Soluble CD40L activates soluble and cell-surface integrin αvβ3, α5β1, and α4β1 by binding to the allosteric ligand-binding site (site 2)
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100399
ISSN1083-351X
AutoresYoko K. Takada, Michiko Shimoda, Emanual Maverakis, Brunie H. Felding, R. Holland Cheng, Yoshikazu Takada,
Tópico(s)T-cell and B-cell Immunology
ResumoCD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants. CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants. CD40L is a type II protein ligand member of the tumor necrosis factor (TNF) superfamily expressed by activated T cells. CD40L is a costimulatory molecule critical in a variety of T cell–antigen presenting cell (APC) interactions, including activating APCs to provide help to cytotoxic T cells (1Schoenberger S.P. Toes R.E. van der Voort E.I. Offringa R. Melief C.J. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.Nature. 1998; 393: 480-483Crossref PubMed Scopus (2087) Google Scholar). It also provides help to B cell to promote class switching (2Korthauer U. Graf D. Mages H.W. Briere F. Padayachee M. Malcolm S. Ugazio A.G. Notarangelo L.D. Levinsky R.J. Kroczek R.A. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM.Nature. 1993; 361: 539-541Crossref PubMed Scopus (628) Google Scholar, 3Schonbeck U. Libby P. The CD40/CD154 receptor/ligand dyad.Cell Mol. Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar). In addition to its transmembrane form, CD40L is also released as a soluble ligand (sCD40L) by proteolytic cleavage, allowing it to interact at more distant sites. CD40L appears to modulate other cell types as well (4Chatzigeorgiou A. Lyberi M. Chatzilymperis G. Nezos A. Kamper E. CD40/CD40L signaling and its implication in health and disease.Biofactors. 2009; 35: 474-483Crossref PubMed Scopus (114) Google Scholar). CD40L can initiate inflammatory and procoagulatory responses in vascular endothelial cells (5Cha J.K. Jeong M.H. Bae H.R. Han J.Y. Jeong S.J. Jin H.J. Lim Y.J. Kim S.H. Kim J.W. Activated platelets induce secretion of interleukin-1beta, monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha and surface expression of intercellular adhesion molecule-1 on cultured endothelial cells.J. Korean Med. Sci. 2000; 15: 273-278Crossref PubMed Scopus (29) Google Scholar, 6Daub K. Langer H. Seizer P. Stellos K. May A.E. Goyal P. Bigalke B. Schonberger T. Geisler T. Siegel-Axel D. Oostendorp R.A. Lindemann S. Gawaz M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells.FASEB J. 2006; 20: 2559-2561Crossref PubMed Scopus (161) Google Scholar, 7Henn V. Slupsky J.R. Grafe M. Anagnostopoulos I. Forster R. Muller-Berghaus G. Kroczek R.A. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.Nature. 1998; 391: 591-594Crossref PubMed Scopus (1660) Google Scholar). Findings such as these have led to the belief that CD40–CD40L interactions play a more general role in immune regulation. Given its diverse functions, it is not surprising that CD40L is critical in a variety of chronic autoimmune and inflammatory diseases, including systemic lupus erythematosus (SLE), diabetes, chronic kidney disease (8Crow M.K. Kirou K.A. Regulation of CD40 ligand expression in systemic lupus erythematosus.Curr. Opin. Rheumatol. 2001; 13: 361-369Crossref PubMed Scopus (54) Google Scholar, 9Delmas Y. Viallard J.F. Solanilla A. Villeneuve J. Pasquet J.M. Belloc F. Dubus I. Dechanet-Merville J. Merville P. Blanco P. Pellegrin J.L. Nurden A.T. Combe C. Ripoche J. Activation of mesangial cells by platelets in systemic lupus erythematosus via a CD154-dependent induction of CD40.Kidney Int. 2005; 68: 2068-2078Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), among others. CD40L functions through its interactions with cell surface proteins CD40, and α5β1 and αIIbβ3 integrins. CD40 belongs to the tumor necrosis factor receptor (TNF-R) family and was first identified and functionally characterized on B lymphocytes (10Grewal I.S. Flavell R.A. CD40 and CD154 in cell-mediated immunity.Annu. Rev. Immunol. 1998; 16: 111-135Crossref PubMed Scopus (1288) Google Scholar). CD40–CD40L interactions play a more general role in immune regulation and stabilize arterial thrombi through binding to integrin αIIbβ3 (11Andre P. Prasad K.S. Denis C.V. He M. Papalia J.M. Hynes R.O. Phillips D.R. Wagner D.D. CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism.Nat. Med. 2002; 8: 247-252Crossref PubMed Scopus (626) Google Scholar). αIIbβ3 recognizes the KGD motif at the N terminus of CD40L (residues 115–117 of CD40L). It has also been reported that CD40L binds to integrin α5β1 and transduces signals through this integrin in a CD40 and αIIbβ3-independent manner. Finally, data suggests that CD40 and integrin α5β1 can bind to CD40L simultaneously (12El Fakhry Y. Alturaihi H. Yacoub D. Liu L. Guo W. Leveille C. Jung D. Khzam L.B. Merhi Y. Wilkins J.A. Li H. Mourad W. Functional interaction of CD154 protein with alpha5beta1 integrin is totally independent from its binding to alphaIIbbeta3 integrin and CD40 molecules.J. Biol. Chem. 2012; 287: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We recently identified vascular integrin αvβ3 as a new receptor for CD40L (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). We localized the α5β1 and αvβ3 binding to the trimeric interface of CD40L. CD40L mutants defective in integrin binding in the predicted integrin-binding site were defective in CD40L/CD40 signaling and acted as antagonists of CD40L/CD40 signaling (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). Furthermore, we demonstrated that CD40L binding to αvβ3 activates αvβ3 in an allosteric manner. Of relevance to our discovery is the finding that eight X-Linked Hyper IgM Syndrome (HIGM1)-causative variants have alterations in the CD40L integrin binding, and they are defective in integrin binding and signaling, suggesting that the loss of integrin binding is related to the defect in CD40L signaling in HIGM1. Also, our previous studies found that several proinflammatory integrin ligands (e.g., CX3CL1, CXCL12, and secreted phospholipase A2 type IIA (sPLA-IIA)) activated integrins by binding to a second ligand-binding site (site 2) in an allosteric manner in addition to binding to a primary site (site 1) (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar, 15Fujita M. Zhu K. Fujita C.K. Zhao M. Lam K.S. Kurth M.J. Takada Y.K. Takada Y. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins alphavbeta3, alpha4beta1, and alpha5beta1.J. Biol. Chem. 2015; 290: 259-271Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16Fujita M. Davari P. Takada Y.K. Takada Y. Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.Biochem. J. 2018; 475: 723-732Crossref PubMed Scopus (9) Google Scholar). The recent discovery on the binding of 25-hydroxycholesterol to integrin site 2 upregulates inflammatory cytokines, TNF, and IL-6, production (17Pokharel S.M. Shil N.K. Gc J.B. Colburn Z.T. Tsai S.Y. Segovia J.A. Chang T.H. Bandyopadhyay S. Natesan S. Jones J.C.R. Bose S. Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response.Nat. Commun. 2019; 10: 1482Crossref PubMed Scopus (18) Google Scholar) indicates that site-2-mediated integrin activation is involved in proinflammatory signaling. Herein, we now present data that integrin α4β1 is a new receptor for CD40L. Notably, we showed that CD40L is an allosteric activator of integrins αvβ3, α5β1, and α4β1. Specifically, we demonstrate that CD40L can bind to an allosteric ligand-binding site (Site 2) (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar, 15Fujita M. Zhu K. Fujita C.K. Zhao M. Lam K.S. Kurth M.J. Takada Y.K. Takada Y. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins alphavbeta3, alpha4beta1, and alpha5beta1.J. Biol. Chem. 2015; 290: 259-271Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16Fujita M. Davari P. Takada Y.K. Takada Y. Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.Biochem. J. 2018; 475: 723-732Crossref PubMed Scopus (9) Google Scholar), which is distinct from αvβ3's classical ligand-binding site (site 1). Also, four HIGM1 mutants are clustered in the site-2-binding site of CD40L, indicating that site-2-binding is potentially involved in CD40L/CD40 signaling. We propose that CD40L acts as a ligand and an allosteric activator of several integrins and mediates proinflammatory signaling independent of CD40. We further propose that this may be a common mechanism for a group of proinflammatory proteins and that CD40L-induced integrin activation is required for CD40L signaling. Previous studies showed that several proinflammatory integrin ligands activated integrins in an allosteric manner (see Introductions). We hypothesized that another proinflammatory cytokine CD40L activates integrins. Previous studies showed that soluble integrin αvβ3 bound to many ligands including the fibrinogen γ-chain C-terminal domain with truncation at the C terminus (γC399tr) (See Introduction). γC399tr specifically binds to site 1 of αvβ3, but not to site 2 (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar). We studied if sCD40L enhances the binding of soluble αvβ3 to γC399tr. γC399tr was immobilized and incubated with soluble αvβ3 in the presence of 1 mM Ca2+ to keep αvβ3 inactive (Fig. 1A). sCD40L enhanced binding of soluble αvβ3 to immobilized γC399tr in a dose-dependent manner. Activation is defined by the increase in binding of soluble integrin αvβ3 to immobilized ligand (γC399tr) by soluble activators (sCD40L). These findings suggest that CD40L activates integrin αvβ3 in an allosteric manner. We next studied if sCD40L activates integrin αvβ3 on the cell surface using CHO cells (CD40-negative) that express recombinant αvβ3 (β3-CHO cells). β3-CHO cells were incubated with sCD40L and FITC-labeled γC399tr in the assays medium with 1 mM Ca2+ to keep αvβ3 inactive and bound FITC-labeled γC399tr was measured in flow cytometry. sCD40L markedly enhanced the binding of γC399tr, indicating that sCD40L activated αvβ3 on the cell surface (Fig. 1B). The activation of αvβ3 by sCD40L was dose-dependent (up to 50 μg/ml) and can be detected at 6 μg/ml. The binding of FITC-labeled γC399tr shown by mean fluorescent intensity (MFI) correlated well with the sCD40L concentrations (Fig. 1D). Although high concentrations of sCD40L were required to activate soluble integrins, it is likely that integrin activation by sCD40L is biologically relevant, since CD40L is a membrane-bound protein and is highly concentrated on the cell surface. We previously showed that several integrin ligands (e.g., CX3CL1, CXCL12, and sPLA2-IIA) bound to an allosteric site (site 2) and activated several integrins (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar, 15Fujita M. Zhu K. Fujita C.K. Zhao M. Lam K.S. Kurth M.J. Takada Y.K. Takada Y. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins alphavbeta3, alpha4beta1, and alpha5beta1.J. Biol. Chem. 2015; 290: 259-271Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16Fujita M. Davari P. Takada Y.K. Takada Y. Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.Biochem. J. 2018; 475: 723-732Crossref PubMed Scopus (9) Google Scholar). We performed docking simulation of interaction between αvβ3 (1JV2.pdb, with closed headpiece) and CD40L monomer (1ALY.pdb) using Autodock3. The simulation predicts that monomeric CD40L binds to site 2 well (docking energy –20.5 kcal/mol) (Fig. 1D). We previously reported that the peptide from site 2 of β3 (QPNDGQSHVGSDNHYSASTTM, residues 267–287 of β3, Cys-273 is changed to S, fused to GST) directly bound to CX3CL1, sPLA2-IIA, and CXCL12 and the peptide suppressed integrin activation by these activators, suggesting that they directly bind to site 2 and mediate integrin activation (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar, 15Fujita M. Zhu K. Fujita C.K. Zhao M. Lam K.S. Kurth M.J. Takada Y.K. Takada Y. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins alphavbeta3, alpha4beta1, and alpha5beta1.J. Biol. Chem. 2015; 290: 259-271Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16Fujita M. Davari P. Takada Y.K. Takada Y. Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.Biochem. J. 2018; 475: 723-732Crossref PubMed Scopus (9) Google Scholar). To prove if CD40L binds to site 2, we tested if CD40L binds to peptides derived from site 2. Linear site 2 peptides did not show good binding to CD40L (not shown). We thus designed disulfide-linked cyclic 28-mer peptides by introducing two Cys residues at both ends to enhance affinity and stability of the peptides. To predict the positions of the two Cys residues that do not affect the peptide conformation of the peptides we used the Disulfide by Design-2 (DbD2) software (18Craig D.B. Dombkowski A.A. Disulfide by Design 2.0: A web-based tool for disulfide engineering in proteins.BMC Bioinformatics. 2013; 14: 346Crossref PubMed Scopus (168) Google Scholar). We found that cyclic site 2 peptide from β3 (C260-RLAGIVQPNDGQSHVGSDNHYSASTTMC288) and the corresponding β1 peptide bound to sCD40L, suggesting that sCD40L binds to site 2 (Fig. 2A). The position of cyclic site 2 peptide is shown in Figure 2B. These findings suggest that sCD40L activates integrins by binding to site 2. The docking simulation predicts that four HIGM1 mutations (S128R/E129G, K143T, G144E, and L155P) are clustered in the predicted site-2-binding interface of CD40L (Fig. 2B). Amino acid residues involved in the predicted CD40L-αvβ3 (closed form) interaction are shown in Table 1. We studied if these HIGM1 mutations affect CD40L-induced αvβ3 activation. Notably, the K143T and G144E mutants were defective in activating soluble αvβ3 (Fig. 2C) and were defective in activating cell surface αvβ3 (Fig. 2D). These finding are consistent with the docking model that these mutations (K143T and G144E) are located in the predicted site-2-binding site of CD40L (Fig. 2B).Table 1Amino acid residues involved in the predicted CD40L-αvβ3 (closed form) interactionCD40LαVβ3Glu129, Ala130, Ser131, Lys133, Thr134, Thr135, Glu142, Lys143, Gly144, Tyr145, Tyr146, Leu155, Lys159, Phe177, Cys178, Ser179, Asn180, Arg181, Ala183, Ser184, Pro217, Cys218, Gln220, Pro244, Ser245, Gln246, Val247, Ser248, His249, Gly250, Thr251,Pro14, Glu15, Asn44, Thr45, Thr46, Gln47, Pro48, Gly49, Ile50, Val51, Glu52, Gln68, Gly76, Asn77, Arg78, Asp79, Ala81, Lys82, Asp83, Asp84, Pro85, Glu87, Phe88, Lys89, Ser90, His91, Arg122,Met165, Ser168, Glu171, Glu174, Asn175, Pro186, Asp278, His280, Tyr281, Ser282, Ala283, Ser284, Thr285, Thr286The amino acid residues in CD40L that are mutated in HIGM1 are in bold.The amino acid residues in β3 that are involved in site 2 peptide are underlined. Open table in a new tab The amino acid residues in CD40L that are mutated in HIGM1 are in bold. The amino acid residues in β3 that are involved in site 2 peptide are underlined. Previous studies identified amino acid residues in the trimeric interface that are involved in binding to the classical ligand-binding site (site 1) of activated integrins (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). Consistently, the two CD40L mutations Y170E and G252E in the trimeric interface are defective in binding to site 1 (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar), but activated soluble αvβ3 (Fig. 2E). This finding is consistent with the model that site-1-binding and site-2-binding sites are distinct. Previous studies showed that integrin α5β1 binds to CD40L and induces signals independent of CD40 (12El Fakhry Y. Alturaihi H. Yacoub D. Liu L. Guo W. Leveille C. Jung D. Khzam L.B. Merhi Y. Wilkins J.A. Li H. Mourad W. Functional interaction of CD154 protein with alpha5beta1 integrin is totally independent from its binding to alphaIIbbeta3 integrin and CD40 molecules.J. Biol. Chem. 2012; 287: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We previously identified the binding site for α5β1 in the trimeric interface of CD40L, indicating that α5β1 and αvβ3-binding sites overlap in the trimeric interface of CD40L (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). We hypothesized that CD40L activates α5β1 as well in an allosteric manner. We confirmed that biotinylated soluble α5β1 binds to the fibronectin fragment (FN8-11), a specific ligand to α5β1, in ELISA-type activation assays in the presence of 1 mM Mn+2 (Fig. 3A). FN8-11 binds to site 1, but does not bind to site 2 (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar). We studied if sCD40L activates soluble α5β1. Activation is defined by the increase in integrin binding to immobilized FN8-11 by soluble sCD40L. FN8-11 was immobilized and incubated with biotinylated soluble α5β1 in the presence of 1 mM Ca2+. sCD40L enhanced the binding of soluble α5β1 to immobilized FN8-11 in a dose-dependent manner (Fig. 3B). These findings indicate that sCD40L activates soluble integrin α5β1 in cell-free conditions. We showed that sCD40L activated cell-surface α5β1 in CHO cells (α5β1+, CD40-) using FITC-labeled FN8-11 in a dose-dependent manner (Fig. 3B). The levels of α5β1 activation by sCD40L in MFI and sCD40L concentrations correlated well (Fig. 3, C and D). We studied if four HIGM1 mutants can activate cell-surface α5β1 in CHO cells. They were defective in activating α5β1 and the K143T was the most defective (Fig. 3E). These findings suggest that sCD40L binds to site 2 of α5β1 in a manner similar to that of αvβ3 and activates α5β1. CD40L is primarily expressed in activated CD4+ T cells, but αvβ3 and α5β1 are not major integrins in T cells. sCD40L binds to site 1 of activated αvβ3 and α5β1 (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). It is unclear if integrin α4β1, which is expressed primarily in immune-competent cells, interacts with CD40L. We used biotinylated soluble α4β1 to study if α4β1 binds to CD40L. We found that soluble α4β1 activated by 1 mM Mn2+ bound to immobilized WT sCD40L in a dose-dependent manner (Fig. 4A), indicating that CD40L is a ligand for α4β1 that binds to site 1. We then studied if sCD40L activates α4β1 by binding to site 2. A fibronectin fragment H120, a ligand specific to α4β1, binds to site 1, but does not bind to site 2 (14Fujita M. Takada Y.K. Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.PLoS One. 2014; 9: e96372Crossref PubMed Scopus (17) Google Scholar). Activation is defined by the increase in the binding of soluble integrin α4β1 to immobilized H120 by sCD40L. We found that sCD40L enhanced the binding of soluble α4β1 to H120 in a dose-dependent manner in the presence of 1 mM Ca2+ (Fig. 4B). Also, sCD40L enhanced the binding of FITC-labeled H120 to CHO cells that express recombinant α4β1 (α4-CHO cells) in the presence of 1 mM Ca2+ (Fig. 4C). The α4β1 activation (MFI) correlated well with the sCD40L concentrations, indicating that sCD40L activated cell-surface α4β1 (Fig. 4D). We studied if the four HIGM1 mutants that are clustered in the predicted site-2-binding site of CD40L can activate α4β1 on α4-CHO cells. The HIGM1 mutants were defective in activating cell surface α4β1. The K143T and G144E mutants were the most defective in activating α4β1 among four HIGM1 mutants (Fig. 4E), indicating that site-2-binding interface in α4β1 is similar to those of αvβ3 and α5β1. These findings suggest that α4β1 is a new receptor for CD40L. CD40L binds to site 2 and activates α4β1 in a manner similar to those of αvβ3 and α5β1. We further characterized the four HIGM1 mutants to determine the role of site 2 in CD40L signaling. They all bound to CD40 in ELISA (Fig. 5A). We tested their ability to bind to activated soluble αvβ3 in ELISA in the presence of 1 mM Mn2+, which reflects their ability to bind to site 1. S128R/E129G and L155P were defective and K143T and G144E were partially defective in binding to activated integrin αvβ3, suggesting that K143T and G144E are still able to bind to site 1 (Fig. 5B). S128R/E129G and L155P did not bind to activated αvβ3 (to site 1) although these mutations are not in the site-1-binding site in the trimeric interface. It is possible that these mutations induced conformational changes in the trimeric interface. We determined the ability of HIGM1 mutants to induce NF-kB activation in HEK293 reporter cells. The four mutants were all defective in inducing NF-kB activation in reporter cells, except that K143T is slightly active (Fig. 5C). K143T at high concentration (5 μg/ml) induced substantial NF-kB activation (Fig. 5D). We tested if excess (20-fold) mutants can suppress NF-kB activation by WT sCD40L. We found that all mutants tested suppressed NF-kB activation by WT sCD40L (Fig. 5D), indicating that they act as antagonists. We previously showed that eight other HIGM1 mutants, which are clustered in the trimeric interface, were defective in integrin binding (to site 1). Their defect in site 1 binding is likely related to their defect in CD40L signaling (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). The two mutants, K143T and G144E, still bind to site 1 but defective in integrin activation by binding to site 2. We thus propose that the binding of CD40L to site 2 is required for NF-kB activation. In the present study, we showed that sCD40L activated soluble and cell-surface integrins αvβ3, α5β1, and α4β1 in a dose-dependent manner. These findings indicate that this activation does not require inside-out signaling. Since cyclic site 2 peptide bound to sCD40L, it is suggested that sCD40L bound to site 2. Docking simulation using inactive/close headpiece αvβ3 as a target predicts that CD40L binds to site 2 and four HIGM1 mutants are clustered in the predicted site-2-binding site in CD40L (Fig. 6A). We showed that the HIGM1 mutants (particularly K143T and G144E) are defective in activating integrins, consistent with the prediction. The present study showed that sCD40L is a new allosteric activator of integrins by binding to site 2. The four HIGM1 mutants, including K143T and G144E, were defective in activating integrins αvβ3, α5β1, and α4β1 because they are defective in site 2 binding. This suggests that the defective binding of CD40L to site 2 is related to defective CD40L/CD40 signaling. Since integrins on normal blood cells (e.g., B cells) are not activated, it is possible that CD40L-mediated integrin activation by binding to site 2 is required for CD40L/CD40 signaling, in addition to site 1 binding. Previous study showed that eight HIGM1 mutants clustered in the trimeric interface are defective in integrin binding and in signaling because they are defective in site 1 binding (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar), indicating that the defect in integrin binding to site 1 is related to defective CD40L/CD40 signaling. We thus propose that CD40L-mediated integrin activation by binding to site 2 is also required for CD40L signaling. Since integrins are not activated in normal leukocytes, integrin activation by this mechanism will facilitate signaling by CD40L. It is well known that high concentrations of sCD40L (>1000-fold) are required to induce CD40L/CD40 signaling compared with membrane-bound CD40L. In our previous study, sCD40L at >1 μg/ml was needed to achieve maximal NF-kB activation in HEK293 reporter cells (13Takada Y.K. Yu J. Shimoda M. Takada Y. Integrin binding to the trimeric interface of CD40L plays a critical role in CD40/CD40L signaling.J. Immunol. 2019; 203: 1383-1391Crossref PubMed Scopus (13) Google Scholar). We proposed that CD40L/CD40 signaling requires direct integrin binding to CD40L (and s
Referência(s)