the Golgi (14Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15Zhang X. Wang G. Dupré D.J. Feng Y. Robitaille M. Lazartigues E. Feng Y.H. Hébert T.E. Wu G. J. Pharmacol. Exp. Ther. 2009; 330: 109-117Crossref PubMed Scopus (31) Google Scholar, 16Filipeanu C.M. Zhou F. Fugetta E.K. Wu G. Mol. Pharmacol. 2006; 69: 1571-1578Crossref PubMed Scopus (48) Google Scholar, 17Filipeanu C.M. Zhou F. Claycomb W.C. Wu G. J. Biol. Chem. 2004; 279: 41077-41084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 18Dong C. Zhang X. Zhou F. Dou H. Duvernay M.T. Zhang P. Wu G. J. Pharmacol. Exp. Ther. 2010; 333: 174-183Crossref PubMed Scopus (36) Google Scholar, 19Dong C. Yang L. Zhang X. Gu H. Lam M.L. Claycomb W.C. Xia H. Wu G. J. Biol. Chem. 2010; 285: 20369-20380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Dong C. Wu G. Cell Signal. 2007; 19: 2388-2399Crossref PubMed Scopus (49) Google Scholar, 21Dong C. Wu G. J. Biol. Chem. 2006; 281: 38543-38554Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). More recently, we have demonstrated that Rab8 and ARF1 directly interact with the receptors to modulate receptor cell surface transport (18Dong C. Zhang X. Zhou F. Dou H. Duvernay M.T. Zhang P. Wu G. J. Pharmacol. Exp. Ther. 2010; 333: 174-183Crossref PubMed Scopus (36) Google Scholar, 19Dong C. Yang L. Zhang X. Gu H. Lam M.L. Claycomb W.C. Xia H. Wu G. J. Biol. Chem. 2010; 285: 20369-20380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). An essential role for the C terminus (CT) in the ER-to-cell surface transport has been described for a number of GPCRs and indeed, several highly conserved motifs, which control receptor export trafficking, have been identified in the CT (22Tetsuka M. Saito Y. Imai K. Doi H. Maruyama K. Endocrinology. 2004; 145: 3712-3723Crossref PubMed Scopus (70) Google Scholar, 23Robert 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, 24Schülein R. Hermosilla R. Oksche A. Dehe M. Wiesner B. Krause G. Rosenthal W. Mol. Pharmacol. 1998; 54: 525-535Crossref PubMed Scopus (133) Google Scholar, 25Bermak J.C. Li M. Bullock C. Zhou Q.Y. Nat. Cell Biol. 2001; 3: 492-498Crossref PubMed Scopus (232) Google Scholar, 26Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 27Tai 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, 28Gáborik Z. Mihalik B. Jayadev S. Jagadeesh G. Catt K.J. Hunyady L. FEBS Lett. 1998; 428: 147-151Crossref PubMed Scopus (31) Google Scholar, 29Venkatesan S. Petrovic A. Locati M. Kim Y.O. Weissman D. Murphy P.M. J. Biol. Chem. 2001; 276: 40133-40145Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However the molecular mechanisms remain poorly defined. To address this issue, we sought to identify proteins interacting with the α2B-AR CT. We report here that the α2B-AR CT directly interacts with α- and β-tubulin. More importantly, the basic Arg residues in the CT not only mediate α2B-AR interaction with tubulin but also are required for receptor export from the ER to the cell surface. These data provide the first evidence implicating that the cargo GPCRs may directly contact with microtubules to coordinate their cell surface transport. Rat α2B-AR in vector pcDNA3 was kindly provided by Dr. Stephen M. Lanier (Medical University of South Carolina). The dominant negative arrestin-3 mutant Arr3-(201–409) and the dominant negative dynamin mutant DynK44A were provided by Dr. Jeffrey L. Benovic (Thomas Jefferson University). Antibodies against phospho-ERK1/2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-ERK1/2 antibody detecting total ERK1/2 expression was from Cell Signaling Technology, Inc. (Beverly, MA). The β-tubulin antibody was purchased from Abcam (Cambridge, MA). The α-tubulin antibody (DM1A), UK14304, and rauwolscine were obtained from Sigma-Aldrich. 4% crosslinked agarose beads were purchased from Agarose Bead Technologies (Tampa, FL). The ER marker pDsRed2-ER was from BD Biosciences (Palo Alto, CA). The α2B-AR C-terminal peptide NH2-NQDFRRAFRRILCRPWTQTGW-COOH, C-terminal peptide mutant (in which three Arg at positions 437, 441, and 446 were mutated to Glu) NH2-NQDFERAFERILCEPWTQTGW-COOH, and third intracellular loop (ICL3) peptide NH2-GKNVGVASGQWWRRRTQLSRE-OH were synthesized, purified by HPLC to >75% and directly conjugated to agarose beads by Biosynthesis Inc. (Lewisville, TX). Purified bovine tubulin was purchased from Cytoskeleton Inc. (Denver, CO). [3H]RX821002 (specific activity = 41 Ci/mmol) was purchased from PerkinElmer (Waltham, MA). Penicillin-streptomycin, l-glutamine, and trypsin-EDTA were from Invitrogen (Rockville, MD). All other materials were obtained as described elsewhere (30Duvernay M.T. Dong C. Zhang X. Robitaille M. Hébert T.E. Wu G. Traffic. 2009; 10: 552-566Crossref PubMed Scopus (50) Google Scholar, 31Duvernay M.T. Dong C. Zhang X. Zhou F. Nichols C.D. Wu G. Mol. Pharmacol. 2009; 75: 751-761Crossref PubMed Scopus (60) Google Scholar, 32Dong C. Zhou F. Fugetta E.K. Filipeanu C.M. Wu G. Cell Signal. 2008; 20: 1035-1043Crossref PubMed Scopus (35) Google Scholar). α2B-AR tagged with green fluorescent protein (GFP) at its CT (α2B-AR-GFP) was generated as described previously (14Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Glutathione S-transferase (GST) fusion protein constructs coding the α2B-AR CT were generated in the pGEX-4T-1 vector as described previously (19Dong C. Yang L. Zhang X. Gu H. Lam M.L. Claycomb W.C. Xia H. Wu G. J. Biol. Chem. 2010; 285: 20369-20380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). All mutants were made with the QuickChange site-directed mutagenesis kit. The sequence of each construct was confirmed by restriction mapping and nucleotide sequence analysis. HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. Transient transfection of the cells was carried out using Lipofectamine 2000 reagent (Invitrogen) as described previously (14Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 30Duvernay M.T. Dong C. Zhang X. Robitaille M. Hébert T.E. Wu G. Traffic. 2009; 10: 552-566Crossref PubMed Scopus (50) Google Scholar). For intact cell ligand binding and ERK1/2 activation, HEK293 cells were cultured in 6-well dishes and transfected with 1.0 μg of plasmid. For fluorescence microscopy, HEK293 cells were cultured in 6-well dishes transfected with 0.5 μg of plasmid. Transfection efficiency was estimated to be greater than 80% based on the GFP fluorescence. Rat brains were homogenized in buffer containing 50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 5 mm EGTA, 9 mm KCl, 2.5 mm MgCl2, 1% Triton at a ratio of 3 ml of buffer to 1 g of rat brain tissue. After homogenization, lysates were centrifuged at 100,000 × g for 1 h at 4 °C, and the supernatant was then collected. The total cytosolic extracts were pre-cleared three times with 2 ml of blank agarose beads for 2 h at 4 °C. 4 ml of pre-cleared rat brain cytosolic extract (∼20 mg) were then incubated with l ml of the α2B-AR CT-conjugated agarose beads with gentle rotation overnight at 4 °C. The resin was washed three times with 4 ml of homogenization buffer at 4 °C, and bound proteins were then eluted with 1 ml of denaturing buffer (7 m urea, 2 m thiourea, 4% Chaps, 30 mm Tris-HCl pH 8.5, 20% glycerol). The eluted proteins were separated by 2-dimensional gels and stained with Sypro Ruby. Images were then captured using a Typhoon 9400 Variable Mode Imager. Proteins of interest were picked using the Ettan Spot Handling Work station (GE Healthcare), digested with trypsin and identified by LTQ electrospray mass spectrometry. The identity of the sequences was then revealed by matching the spectrum against a database of previously generated spectra with MASCOT as described (33Simon V. Guidry J. Gettys T.W. Tobin A.B. Lanier S.M. J. Biol. Chem. 2006; 281: 40310-40320Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The GST-fusion proteins were expressed in bacteria and purified using a glutathione-affinity matrix as described previously (18Dong C. Zhang X. Zhou F. Dou H. Duvernay M.T. Zhang P. Wu G. J. Pharmacol. Exp. Ther. 2010; 333: 174-183Crossref PubMed Scopus (36) Google Scholar, 19Dong C. Yang L. Zhang X. Gu H. Lam M.L. Claycomb W.C. Xia H. Wu G. J. Biol. Chem. 2010; 285: 20369-20380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Immobilized fusion proteins were either used immediately or stored at 4 °C for no longer than 3 days. Each batch of fusion protein used in experiments was first analyzed by Coomassie Blue staining following SDS-PAGE. Tubulin purified from bovine brain was reconstituted in general tubulin buffer (G-PEM: 80 mm PIPES, pH 6.9, 2 mm MgCl2, 0.5 mm EGTA, 50 mm GTP). Tubulin lacking the acidic CT (tubulin S) was prepared by limited proteolysis of rat tubulin with subtilisin as described (34Rostovtseva T.K. Sheldon K.L. Hassanzadeh E. Monge C. Saks V. Bezrukov S.M. Sackett D.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18746-18751Crossref PubMed Scopus (286) Google Scholar, 35Knipling L. Hwang J. Wolff J. Cell Motil. Cytoskeleton. 1999; 43: 63-71Crossref PubMed Scopus (34) Google Scholar). 2 μl of GST fusion proteins bound to glutathione-Sepharose beads were incubated with 1 μg of purified tubulin (unless otherwise stated) or tubulin S in G-PEM plus 2% Nonidet P-40 and 100 mm NaCl. 10 μl of GST fusion proteins bound to glutathione-Sepharose beads were incubated with 100 μg of rat brain cytosolic extracts in homogenization buffer plus 100 mm NaCl. To determine the effect of salt on the interaction with tubulin, the interaction was carried out in buffer containing increasing concentrations of NaCl from 0 to 400 mm. Incubations were carried out at room temperature for 1 h. The resin was then washed three times with binding buffer. The bound proteins were solubilized in 20 μl of 2× SDS-gel buffer and separated by 10% SDS-PAGE. The bound tubulin was detected by Western blotting with either α-tubulin or β-tubulin specific antibodies. The membranes were stained with Amido Black to confirm equal input of fusion proteins into each reaction. As the concentrations of tubulin and tubulin S in GST pull-down assays was lower than that required for polymerization, both tubulin and tubulin S are likely dimeric, but not polymerized. HEK293 cells cultured on 100-mm dishes were transfected with 2 μg of HA-tagged α2B-AR or its mutant 3R-3A in which Arg-437, Arg-441, and Arg-446 were mutated to Ala for 36 h. The cells were washed twice with PBS, harvested and lysed with 500 μl of lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet 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 3 μg of anti-HA antibodies overnight at 4 °C with gentle rotation followed by an incubation with 50 μl of protein G-Sepharose 4B beads for 5 h. Resin was collected by centrifugation and washed three times each with 500 μl of lysis buffer without SDS. Immunoprecipitated proteins were eluted with 100 μl of 1× SDS gel loading buffer and separated by SDS-PAGE. α2B-AR and tubulin in the immunoprecipitates were detected by Western blotting using anti-HA and anti-tubulin antibodies, respectively. Cell-surface expression of α2B-AR in HEK293 cells was measured by ligand binding on intact cells using [3H]RX821002 as described previously (30Duvernay M.T. Dong C. Zhang X. Robitaille M. Hébert T.E. Wu G. Traffic. 2009; 10: 552-566Crossref PubMed Scopus (50) Google Scholar, 31Duvernay M.T. Dong C. Zhang X. Zhou F. Nichols C.D. Wu G. Mol. Pharmacol. 2009; 75: 751-761Crossref PubMed Scopus (60) Google Scholar). HEK293 cells cultured on 6-well dishes were transfected with α2B-AR or its mutants as described above. After transfection for 6 h the cells were split into 12-well dishes pre-coated with poly-l-lysine at a density of 5 × 105 cells/well. After transfection 48 h, the cells were incubated with [3H]RX821002 at a concentration of 20 nm in a total of 400 μl for 90 min at room temperature. The cells were washed twice with ice-cold DMEM, and the retained radioligand was then extracted by digesting the cells in 300 μl of 1 m NaOH for 2 h. The amount of radioactivity retained was measured by liquid scintillation spectrometry. For measurement of α2B-AR internalization, HEK293 cells were cultured 6-well dishes and transfected with 0.5 μg of α2B-AR and 1 μg of arrestin-3 plus 1 μg of empty pcDNA3.1(-) vector, Arr3-(201–409), DynK44A, or Rab5S34N for 24 h. After starvation for 3 h, the cells were stimulated with epinephrine at a concentration of 100 μm for 1 h. The cells were washed three times with cold PBS and α2B-AR expression at the cell surface was measured by intact cell ligand binding as described above. To determine the effect of drug treatment on the cell surface expression of α2B-AR, HEK293 cell transfected with α2B-AR were incubated with GM132 (20 μm), NH4Cl (20 mm), or chloroquine (100 μm) for 6 h at 37C°. For measurement of α2B-AR expression at the cell surface, HEK293 cells were transfected with HA-tagged receptor for 36–48 h. The cells were collected, suspended in PBS containing 1% FBS at a density of 4 × 106 cells/ml and incubated with high affinity anti-HA-fluorescein (3F10) at a final concentration of 2 μg/ml for 30 min at 4 °C. After washing twice with 0.5 ml of PBS/1% FBS, the cells were resuspended, and the fluorescence was analyzed on a flow cytometer (Dickinson FACSCalibur) as described (30Duvernay M.T. Dong C. Zhang X. Robitaille M. Hébert T.E. Wu G. Traffic. 2009; 10: 552-566Crossref PubMed Scopus (50) Google Scholar). For fluorescence microscopic analysis of α2B-AR subcellular distribution, HEK293 cells were grown on coverslips pre-coated with poly-l-lysine in 6-well plates and transfected with 500 ng of α2B-AR-GFP for 36 to 48 h. For co-localization of α2B-AR with Sec24, HEK293 cells were transfected with 100 ng of α2B-AR-GFP and 400 ng of Sar1H79G. For co-localization of α2B-AR with the ER marker DsRed2-ER, HEK293 cells were transfected with 100 ng of α2B-AR-GFP and 100 ng of pDsRed2-ER. The cells were fixed with 4% paraformaldehyde-4% sucrose mixture in PBS for 15 min. The coverslips were mounted, and fluorescence was detected with a Leica DMRA2 epifluorescent microscope. Images were deconvolved using SlideBook software and the nearest neighbor deconvolution algorithm (Intelligent Imaging Innovations, Denver, CO) as described previously (30Duvernay M.T. Dong C. Zhang X. Robitaille M. Hébert T.E. Wu G. Traffic. 2009; 10: 552-566Crossref PubMed Scopus (50) Google Scholar). HEK293 cells were cultured in 6-well dishes and transfected with 0.5 μg of α2B-AR or its mutant. At 6–8 h after transfection, the cells were split into 6-well dishes and cultured for additional 36 h. The cells were starved for at least 3 h and then stimulated with 1 μm UK14304 for 5 min. Stimulation was terminated by addition of 1× SDS gel loading buffer. After solubilizing the cells, 20 μl of total cell lysates were separated by 12% SDS-PAGE. ERK1/2 activation was determined by measuring the levels of phosphorylation of ERK1/2 with phosphospecific ERK1/2 antibodies by immunoblotting (14Wu G. Zhao G. He Y. J. Biol. Chem. 2003; 278: 47062-47069Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). 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. To search for proteins interacting with the α2B-AR CT (Fig. 1A), the CT peptide was synthesized, directly conjugated to agarose beads and incubated with the brain cytosolic extracts. The bound proteins were eluted, separated by two-dimensional gels, and identified by LTQ electrospray mass spectrometry. The most predominant proteins pulled out from the rat brain lysates were tubulin isoforms (Fig. 1B). Western blot analysis using monoclonal α-tubulin-specific antibodies further revealed that tubulin was pulled out by the CT-conjugated agarose matrix. In contrast, a control peptide derived from the ICL3 of α2B-AR did not pull down detectable tubulin from brain lysates (Fig. 1C). We next used GST pull-down assays to confirm the interaction between the α2B-AR CT and tubulin. The GST fusion protein encoding the α2B-AR CT, but not GST, strongly interacted with tubulin from total brain extracts (Fig. 2, A and B). To determine if the α2B-AR CT could directly interact with tubulin, the GST fusion proteins were incubated with increasing concentrations of purified bovine brain tubulin. The GST-α2B-AR CT was able to bind to purified α- and β-tubulin (Fig. 2C). These data indicate that the α2B-AR CT directly interacts with tubulin. Next, we identified specific residues in the α2B-AR CT responsible for interacting with tubulin. In the first series of experiments, each non-basic residue in the CT was mutated to Ala, whereas 5 Arg residues were mutated to Glu together (5R-5E), which will presumably preserve the amphipathic character of the α-helix 8, but reverse the charge on the face of the helix. The abilities of mutated CT to interact with purified tubulin and brain extracts were determined in the GST fusion protein pull-down assay. Mutation of the non-basic residues had variable effects on the affinity of the α2B-AR CT for tubulin, but was insufficient to abolish the interaction. Simultaneous mutation of the 5 Arg residues to Glu, on the other hand, abolished the interaction of the α2B-AR CT with purified tubulin (Fig. 3A). Furthermore, mutation of the 5 Arg residues also markedly inhibited the α2B-AR CT interaction with tubulin from the brain extracts (Fig. 3B). These data indicate that Arg residues are the main determinant of the interaction between the α2B-AR CT and tubulin. In the second series of experiments, each Arg residue in the α2B-AR CT was individually mutated to Glu and the effect on the CT interaction with tubulin was measured. Surprisingly, individual mutation of Arg-437, Arg-441, and Arg-446 or simultaneous mutation of all three Arg residues together almost completely blocked the CT interaction with tubulin, whereas mutation of Arg-438 and Arg-442 only partially inhibited the interaction (Fig. 3C). These data demonstrate that the five Arg residues in the CT unequally contribute to α2B-AR interaction with tubulin, and Arg residues at positions of 437, 441, and 446 play a crucial role in mediating α2B-AR interaction with tubulin. In the third series of experiments, we determined if these Arg residues mediate α2B-AR interaction with tubulin in cells. HEK293 cells were transiently transfected with the empty vector (control), HA-tagged α2B-AR or its mutant in which Arg-437, Arg-441, and Arg-446 were mutated to Glu (3R-3E) and their interaction with tubulin was determined by co-immunoprecipitation using anti-HA antibodies. The amount of tubulin was much more in the immunoprecipitates from cells expressing wild-type α2B-AR than from cells expressing α2B-AR mutant 3R-3E and control cells (Fig. 3D). These data suggest that α2B-AR may physically associate with tubulin via C-terminal Arg residues in a cellular context. Our preceding data have identified Arg residues as tubulin binding sites in the α2B-AR CT. To define the α2B-AR binding domain in tubulin, we focused on the CT of tubulin as it is negatively charged containing multiple acidic residues. To determine if the CT of tubulin interacts with α2B-AR, we generated tubulin S in which the CT of tubulin was removed and determined its ability to interact with the α2B-AR CT. GST fusion protein pull-down assay shown that tubulin S did not interact with the α2B-AR CT (Fig. 4A). These data indicate that the CT of tubulin is the α2B-AR binding site. We then determined the effect of increasing concentrations of NaCl on α2B-AR CT interaction with tubulin. The maximal levels of tubulin were pulled down in the incubation buffer containing 0, 5, and 10 mm NaCl. When NaCl concentration was increased beyond 10 mm, the amount of tubulin pulled down dropped sharply until there was almost no tubulin detectable at 400 mm NaCl (Fig. 4B). These data further demonstrate that the interaction between α2B-AR and tubulin is ionic in nature. To determine the role of the interaction between α2B-AR and tubulin, we compared the cell surface expression of α2B-AR with its mutants in which the five Arg residues were mutated to Glu individually or in combination. Mutation of Arg-437, Arg-441, and Arg-446 significantly attenuated the numbers of α2B-AR at the cell surface, whereas mutations of Arg-438 and Arg-442 did not have a clear inhibitory effect. Furthermore, simultaneous mutation of all five Arg residues (5R-5E) (not shown) or three Arg residues at positions 437, 441, and 446 (3R-3E) abolished α2B-AR transport to the cell surface (Fig. 5A). In contrast the cell surface expression, the total α2B-AR expression was very much the same between wild type and mutants (Fig. 5A). These data demonstrate that the positively charged Arg cluster in the CT not only mediates α2B-AR interaction with tubulin but also is required for α2B-AR transport to the cell surface. As the cell surface expression of α2B-AR was measured by intact cell ligand binding, to exclude the possibility that mutation of the C-terminal Arg residues alters the ability of α2B-AR to bind to its ligand, α2B-AR and its mutant 3R-3E were tagged with HA at their N termini and their expression at the cell surface was then measured by flow cytometry following staining with anti-HA antibodies in non-permeabilized cells. Consistent with the data obtained in ligand binding, cell surface expression of the mutant 3R-3E was reduced by 96% as compared with wild-type receptor (Fig. 5B). To eliminate the possibility that attenuated cell surface expression of the α2B-AR mutant 3R-3E is caused by its 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 of the mutant. Arrestin-3, dynamin, and Rab5 have been well demonstrated to modulate endocytic trafficking of GPCRs including α2B-AR (4Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 36DeGraff 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, 37DeGraff 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). Stimulation with 100 μm epinephrine for 1 h reduced the cell surface expression of α2B-AR by 42 ± 2% (n = 5). Co-expression of Arr3-(201–409), DynK44A, and Ra" @default.
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