Ligand-independent CXCR2 Dimerization
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m306815200
ISSN1083-351X
AutoresFlavia Trettel, Sabrina Di Bartolomeo, Clotilde Lauro, Myriam Catalano, Maria Teresa Ciotti, Cristina Limatola,
Tópico(s)Neuropeptides and Animal Physiology
ResumoHomo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product β) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers. Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product β) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers. Homo- or heterodimerization of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR(s), G-protein coupled receptor(s); AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CGNs, cerebellar granule neurons; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GluR, glutamate receptor; HEK, human embryonic kidney; mAb, monoclonal antibody; PI3-kinase, phosphatidylinositol 3-kinase; PTX, pertussis toxin; TM, transmembrane; GABAB, gamma-aminobutyric acid type B. has recently emerged as a constitutive or ligand-induced property of several receptor types. Receptor oligomerization has functional implications in terms of cell surface expression, ligand binding, signaling, and receptor trafficking (1Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar). Many different GPCRs have been proved to undergo homo- or heterodimer formation; these include the receptors for dopamine which can form both homo- (2Ng G.Y. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (246) Google Scholar, 3George S.R. Lee S.P. Varghese G. Zeman P.R. Seeman P. Ng G.Y. O'Dowd B.F. J. Biol. Chem. 1998; 273: 30244-30248Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 4Guo W. Shi L. Javitch J.A. J. Biol. Chem. 2003; 278: 4385-4388Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) and heterodimers with somatostatin receptors (5Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (755) Google Scholar), angiotensin A1 and A2 (6AbdAlla S. Lother H. Abdel-Tawab A.M. Quitterer U. J. Biol. Chem. 2001; 276: 39721-39726Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), adrenergic (7Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 8Xu J. He J. Castleberry A.M. Balusubramanian S. Lau A.G. Hall R.A. J. Biol. Chem. 2003; 278: 10770-10777Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), GABAB (9Jones K. Borowsky B. Tamm J.A. Graig D.A. Durkin M.M. Dai M. Yao W. Johnson M. Gunwaldsen C. Huang L. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek S.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (929) Google Scholar, 10White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foords S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1019) Google Scholar, 11Kaupman K. Malitschek B. Schuler V. Heid J. Froestel W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1018) Google Scholar), δ- and κ-opioid (12Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 13Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (982) Google Scholar, 14Gomes I. Jordan B.A. Gupta A. Trapaizde N. Nagy V. Devi L.A. J. Neurosci. 2000; 20: RC110Crossref PubMed Google Scholar, 15Pfeiffer M. Koch T. Schroder H. Laugsch M. Hollt V. Schulz S. J. Biol. Chem. 2002; 277: 19762-19772Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), rhodopsin (16Fotiadis D. Liang Y. Filipek S. Saperstein D.A. Engel A. Palczewski K. Nature. 2003; 421: 127-128Crossref PubMed Scopus (661) Google Scholar), mGlu (17Romano C. Yang W. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar), vasopressin V2 (18Zhu X. Wess J. Biochemistry. 1998; 37: 15773-15784Crossref PubMed Scopus (175) Google Scholar), vasopressin/ossitocin (19Terillon S. Durroux T. Mouillac B. Breit A. Ayoub M.A. Taulan M. Jockers R. Barberis C. Bouvier M. Mol. Endocrinol. 2003; 17: 677-691Crossref PubMed Scopus (280) Google Scholar), muscarinic (20Avissar S. Amitai G. Sokolovsky M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 156-159Crossref PubMed Scopus (88) Google Scholar), glucagon (21Herberg J.T. Codina J. Rich K.A. Rojas F.J. Iyengar R. J. Biol. Chem. 1984; 259: 9285-9294Abstract Full Text PDF PubMed Google Scholar), Ca2+ (22Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar) sphingosine 1-phosphate (23Van Brocklyn J.R. Behbahani B. Lee N.H. Biochim. Biophys. Acta. 2002; 1582: 89-93Crossref PubMed Scopus (49) Google Scholar), and the chemokine CXCR4, CCR2, and CCR5 receptors (24Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 25Vila-Coro A.J. Rodríguez-Frade J.M. Martín de Ana A. Moreno-Ortíz M.C. Martínez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 26Mellado M. Rodríguez-Frade J.M. Vila-Coro A.J. Fernández S. Martín de Ana A. Jones D.R. Torán J.L. Martínez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar, 27Issafras H. Angers S. Bulenger S. Blanpain C. Parmentier M. Labbe-Jullie C. Bouvier M. Marullo S. J. Biol. Chem. 2002; 277: 34666-34673Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 28Babcock G.J. Farzan M. Sodroski J. J. Biol. Chem. 2003; 278: 3378-3385Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The molecular mechanism of receptor oligomerization varies among different receptors and involves transmembrane (TM) domains as well as extramembrane regions. Cysteine residues in the fourth TM segment are responsible for D2 dopamine receptor dimerization (4Guo W. Shi L. Javitch J.A. J. Biol. Chem. 2003; 278: 4385-4388Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), and disulfide bonds are also involved in the oligomerization of Ca2+ (22Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar), κ-opioid (13Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (982) Google Scholar), sphingosine 1-phosphate (23Van Brocklyn J.R. Behbahani B. Lee N.H. Biochim. Biophys. Acta. 2002; 1582: 89-93Crossref PubMed Scopus (49) Google Scholar), and V2 vasopressin receptors (18Zhu X. Wess J. Biochemistry. 1998; 37: 15773-15784Crossref PubMed Scopus (175) Google Scholar); sequences in the N-terminal extracellular domains are involved in mGluR (17Romano C. Yang W. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar) and CCR5 (24Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 25Vila-Coro A.J. Rodríguez-Frade J.M. Martín de Ana A. Moreno-Ortíz M.C. Martínez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 26Mellado M. Rodríguez-Frade J.M. Vila-Coro A.J. Fernández S. Martín de Ana A. Jones D.R. Torán J.L. Martínez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar, 27Issafras H. Angers S. Bulenger S. Blanpain C. Parmentier M. Labbe-Jullie C. Bouvier M. Marullo S. J. Biol. Chem. 2002; 277: 34666-34673Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) homodimer formation; the C-terminal region is essential for δ-opioid receptor dimerization (12Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). The seventh TM domain controls noncovalent hydrophobic interactions for adrenergic receptors, which also require receptor glycosylation (7Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 8Xu J. He J. Castleberry A.M. Balusubramanian S. Lau A.G. Hall R.A. J. Biol. Chem. 2003; 278: 10770-10777Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Contrasting consequences are also reported for receptor functional properties: in some cases receptor dimerization is essential for receptor function, as demonstrated for GABAB (9Jones K. Borowsky B. Tamm J.A. Graig D.A. Durkin M.M. Dai M. Yao W. Johnson M. Gunwaldsen C. Huang L. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek S.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (929) Google Scholar, 10White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foords S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1019) Google Scholar, 11Kaupman K. Malitschek B. Schuler V. Heid J. Froestel W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1018) Google Scholar) and for the taste receptors (29Nelson G. Chandrashekar J. Hoon M.A. Feng L. Zhao G. Ryba N.J. Zuker C.S. Nature. 2002; 416: 199-202Crossref PubMed Scopus (1168) Google Scholar); for other GPCRs, heterodimerization impairs or changes the features of their activation, as shown for the angiotensin II (6AbdAlla S. Lother H. Abdel-Tawab A.M. Quitterer U. J. Biol. Chem. 2001; 276: 39721-39726Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), the opioid and dopamine (30George S.R. Fan T. Xie Z. Tse R. Tam V. Varghese G. O'Dowd B.F. J. Biol. Chem. 2000; 275: 26128-26135Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 31Scarselli M. Novi F. Schallmach E. Lin R. Baragli A. Colzi A. Griffon N. Corsini G.U. Sokoloff P. Levenson R. Vogel Z. Maggio R. J. Biol. Chem. 2001; 276: 30308-30314Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and for the β-adrenergic receptors (32Lavoie C. Mercier J.F. Salahpour A. Umapathy D. Breit A. Villeneuve L.R. Zhu W.Z. Xiao R.P. Lakatta E.G. Bouvier M. Hébert T.E. J. Biol. Chem. 2002; 277: 35402-35410Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Furthermore, both agonist-dependent and agonist-independent receptor dimerization have been reported. From all of these studies it clearly appears that there are not unique molecular mechanisms of dimerization for GPCRs and that the functional effects of receptor oligomerization are likely to be understood completely in the future. The oligomerization of chemokine receptors has been shown to be relevant for receptor biology (24Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 26Mellado M. Rodríguez-Frade J.M. Vila-Coro A.J. Fernández S. Martín de Ana A. Jones D.R. Torán J.L. Martínez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar, 27Issafras H. Angers S. Bulenger S. Blanpain C. Parmentier M. Labbe-Jullie C. Bouvier M. Marullo S. J. Biol. Chem. 2002; 277: 34666-34673Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The homodimerization of the chemokine receptor CCR5 with a natural genetic mutation found in some ethnic groups (ccr5Δ32) confers some resistance to human immunodeficiency virus type 1 infection because of a sort of loss of function of the heterodimers (24Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Both CCR5 and CXCR4 receptors have been reported to homodimerize, but the role of agonist stimulation on receptor oligomerization is debated (25Vila-Coro A.J. Rodríguez-Frade J.M. Martín de Ana A. Moreno-Ortíz M.C. Martínez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 27Issafras H. Angers S. Bulenger S. Blanpain C. Parmentier M. Labbe-Jullie C. Bouvier M. Marullo S. J. Biol. Chem. 2002; 277: 34666-34673Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 28Babcock G.J. Farzan M. Sodroski J. J. Biol. Chem. 2003; 278: 3378-3385Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). CCR5 receptor oligomerization occurs in the endoplasmic reticulum, consistent with a constitutive receptor assembly under resting conditions (27Issafras H. Angers S. Bulenger S. Blanpain C. Parmentier M. Labbe-Jullie C. Bouvier M. Marullo S. J. Biol. Chem. 2002; 277: 34666-34673Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). CCR2 and CCR5 receptors heterodimerize upon treatment with a combination of chemokines, and heteromeric receptors may induce different functional responses (26Mellado M. Rodríguez-Frade J.M. Vila-Coro A.J. Fernández S. Martín de Ana A. Jones D.R. Torán J.L. Martínez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar). Because chemokine receptor dimerization may add further complexity to the biology of this promiscuous receptor family (33Baggiolini M. Loetscher P. Immunol. Today. 2000; 21: 418-420Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), we decided to investigate the oligomerization of the chemokine receptor CXCR2. We have demonstrated recently that the CXCR2 receptors, expressed by cerebellar neurons or transfected on HEK cells, interact physically and functionally with the AMPA-type glutamate receptors (34Lax P. Limatola C. Fucile S. Trettel F. Di Bartolomeo S. Renzi M. Ragozzino D. Eusebi F. J. Neuroimmunol. 2002; 129: 66-73Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 35Limatola C. Di Bartolomeo S. Trettel F. Lauro C. Ciotti M.T. Mercanti D. Castellani L. Eusebi F. J. Neuroimmunol. 2003; 134: 61-71Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Here, we investigated whether CXCR2 receptors could also form oligomers and what consequences oligomerization might produce on CXCR2 functions. In this paper we describe that CXCR2 forms oligomers when expressed in HEK cells and in native neuronal systems and that oligomer formation is independent of receptor activation by agonist. Attempting to define the molecular regions responsible for receptor oligomerization, we have generated several drastic deletion mutants of CXCR2. We demonstrated that most of these mutants act as dominant negative inhibitors of receptor function, in terms of cell signaling and chemotaxis. Furthermore, CXCR2 receptors mutated in different molecular domains and expressed in HEK cells do not complement the original function. From all these data, we suggest that the active form of the CXCR2 receptor is a dimer and that each individual subunit is activated with an intramolecular mechanism (cis-activation). To our knowledge, this is the first demonstration that CXCR2 receptors function in oligomerized state. Materials—Monoclonal and polyclonal antibodies against human CXCR2 (E2, C19), anti-ERK2, polyclonal antibody against rat CXCR2 (K19), and against GFP (FL-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody against human GluR1 (Ab1504) was purchased from Chemicon International (Temecula, CA). Anti-phospho-Akt (Ser-473), anti-Akt, and anti-phospho-p44/p42 mitogen-activated protein kinase (Thr-202/Tyr-204) mAb were from New England Biolabs. Recombinant rat CXCL2 and human CXCL8 were from Peprotech (London). Transwell cell culture inserts were from BD Biosciences. The BCA protein assay was from Pierce. All culture media and V5 mAbs were purchased from Invitrogen. Generation of CXCR2 Mutants—cDNAs encoding for CXCR2 and CXCR1, cloned in pCEP4 expression vector, were kindly provided by Dr. Massimo Locati (University of Brescia, Italy). cDNA for human GluR1 (flip) was from ATCC (Manassas, VA). To obtain CXCR2-tagged proteins, cDNA encoding for CXCR2 (Swiss-Prot P25025) was subcloned in pEGFP-C3 (Clontech, Palo Alto, CA) expression vector using the primers 5′-ATAACTCGAGATGGAAGATTTTAAC-3′ and 5′-GTCAGAATTCTTAGAGAGTAGTG-3′ and in pcDNA3.1D/V5-His-TOPO (Invitrogen) using the primers 5′-AGACAAGCTTATGGAAGATTTTAAC-3′ and 5′-ATAACTCGAGTGGAGAGTAGTG-3′. C- (Ala-315, Phe-183, Lys-163, Val-142) and N-terminal (Tyr-49, Ala-106, Asp-143) CXCR2 deletion mutants were generated by PCR and cloned into the expression vector pCEP4 (Invitrogen). The following couples of primers were used for the PCRs: 5′-GATCAAGCTTATGGAAGATTTTAAC-3′ and 5′-GATCCTCGAGTTAGGCGTAGATGAG-3′ for CXCR2-A315; 5′-GATCAAGCTTATGGAAGATTTTAAC-3′ and 5′-GATCCTCGAGTTAGAAAAGTAAGACAG-3′ for CXCR2-F183; 5′-GATCAAGCTTATGGAAGATTTTAAC-3′ and 5′-GATCCTCGAGTTATTTGACCAAGTA-3′ for CXCR2-K163; 5′-GATCAAGCTTATGGAAGATTTTAAC-3′ and 5′-GATCCTCGAGTTACACACTGATGCAG-3′ for CXCR2-V142; 5′-GATCAAGCTTATGTATTTTGTGGTC-3′ and 5′-TATCCTCGAGTTAGAGAGTAGTGGA-3′ for Y49-CXCR2; 5′-GATCAAGCTTATGGCCTCCAAGGTG-3′ and 5′-TATCCTCGAGTTAGAGAGTAGTGGA-3′ for A106-CXCR2; 5′-GATCAAGCTTATGGACCGTTACCTG-3′ and 5′-TATCCTCGAGTTAGAGAGTAGTGGA-3′ for D143-CXCR2. The fidelity of all CXCR2 constructs was checked by DNA sequencing, and their expression in HEK cells was confirmed by Western blot and immunofluorescence analysis. GFP-GluR1 fusion proteins were obtained as described previously (35Limatola C. Di Bartolomeo S. Trettel F. Lauro C. Ciotti M.T. Mercanti D. Castellani L. Eusebi F. J. Neuroimmunol. 2003; 134: 61-71Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Cell Transfection and Signaling Studies—HEK 293 (HEK) and human CXCR2-stably transfected HEK (HEK-CXCR2) cells (kindly provided by Dr. Massimo Locati and by Dr. Adit Ben-Baruch, Tel-Aviv University, Israel), were transfected with LipofectAMINE 2000 plus reagent (Invitrogen). Routinely, cells were used for experiments 48 h after transfection. When used for cellular signaling, 18 h after transfection, cells from different transfections were trypsinized and reseeded on 12-well plates. After an additional 6 h cells were serum-starved for 16 h and incubated further for additional 2 h in Locke's buffer (35Limatola C. Di Bartolomeo S. Trettel F. Lauro C. Ciotti M.T. Mercanti D. Castellani L. Eusebi F. J. Neuroimmunol. 2003; 134: 61-71Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). When necessary, cells were preincubated with 100 ng/ml PTX for 16 h or 50 μm LY294002 for 1 h and stimulated with chemokine (120 nm for CXCL8 and 30 nm for CXCL2) in this same buffer. After 10 min, the chemokine-containing medium was removed, and cells were washed with ice-cold phosphate-buffered saline, lysed in Triton X-100 buffer (described below), and analyzed for protein content with a commercial kit. The same amounts of cellular proteins (10–20 μg) were analyzed by SDS-PAGE and Western blot analysis with Abs specific for phospho-ERK1/2, phospho-Akt, ERK2, and Akt. For experiments with cerebellar neurons, cerebellar granule neurons (CGNs) were obtained from 3- or 7-day-old Wistar rats, as described (35Limatola C. Di Bartolomeo S. Trettel F. Lauro C. Ciotti M.T. Mercanti D. Castellani L. Eusebi F. J. Neuroimmunol. 2003; 134: 61-71Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), stimulated with CXCL2 and analyzed as above. Immunoprecipitation—Transiently transfected HEK cells were washed with phosphate-buffered saline and lysed for 15 min on ice in buffer containing 50 mm Tris-HCl, pH 8, 20 mm EDTA, 1% Nonidet P-40, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 mm NaF, and 1 mm phenylmethylsulfonyl fluoride. After centrifugation at 15,000 × g for 20 min at 4 °C, cell lysates were precleared with normal mouse serum or with preimmune rabbit IgG and then incubated at 4 °C for 16 h with 8 μg/ml anti-CXCR2 mAb, 10 μg/ml V5 mAb, or 2 μg/ml anti-GFP polyclonal antibody. The resulting immunocomplexes were separated on SDS-PAGE and analyzed by Western blotting with anti-GluR1 antibody (Ab1504), anti-V5 antibody (V5), or anti-CXCR2 antibody (C19). Expression of Wild-type and Mutant CXCR2 Proteins—HEK cells were transiently transfected with different combinations of CXCR2 constructs and lysed by the addition of hot 2× SDS-Laemmli buffer or by using a Triton X-100 lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 1% Triton X-100, 20 mm sodium pyrophosphate, 20 mm NaF, 1 mm sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 1 mm CaCl2). Expression of wt and mutant CXCR2 was analyzed by Western blotting of SDS-PAGE. MAb E2, recognizing an N-terminal epitope, was used to analyze wt, Ala-315, Phe-183, Lys-163, and Val-142 proteins; polyclonal Ab C19, recognizing a C-terminal epitope, was used to analyze wt, Tyr-49, Ala-106, and Asp-143 proteins. Chemotaxis Assay—CXCL8-induced chemotaxis was investigated in HEK cells transfected with CXCR2 alone or with different combinations of truncated mutants. 48 h after transfection, cells were trypsinized, washed twice in chemotaxis medium (fetal bovine serum-free Dulbecco's modified Eagle's medium plus 0.1% bovine serum and 25 mm Hepes, pH 7.4) and plated (500,000 cells/well) on collagen-pre-coated 12 mm Transwells (12-μm pore size filters) in the same medium. The lower chambers of the Transwell system contained vehicle (water) or 6 nm CXCL8 in chemotaxis medium. After 2 h of incubation at 37 °C, cells were washed with phosphate-buffered saline and treated with trichloroacetic acid on ice for 10 min. Cells adhering to the upper side of the filter were scraped off, and cells on the lower side were stained with a solution containing 50% isopropyl alcohol, 1% formic acid, and 0.5% (w/v) Coomassie Brilliant Blue R-250. Stained cells were visually counted in at least 20 fields using a 20× objective. The chemotactic index is obtained by the ratio between the number of migrating cells in chemokine-treated versus untreated cells for each type of transfection. CXCR2 Forms Dimers in HEK Cells and in Cerebellar Neurons—To examine whether CXCR2 forms oligomers, as already demonstrated for other chemokine receptors, we generated CXCR2 constructs with either a V5 (CXCR2-V5) or a GFP (GFP-CXCR2) tail, at the C- and N-terminal domains, respectively. These constructs were transfected in HEK cells, and the cell lysates, obtained 48 h later, were immunoprecipitated with GFP Ab and analyzed by Western blot for V5 immunoreactivity. Results in Fig. 1A show that the complex CXCR2-V5-GFP-CXCR2 is immunoprecipitated by the GFP Ab only when the two constructs are expressed simultaneously. Similar results were obtained when the immunoprecipitation was performed with V5 Ab, and the corresponding Western blots were analyzed for GFP immunoreactivity (not shown). Coimmunoprecipitation was not detected in a mixture of lysates from cells individually expressing the tagged receptors, excluding the possibility of nonspecific receptor aggregation (not shown). Fig. 1B shows that when HEK cells, transfected with plasmids containing CXCR2 (wt), were lysed with SDS-Laemmli buffer and analyzed by Western blot, both monomeric and oligomeric forms of the receptor became evident. A mutated form of the CXCR2 receptor (Ala-315), obtained by the complete truncation of the intracellular C-terminal tail, has a similar shifted pattern. This supports the view that the high molecular weight bands are composed of aggregated receptors. When CXCR2-transfected cells were treated with tunicamycin and analyzed for receptor oligomerization, we observed the disappearance of glycosylated monomeric CXCR2 and a shift in the putative CXCR2 dimer band (Fig. 1C). This indicates that CXCR2 receptors dimerize before they appear on the plasma membrane, during the biosynthetic pathway. To investigate whether receptor oligomer formation could be modulated by receptor activation, transfected cells were treated with the specific agonist CXCL8 for 5 min and analyzed as above. Fig. 1D indicates that the formation of CXCR2 oligomers (wt or Ala-315) was not affected by receptor activation, showing that CXCR2 oligomer formation was ligand-independent. CXCR2 dimers were resistant to membrane solubilization with detergent, being present both in cell lysates obtained with Triton X-100 buffer and with hot SDS buffer and were also resistant to reducing conditions because the Laemmli buffer we used always contained 50 mm dithiothreitol (see "Experimental Procedures"). We have described the functional expression of CXCR2 in rat CGNs previously (36Limatola C. Mileo A.M. Giovannelli A. Vacca F. Ciotti M.T. Mercanti D. Santoni A. Eusebi F. J. Biol. Chem. 1999; 274: 36537-36543Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Lysates obtained from 8-day cultured neurons, analyzed by Western blot with a polyclonal CXCR2 Ab (K19), revealed a main band at 40 kDa and an additional protein at about 78 kDa, which likely represents CXCR2 dimers (Fig. 1E). To establish the receptor domain(s) directly involved in CXCR2 receptor oligomerization, deletion mutants were generated at the C- or N-terminal regions (see Fig. 2) and tested for dimer formation by Western blot analysis. For each kind of transfection, protein expression was also confirmed by immunofluorescence (not shown). Fig. 3A (right side, and Fig. 1B) shows that when the whole intracellular C-terminal (CXCR2-A315) region was deleted, the truncated CXCR2 receptors retained their ability to form homodimers, as evidenced by the immunological detection of both the monomeric deleted receptors and additional proteins with molecular weights compatible with truncated dimers. Progressive deletions at the C-terminal region led to the generation of receptors truncated at the amino acids Phe-183, Lys-163 and Val-142, as detailed in Fig. 2. Dimeric forms of CXCR2-F183 were detectable (Fig. 3A, left side), whereas CXCR2-V142 was only present as monomer (shown in Fig. 3B). Interestingly, the C-terminally deleted mutants Ala-315, Phe-183, and Val-142, when coexpressed together with the wt CXCR2, retained the ability to bind the wt receptors, as evidenced by the bands with molecular weight corresponding to the sum of the wt and deleted receptor types (here called "heterodimers" wt/A315 and wt/F183, Fig. 3A and wt/V142, Fig. 3B). Interestingly, the C-terminal mutant CXCR2-K163 also retained the ability to hetero- and homodimerize, as demonstrated in Fig. 3C.Fig. 3Analysis of the homo- and hetero-oligomerization of the CXCR2 rece
Referência(s)