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Comodulation of CXCR4 and CD26 in Human Lymphocytes

2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês

10.1074/jbc.m004586200

1083-351X

Carolina Herrera, Chikao Morimoto, Julià Blanco, Josefa Mallol, Fernando Arenzana, Carmen Lluís, Rafael Franco,

Cell Adhesion Molecules Research

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4+) and B (CD4−) cell lines. Upon induction with stromal cell-derived factor 1α (SDF-1α), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1α−mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1α, the CXCR4·CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1α-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1α and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection. We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4+) and B (CD4−) cell lines. Upon induction with stromal cell-derived factor 1α (SDF-1α), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1α−mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1α, the CXCR4·CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1α-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1α and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection. human immunodeficiency virus adenosine deaminase CXC chemokine receptor stromal-derived factor fluorescein isothiocyanate tetramethylrhodamine isothiocyanate monoclonal antibody phosphate-buffered saline phorbol 12-myristate 13-acetate CD26 is a widely distributed lymphocyte activation antigen that displays dipeptidyl peptidase IV activity in its extracellular domain. The role of CD26 in HIV1infection has been controversial. Its postulated role as coreceptor for HIV entry (1Callebaut C. Krust B. Jacotot E. Hovanessian A.G. Science. 1993; 262: 2045-2050Crossref PubMed Scopus (207) Google Scholar) was rapidly contested (2Broder C.C. Nussbaum O. Gutheil W.G. Bachovchin W.W. Berger E.A. Science. 1994; 264: 1156-1165Crossref PubMed Scopus (48) Google Scholar, 3Morimoto C. Lord C.I. Zhang C. Duke-Cohan J.S. Letvin N.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9960-9964Crossref PubMed Scopus (73) Google Scholar). However, it has been more recently shown that the expression of CD26 may modulate HIV infection by direct effects of dipeptidyl peptidase IV activity on the antiviral activity of several chemokines or by unknown mechanisms independent of peptidase activity (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 5Oravecz T. Roderiquez G. Koffi J. Wang J. Ditto M. Bou-Habib D.C. Lusso P. Norcross M.A. Nat. Med. 1995; 1: 919-926Crossref PubMed Scopus (61) Google Scholar, 6Callebaut C. Jacotot E. Blanco J. Krust B. Hovanessian A.G. Exp. Cell Res. 1998; 241: 352-362Crossref PubMed Scopus (22) Google Scholar, 7Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar).In fact, CD26 can cleave out NH2-terminal dipeptides from a protein with either l-proline or l-alanine at the penultimate position and several chemokines can be processed by CD26 (see Ref. 8De Meester I. Korom S. Van Damme J. Scharpé S. Immunol. Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar for review). For some chemokines proteolysis by CD26 does not affect the physiological activity. In contrast, truncation of RANTES (regulated on activation normal T cell expressed and secreted) by CD26 leads to higher receptor selectivity and anti-HIV activity (9Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar,10Proost P. de Meester I. Schols D. Struyf S. Lambeir A.-M. Wuyts A. Opdenakker G. De Clercq E. Scharpé S. van Damme J. J. Biol. Chem. 1998; 273: 7222-7227Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Stromal cell-derived factor 1α (SDF-1α) also loses the NH2-terminal Lys-Pro dipeptide by the action of CD26. This processing significantly reduces the chemotactic and anti-HIV-1 activity of SDF-1α (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 11Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar).Besides its enzymatic activity, CD26 interacts physically with membrane-bound signal-transduction molecules. Thus, the group of Morimoto has reported that CD26 associates to CD45, a membrane-linked protein tyrosine phosphatase (12Torimoto Y. Dang N.H. Vivier E. Tanaka T. Schlossman S.F. Morimoto C. J. Immunol. 1991; 147: 2514-2517PubMed Google Scholar) and adenosine deaminase (ADA; Ref.13Kameoka J. Tanaka T. Nojima Y. Schlossman S.F. Morimoto C. Science. 1993; 261: 466-469Crossref PubMed Scopus (448) Google Scholar), which is a cytosolic globular protein that also appears on the surface of lymphocytes (14Arán J.M. Colomer D. Matutes E. Vives-Corrons J.L. Franco R. J. Histochem. Cytochem. 1991; 39: 1001-1008Crossref PubMed Scopus (62) Google Scholar). It has been shown that the binding of iodinated ADA to CD26 is inhibited by HIV-1 particles and by the viral envelope glycoprotein gp120, with IC50 values in the nanomolar range. This inhibition is mediated by the C3 region of the gp120 protein and occurs in human T and B cell lines by a mechanism modulated by CD4 and CXCR4 expression (15Valenzuela A. Blanco J. Callebaut C. Jacotot E. Lluis C. Hovanessian A.G. Franco R. J. Immunol. 1997; 158: 3721-3729PubMed Google Scholar, 16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar).Given the functional relationship between dipeptidyl peptidase IV activity of CD26 and CXCR4, and the role of CXCR4 in gp120-mediated inhibition of ADA binding to CD26, we have studied the distribution of these proteins on the surface of human lymphocytes. In this paper we present evidence of a colocalization, coimmunoprecipitation, and comodulation of CD26 and CXCR4. On the other hand, there is a redistribution of these cell surface proteins by the action of gp120 or SDF-1α, which is the natural ligand of CXCR4. gp120-induced redistribution of CXCR4 and CD26 also affects cell surface ADA.DISCUSSIONIn this paper we demonstrate for the first time that CD26 and CXCR4 interact in primary lymphocytes from human blood and also in T and B cell lines. The CXCR4 natural ligand SDF-1α has the conserved NH2-X-Pro sequence (where X is any amino acid) at the NH2 terminus, which is a consensus sequence for substrates of dipeptidyl peptidase IV activity of CD26. It has been demonstrated that SDF-1α can be processed by CD26 (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 9Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 11Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar,32Iwata S. Yamaguchi N. Munakata Y. Ikushima H. Lee J.F. Hosono O. Schlossman S.F. Morimoto C. Int. Immunol. 1999; 11: 417-426Crossref PubMed Scopus (88) Google Scholar). Therefore the CD26·CXCR4 complex is likely a functional unit in which the expression of CD26 on the cell surface may modulate SDF-1α-induced chemotaxis. Since SDF-1α is a natural antiviral agent (33Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1748) Google Scholar, 34Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.-L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1476) Google Scholar), the expression of CD26 can also modulate the activity of this substrate of CXCR4 against T-tropic strains of HIV.The fact that SDF-1α induces the cointernalization of CXCR4 and CD26 in all cells tested (Figs. 3 and 4) is also relevant. The SDF-1α-induced internalization of the CD26/CXCR4 module was not CD4-dependent, as we could detect that the level of expression of both molecules on the cell surface decreased with SDF-1α incubation in T, but also in B, cells. The specificity and physiological relevance of CD26/CXCR4 cointernalization was demonstrated by the lack of internalization in the presence of the antagonist P2G in primary lymphocytes and in cell lines. The cells expressing a chemokine receptor lacking the COOH-terminal part were also a suitable model to assess the relevance of the interaction. In fact, the lack of the cytoplasmic tail, which is known to prevent SDF-1α−induced down-regulation of CXCR4, also prevents internalization of CD26. These results indicate that treatment of lymphocytes with chemokines or agents that activate protein kinase C leads to the simultaneous internalization of CXCR4 and CD26, probably via the same endocytic pathway, as we have demonstrated for other interacting cell surface proteins (35Saura C.A. Mallol J. Canela E.I. Lluis C. Franco R. J. Biol. Chem. 1998; 273: 17610-17617Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). SDF-1α-induced internalization of CXCR4 is mediated by coated vesicles (17Amara A. Le Gall S. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar). According to this, the blockade of the cointernalization by sucrose and by acetic acid further suggests that endocytosis of CXCR4 and CD26 follows the same endocytic pathway mediated by coated vesicles. Interestingly, after a long period of chemokine treatment, the codistribution of the two proteins inside the cell is lost. At 1 h of treatment CXCR4 is found homogeneously distributed near the plasma membrane (Fig. 3), which would fit with the rapid recycling reported for this chemokine receptor. In contrast, internalized CD26 is clustered in intracellular vesicles, thus confirming that the traffic of the two proteins after the cointernalization follows different routes. As reported previously, ligand-induced internalization of CXCR4 is not mediated by Gi proteins (17Amara A. Le Gall S. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar). The assays performed in the presence of pertussis toxin indicate that blocking of G-protein-mediated signaling does not prevent the cointernalization. This interesting finding is evidence for a Gi protein-independent signaling pathway that regulates the SDF-1α-induced simultaneous internalization of the two proteins. To our knowledge this is the second example of ligand-induced cointernalization of a receptor and the enzyme that inactivates the ligand for the receptor (35Saura C.A. Mallol J. Canela E.I. Lluis C. Franco R. J. Biol. Chem. 1998; 273: 17610-17617Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In these models of interacting proteins, the existence of membrane-bound and soluble forms of degrading enzymes is relevant. Thus, in addition to membrane-bound enzyme, soluble forms of CD26 are able to inactivate SDF-1α (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 7Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar). While the soluble enzyme could regulate circulating concentrations of SDF-1, the cell-bound enzyme should be responsible for the control of local changes in ligand concentration. Cointernalization of CD26 with CXCR4 might represent a second mechanism of control of CXCR4 signaling. Consistent with the results shown here, it has been reported that peripheral blood lymphocytes migrating toward SDF-1α show low expression of CXCR4 and CD26. This finding can be a consequence of a higher response to SDF-1α from CD26lowcells, but it could reflect a SDF-1 α-induced cointernalization of CD26 and CXCR4 (see Fig. 3).Iyengar et al. (36Iyengar S. Hildreth J.E.K. Schwartz D.H. J. Virol. 1998; 72: 5251-5255Crossref PubMed Google Scholar) have described that in peripheral blood mononuclear cells the addition of gp120 cause cocapping of CD4 and CXCR4 with subsequent pseudopodium formation. On the other hand, Feitoet al. (37Feito M.J. Bragardo M. Buonfiglio D. Bonissoni S. Bottarel F. Malavasi F. Dianzani U. Int. Immunol. 1997; 9: 1141-1147Crossref PubMed Scopus (18) Google Scholar) described that gp120 from the syncytium-induced HIV-1 strain increased the cocapping of CD4 with some proteins, CD26 included. We here demonstrate that CD26·CXCR4 complexes are coredistributed into gp120-induced pseudopodia. Taken together, these results suggest that the viral envelope glycoprotein gp120 induces the formation in T lymphocytes of pseudopodia in which the main virus receptor, the CD4, the coreceptor CXCR4, and the CD26 coexist.Although the importance of CD4 and chemokine receptors in the process of HIV entry has been extensively described, the role of CD26 in this process is still not well understood. Previous results of our laboratory (15Valenzuela A. Blanco J. Callebaut C. Jacotot E. Lluis C. Hovanessian A.G. Franco R. J. Immunol. 1997; 158: 3721-3729PubMed Google Scholar) described the inhibition of the binding of125I-labeled ADA to the CD26 by HIV-1 envelope gp120 and viral particles, suggesting that gp120 recognizes the ADA binding site of CD26. gp120 blocks binding of ADA to CD26 in a CXCR4-dependent manner (16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar). It is possible that gp120 displaces ADA binding to CD26 in CD4-negative cells because of the presence of CXCR4. These results support the hypothesis (see Ref. 16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar) that gp120 has to bind to CXCR4 prior to its interaction with CD26 and the disruption of the ADA/CD26 interaction. In this report we confirm the gp120-induced inhibition of ADA binding to CD26, but we also demonstrate that gp120 provokes the release of endogenous ADA bound to CD26, and this happens without changes in the expression of cell surface CD26 (Fig. 11). These data would suggest that gp120 and ADA share a common binding site in CD26. As shown in Fig.9 B, when the FITC-conjugated gp120 incubation was done using nonfixed cells, the viral glycoprotein could produce some membrane protein rearrangements displacing cell surface ADA from CD26. However, when cells were fixed before the incubation with gp120, a marked colocalization between the viral glycoprotein and the cell surface ADA was observed (Fig. 9 A). It should be noted that fixation avoids any cell membrane protein mobility or redistribution. These results led to hypothesize the existence of a gp120 binding domain in CD26 distinct from the ADA binding site. This indicates that the gp120-mediated inability of ADA to bind to CD26 would be a consequence of a first interaction of gp120 with a distinct epitope in CD26. We detected two anti-CD26 mAb, 4H12 and 202.36p, raised against a different epitope from ADA binding center that decrease the binding degree of gp120. Both antibodies are, also, less effective in labeling CD26 when cells are incubated with the viral glycoprotein gp120. These results correlate with the hypothesis of an interaction of gp120 with CD26 at an epitope different from the ADA binding domain, and they also indicate a recognition of cell surface CD26 by the viral envelope protein gp120.Comodulation of CXCR4 and CD26 by SDF-1α, gp120-induced formation of pseudopodia in which ADA is not present, and the role of ADA in the development and function of the immune system (38Franco R. Valenzuela A. Lluis C. Blanco J. Immunol. Rev. 1998; 161: 27-42Crossref PubMed Scopus (155) Google Scholar) suggest that the ADA·CD26 and the CXCR4·CD26·ADA complex is important for the functionality of human lymphocytes. CD26 is a widely distributed lymphocyte activation antigen that displays dipeptidyl peptidase IV activity in its extracellular domain. The role of CD26 in HIV1infection has been controversial. Its postulated role as coreceptor for HIV entry (1Callebaut C. Krust B. Jacotot E. Hovanessian A.G. Science. 1993; 262: 2045-2050Crossref PubMed Scopus (207) Google Scholar) was rapidly contested (2Broder C.C. Nussbaum O. Gutheil W.G. Bachovchin W.W. Berger E.A. Science. 1994; 264: 1156-1165Crossref PubMed Scopus (48) Google Scholar, 3Morimoto C. Lord C.I. Zhang C. Duke-Cohan J.S. Letvin N.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9960-9964Crossref PubMed Scopus (73) Google Scholar). However, it has been more recently shown that the expression of CD26 may modulate HIV infection by direct effects of dipeptidyl peptidase IV activity on the antiviral activity of several chemokines or by unknown mechanisms independent of peptidase activity (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 5Oravecz T. Roderiquez G. Koffi J. Wang J. Ditto M. Bou-Habib D.C. Lusso P. Norcross M.A. Nat. Med. 1995; 1: 919-926Crossref PubMed Scopus (61) Google Scholar, 6Callebaut C. Jacotot E. Blanco J. Krust B. Hovanessian A.G. Exp. Cell Res. 1998; 241: 352-362Crossref PubMed Scopus (22) Google Scholar, 7Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar). In fact, CD26 can cleave out NH2-terminal dipeptides from a protein with either l-proline or l-alanine at the penultimate position and several chemokines can be processed by CD26 (see Ref. 8De Meester I. Korom S. Van Damme J. Scharpé S. Immunol. Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar for review). For some chemokines proteolysis by CD26 does not affect the physiological activity. In contrast, truncation of RANTES (regulated on activation normal T cell expressed and secreted) by CD26 leads to higher receptor selectivity and anti-HIV activity (9Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar,10Proost P. de Meester I. Schols D. Struyf S. Lambeir A.-M. Wuyts A. Opdenakker G. De Clercq E. Scharpé S. van Damme J. J. Biol. Chem. 1998; 273: 7222-7227Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Stromal cell-derived factor 1α (SDF-1α) also loses the NH2-terminal Lys-Pro dipeptide by the action of CD26. This processing significantly reduces the chemotactic and anti-HIV-1 activity of SDF-1α (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 11Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar). Besides its enzymatic activity, CD26 interacts physically with membrane-bound signal-transduction molecules. Thus, the group of Morimoto has reported that CD26 associates to CD45, a membrane-linked protein tyrosine phosphatase (12Torimoto Y. Dang N.H. Vivier E. Tanaka T. Schlossman S.F. Morimoto C. J. Immunol. 1991; 147: 2514-2517PubMed Google Scholar) and adenosine deaminase (ADA; Ref.13Kameoka J. Tanaka T. Nojima Y. Schlossman S.F. Morimoto C. Science. 1993; 261: 466-469Crossref PubMed Scopus (448) Google Scholar), which is a cytosolic globular protein that also appears on the surface of lymphocytes (14Arán J.M. Colomer D. Matutes E. Vives-Corrons J.L. Franco R. J. Histochem. Cytochem. 1991; 39: 1001-1008Crossref PubMed Scopus (62) Google Scholar). It has been shown that the binding of iodinated ADA to CD26 is inhibited by HIV-1 particles and by the viral envelope glycoprotein gp120, with IC50 values in the nanomolar range. This inhibition is mediated by the C3 region of the gp120 protein and occurs in human T and B cell lines by a mechanism modulated by CD4 and CXCR4 expression (15Valenzuela A. Blanco J. Callebaut C. Jacotot E. Lluis C. Hovanessian A.G. Franco R. J. Immunol. 1997; 158: 3721-3729PubMed Google Scholar, 16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar). Given the functional relationship between dipeptidyl peptidase IV activity of CD26 and CXCR4, and the role of CXCR4 in gp120-mediated inhibition of ADA binding to CD26, we have studied the distribution of these proteins on the surface of human lymphocytes. In this paper we present evidence of a colocalization, coimmunoprecipitation, and comodulation of CD26 and CXCR4. On the other hand, there is a redistribution of these cell surface proteins by the action of gp120 or SDF-1α, which is the natural ligand of CXCR4. gp120-induced redistribution of CXCR4 and CD26 also affects cell surface ADA. DISCUSSIONIn this paper we demonstrate for the first time that CD26 and CXCR4 interact in primary lymphocytes from human blood and also in T and B cell lines. The CXCR4 natural ligand SDF-1α has the conserved NH2-X-Pro sequence (where X is any amino acid) at the NH2 terminus, which is a consensus sequence for substrates of dipeptidyl peptidase IV activity of CD26. It has been demonstrated that SDF-1α can be processed by CD26 (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 9Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 11Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar,32Iwata S. Yamaguchi N. Munakata Y. Ikushima H. Lee J.F. Hosono O. Schlossman S.F. Morimoto C. Int. Immunol. 1999; 11: 417-426Crossref PubMed Scopus (88) Google Scholar). Therefore the CD26·CXCR4 complex is likely a functional unit in which the expression of CD26 on the cell surface may modulate SDF-1α-induced chemotaxis. Since SDF-1α is a natural antiviral agent (33Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1748) Google Scholar, 34Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.-L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1476) Google Scholar), the expression of CD26 can also modulate the activity of this substrate of CXCR4 against T-tropic strains of HIV.The fact that SDF-1α induces the cointernalization of CXCR4 and CD26 in all cells tested (Figs. 3 and 4) is also relevant. The SDF-1α-induced internalization of the CD26/CXCR4 module was not CD4-dependent, as we could detect that the level of expression of both molecules on the cell surface decreased with SDF-1α incubation in T, but also in B, cells. The specificity and physiological relevance of CD26/CXCR4 cointernalization was demonstrated by the lack of internalization in the presence of the antagonist P2G in primary lymphocytes and in cell lines. The cells expressing a chemokine receptor lacking the COOH-terminal part were also a suitable model to assess the relevance of the interaction. In fact, the lack of the cytoplasmic tail, which is known to prevent SDF-1α−induced down-regulation of CXCR4, also prevents internalization of CD26. These results indicate that treatment of lymphocytes with chemokines or agents that activate protein kinase C leads to the simultaneous internalization of CXCR4 and CD26, probably via the same endocytic pathway, as we have demonstrated for other interacting cell surface proteins (35Saura C.A. Mallol J. Canela E.I. Lluis C. Franco R. J. Biol. Chem. 1998; 273: 17610-17617Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). SDF-1α-induced internalization of CXCR4 is mediated by coated vesicles (17Amara A. Le Gall S. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar). According to this, the blockade of the cointernalization by sucrose and by acetic acid further suggests that endocytosis of CXCR4 and CD26 follows the same endocytic pathway mediated by coated vesicles. Interestingly, after a long period of chemokine treatment, the codistribution of the two proteins inside the cell is lost. At 1 h of treatment CXCR4 is found homogeneously distributed near the plasma membrane (Fig. 3), which would fit with the rapid recycling reported for this chemokine receptor. In contrast, internalized CD26 is clustered in intracellular vesicles, thus confirming that the traffic of the two proteins after the cointernalization follows different routes. As reported previously, ligand-induced internalization of CXCR4 is not mediated by Gi proteins (17Amara A. Le Gall S. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar). The assays performed in the presence of pertussis toxin indicate that blocking of G-protein-mediated signaling does not prevent the cointernalization. This interesting finding is evidence for a Gi protein-independent signaling pathway that regulates the SDF-1α-induced simultaneous internalization of the two proteins. To our knowledge this is the second example of ligand-induced cointernalization of a receptor and the enzyme that inactivates the ligand for the receptor (35Saura C.A. Mallol J. Canela E.I. Lluis C. Franco R. J. Biol. Chem. 1998; 273: 17610-17617Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In these models of interacting proteins, the existence of membrane-bound and soluble forms of degrading enzymes is relevant. Thus, in addition to membrane-bound enzyme, soluble forms of CD26 are able to inactivate SDF-1α (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 7Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar). While the soluble enzyme could regulate circulating concentrations of SDF-1, the cell-bound enzyme should be responsible for the control of local changes in ligand concentration. Cointernalization of CD26 with CXCR4 might represent a second mechanism of control of CXCR4 signaling. Consistent with the results shown here, it has been reported that peripheral blood lymphocytes migrating toward SDF-1α show low expression of CXCR4 and CD26. This finding can be a consequence of a higher response to SDF-1α from CD26lowcells, but it could reflect a SDF-1 α-induced cointernalization of CD26 and CXCR4 (see Fig. 3).Iyengar et al. (36Iyengar S. Hildreth J.E.K. Schwartz D.H. J. Virol. 1998; 72: 5251-5255Crossref PubMed Google Scholar) have described that in peripheral blood mononuclear cells the addition of gp120 cause cocapping of CD4 and CXCR4 with subsequent pseudopodium formation. On the other hand, Feitoet al. (37Feito M.J. Bragardo M. Buonfiglio D. Bonissoni S. Bottarel F. Malavasi F. Dianzani U. Int. Immunol. 1997; 9: 1141-1147Crossref PubMed Scopus (18) Google Scholar) described that gp120 from the syncytium-induced HIV-1 strain increased the cocapping of CD4 with some proteins, CD26 included. We here demonstrate that CD26·CXCR4 complexes are coredistributed into gp120-induced pseudopodia. Taken together, these results suggest that the viral envelope glycoprotein gp120 induces the formation in T lymphocytes of pseudopodia in which the main virus receptor, the CD4, the coreceptor CXCR4, and the CD26 coexist.Although the importance of CD4 and chemokine receptors in the process of HIV entry has been extensively described, the role of CD26 in this process is still not well understood. Previous results of our laboratory (15Valenzuela A. Blanco J. Callebaut C. Jacotot E. Lluis C. Hovanessian A.G. Franco R. J. Immunol. 1997; 158: 3721-3729PubMed Google Scholar) described the inhibition of the binding of125I-labeled ADA to the CD26 by HIV-1 envelope gp120 and viral particles, suggesting that gp120 recognizes the ADA binding site of CD26. gp120 blocks binding of ADA to CD26 in a CXCR4-dependent manner (16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar). It is possible that gp120 displaces ADA binding to CD26 in CD4-negative cells because of the presence of CXCR4. These results support the hypothesis (see Ref. 16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar) that gp120 has to bind to CXCR4 prior to its interaction with CD26 and the disruption of the ADA/CD26 interaction. In this report we confirm the gp120-induced inhibition of ADA binding to CD26, but we also demonstrate that gp120 provokes the release of endogenous ADA bound to CD26, and this happens without changes in the expression of cell surface CD26 (Fig. 11). These data would suggest that gp120 and ADA share a common binding site in CD26. As shown in Fig.9 B, when the FITC-conjugated gp120 incubation was done using nonfixed cells, the viral glycoprotein could produce some membrane protein rearrangements displacing cell surface ADA from CD26. However, when cells were fixed before the incubation with gp120, a marked colocalization between the viral glycoprotein and the cell surface ADA was observed (Fig. 9 A). It should be noted that fixation avoids any cell membrane protein mobility or redistribution. These results led to hypothesize the existence of a gp120 binding domain in CD26 distinct from the ADA binding site. This indicates that the gp120-mediated inability of ADA to bind to CD26 would be a consequence of a first interaction of gp120 with a distinct epitope in CD26. We detected two anti-CD26 mAb, 4H12 and 202.36p, raised against a different epitope from ADA binding center that decrease the binding degree of gp120. Both antibodies are, also, less effective in labeling CD26 when cells are incubated with the viral glycoprotein gp120. These results correlate with the hypothesis of an interaction of gp120 with CD26 at an epitope different from the ADA binding domain, and they also indicate a recognition of cell surface CD26 by the viral envelope protein gp120.Comodulation of CXCR4 and CD26 by SDF-1α, gp120-induced formation of pseudopodia in which ADA is not present, and the role of ADA in the development and function of the immune system (38Franco R. Valenzuela A. Lluis C. Blanco J. Immunol. Rev. 1998; 161: 27-42Crossref PubMed Scopus (155) Google Scholar) suggest that the ADA·CD26 and the CXCR4·CD26·ADA complex is important for the functionality of human lymphocytes. In this paper we demonstrate for the first time that CD26 and CXCR4 interact in primary lymphocytes from human blood and also in T and B cell lines. The CXCR4 natural ligand SDF-1α has the conserved NH2-X-Pro sequence (where X is any amino acid) at the NH2 terminus, which is a consensus sequence for substrates of dipeptidyl peptidase IV activity of CD26. It has been demonstrated that SDF-1α can be processed by CD26 (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 9Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 11Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar,32Iwata S. Yamaguchi N. Munakata Y. Ikushima H. Lee J.F. Hosono O. Schlossman S.F. Morimoto C. Int. Immunol. 1999; 11: 417-426Crossref PubMed Scopus (88) Google Scholar). Therefore the CD26·CXCR4 complex is likely a functional unit in which the expression of CD26 on the cell surface may modulate SDF-1α-induced chemotaxis. Since SDF-1α is a natural antiviral agent (33Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1748) Google Scholar, 34Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.-L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1476) Google Scholar), the expression of CD26 can also modulate the activity of this substrate of CXCR4 against T-tropic strains of HIV. The fact that SDF-1α induces the cointernalization of CXCR4 and CD26 in all cells tested (Figs. 3 and 4) is also relevant. The SDF-1α-induced internalization of the CD26/CXCR4 module was not CD4-dependent, as we could detect that the level of expression of both molecules on the cell surface decreased with SDF-1α incubation in T, but also in B, cells. The specificity and physiological relevance of CD26/CXCR4 cointernalization was demonstrated by the lack of internalization in the presence of the antagonist P2G in primary lymphocytes and in cell lines. The cells expressing a chemokine receptor lacking the COOH-terminal part were also a suitable model to assess the relevance of the interaction. In fact, the lack of the cytoplasmic tail, which is known to prevent SDF-1α−induced down-regulation of CXCR4, also prevents internalization of CD26. These results indicate that treatment of lymphocytes with chemokines or agents that activate protein kinase C leads to the simultaneous internalization of CXCR4 and CD26, probably via the same endocytic pathway, as we have demonstrated for other interacting cell surface proteins (35Saura C.A. Mallol J. Canela E.I. Lluis C. Franco R. J. Biol. Chem. 1998; 273: 17610-17617Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). SDF-1α-induced internalization of CXCR4 is mediated by coated vesicles (17Amara A. Le Gall S. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar). According to this, the blockade of the cointernalization by sucrose and by acetic acid further suggests that endocytosis of CXCR4 and CD26 follows the same endocytic pathway mediated by coated vesicles. Interestingly, after a long period of chemokine treatment, the codistribution of the two proteins inside the cell is lost. At 1 h of treatment CXCR4 is found homogeneously distributed near the plasma membrane (Fig. 3), which would fit with the rapid recycling reported for this chemokine receptor. In contrast, internalized CD26 is clustered in intracellular vesicles, thus confirming that the traffic of the two proteins after the cointernalization follows different routes. As reported previously, ligand-induced internalization of CXCR4 is not mediated by Gi proteins (17Amara A. Le Gall S. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar). The assays performed in the presence of pertussis toxin indicate that blocking of G-protein-mediated signaling does not prevent the cointernalization. This interesting finding is evidence for a Gi protein-independent signaling pathway that regulates the SDF-1α-induced simultaneous internalization of the two proteins. To our knowledge this is the second example of ligand-induced cointernalization of a receptor and the enzyme that inactivates the ligand for the receptor (35Saura C.A. Mallol J. Canela E.I. Lluis C. Franco R. J. Biol. Chem. 1998; 273: 17610-17617Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In these models of interacting proteins, the existence of membrane-bound and soluble forms of degrading enzymes is relevant. Thus, in addition to membrane-bound enzyme, soluble forms of CD26 are able to inactivate SDF-1α (4Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P. De Clercq E. De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 7Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar). While the soluble enzyme could regulate circulating concentrations of SDF-1, the cell-bound enzyme should be responsible for the control of local changes in ligand concentration. Cointernalization of CD26 with CXCR4 might represent a second mechanism of control of CXCR4 signaling. Consistent with the results shown here, it has been reported that peripheral blood lymphocytes migrating toward SDF-1α show low expression of CXCR4 and CD26. This finding can be a consequence of a higher response to SDF-1α from CD26lowcells, but it could reflect a SDF-1 α-induced cointernalization of CD26 and CXCR4 (see Fig. 3). Iyengar et al. (36Iyengar S. Hildreth J.E.K. Schwartz D.H. J. Virol. 1998; 72: 5251-5255Crossref PubMed Google Scholar) have described that in peripheral blood mononuclear cells the addition of gp120 cause cocapping of CD4 and CXCR4 with subsequent pseudopodium formation. On the other hand, Feitoet al. (37Feito M.J. Bragardo M. Buonfiglio D. Bonissoni S. Bottarel F. Malavasi F. Dianzani U. Int. Immunol. 1997; 9: 1141-1147Crossref PubMed Scopus (18) Google Scholar) described that gp120 from the syncytium-induced HIV-1 strain increased the cocapping of CD4 with some proteins, CD26 included. We here demonstrate that CD26·CXCR4 complexes are coredistributed into gp120-induced pseudopodia. Taken together, these results suggest that the viral envelope glycoprotein gp120 induces the formation in T lymphocytes of pseudopodia in which the main virus receptor, the CD4, the coreceptor CXCR4, and the CD26 coexist. Although the importance of CD4 and chemokine receptors in the process of HIV entry has been extensively described, the role of CD26 in this process is still not well understood. Previous results of our laboratory (15Valenzuela A. Blanco J. Callebaut C. Jacotot E. Lluis C. Hovanessian A.G. Franco R. J. Immunol. 1997; 158: 3721-3729PubMed Google Scholar) described the inhibition of the binding of125I-labeled ADA to the CD26 by HIV-1 envelope gp120 and viral particles, suggesting that gp120 recognizes the ADA binding site of CD26. gp120 blocks binding of ADA to CD26 in a CXCR4-dependent manner (16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar). It is possible that gp120 displaces ADA binding to CD26 in CD4-negative cells because of the presence of CXCR4. These results support the hypothesis (see Ref. 16Blanco J. Valenzuela A. Herrera C. Lluis C. Hovanessian A.G. Franco R. FEBS Lett. 2000; 477: 123-128Crossref PubMed Scopus (30) Google Scholar) that gp120 has to bind to CXCR4 prior to its interaction with CD26 and the disruption of the ADA/CD26 interaction. In this report we confirm the gp120-induced inhibition of ADA binding to CD26, but we also demonstrate that gp120 provokes the release of endogenous ADA bound to CD26, and this happens without changes in the expression of cell surface CD26 (Fig. 11). These data would suggest that gp120 and ADA share a common binding site in CD26. As shown in Fig.9 B, when the FITC-conjugated gp120 incubation was done using nonfixed cells, the viral glycoprotein could produce some membrane protein rearrangements displacing cell surface ADA from CD26. However, when cells were fixed before the incubation with gp120, a marked colocalization between the viral glycoprotein and the cell surface ADA was observed (Fig. 9 A). It should be noted that fixation avoids any cell membrane protein mobility or redistribution. These results led to hypothesize the existence of a gp120 binding domain in CD26 distinct from the ADA binding site. This indicates that the gp120-mediated inability of ADA to bind to CD26 would be a consequence of a first interaction of gp120 with a distinct epitope in CD26. We detected two anti-CD26 mAb, 4H12 and 202.36p, raised against a different epitope from ADA binding center that decrease the binding degree of gp120. Both antibodies are, also, less effective in labeling CD26 when cells are incubated with the viral glycoprotein gp120. These results correlate with the hypothesis of an interaction of gp120 with CD26 at an epitope different from the ADA binding domain, and they also indicate a recognition of cell surface CD26 by the viral envelope protein gp120. Comodulation of CXCR4 and CD26 by SDF-1α, gp120-induced formation of pseudopodia in which ADA is not present, and the role of ADA in the development and function of the immune system (38Franco R. Valenzuela A. Lluis C. Blanco J. Immunol. Rev. 1998; 161: 27-42Crossref PubMed Scopus (155) Google Scholar) suggest that the ADA·CD26 and the CXCR4·CD26·ADA complex is important for the functionality of human lymphocytes. We acknowledge the technical help received from Jaume Comas and Ma del Rosario González (flow cytometry) and from Susana Castel (confocal microscopy section).