Molecular mechanism of α2β1 integrin interaction with human echovirus 1
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.326
ISSN1460-2075
AutoresJohanna Jokinen, Daniel White, Maria Salmela, Mikko Huhtala, Jarmo Käpylä, Kalle Sipilä, J. Santeri Puranen, Liisa Nissinen, Pasi Kankaanpää, Varpu Marjomäki, Timo Hyypiä, Mark S. Johnson, Jyrki Heino,
Tópico(s)Immune Response and Inflammation
ResumoArticle19 November 2009free access Molecular mechanism of α2β1 integrin interaction with human echovirus 1 Johanna Jokinen Johanna Jokinen Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Daniel J White Daniel J White Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland Search for more papers by this author Maria Salmela Maria Salmela Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Mikko Huhtala Mikko Huhtala Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Search for more papers by this author Jarmo Käpylä Jarmo Käpylä Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Kalle Sipilä Kalle Sipilä Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author J Santeri Puranen J Santeri Puranen Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Search for more papers by this author Liisa Nissinen Liisa Nissinen Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Pasi Kankaanpää Pasi Kankaanpää Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Varpu Marjomäki Varpu Marjomäki Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland Search for more papers by this author Timo Hyypiä Timo Hyypiä Department of Virology, University of Turku, Turku, Finland Search for more papers by this author Mark S Johnson Mark S Johnson Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Search for more papers by this author Jyrki Heino Corresponding Author Jyrki Heino Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Johanna Jokinen Johanna Jokinen Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Daniel J White Daniel J White Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland Search for more papers by this author Maria Salmela Maria Salmela Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Mikko Huhtala Mikko Huhtala Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Search for more papers by this author Jarmo Käpylä Jarmo Käpylä Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Kalle Sipilä Kalle Sipilä Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author J Santeri Puranen J Santeri Puranen Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Search for more papers by this author Liisa Nissinen Liisa Nissinen Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Pasi Kankaanpää Pasi Kankaanpää Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Varpu Marjomäki Varpu Marjomäki Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland Search for more papers by this author Timo Hyypiä Timo Hyypiä Department of Virology, University of Turku, Turku, Finland Search for more papers by this author Mark S Johnson Mark S Johnson Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Search for more papers by this author Jyrki Heino Corresponding Author Jyrki Heino Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Author Information Johanna Jokinen1, Daniel J White2, Maria Salmela1, Mikko Huhtala3, Jarmo Käpylä1, Kalle Sipilä1, J Santeri Puranen3, Liisa Nissinen1, Pasi Kankaanpää1, Varpu Marjomäki2, Timo Hyypiä4, Mark S Johnson3 and Jyrki Heino 1 1Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland 2Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland 3Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland 4Department of Virology, University of Turku, Turku, Finland *Corresponding author. Department of Biochemistry and Food Chemistry, University of Turku, Turku 20014, Finland. Tel.: +358 2 333 6879; Fax: +358 2 333 6860; E-mail: [email protected] The EMBO Journal (2010)29:196-208https://doi.org/10.1038/emboj.2009.326 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of α2β1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the α2I domain. Furthermore, a mutation E336A in the α2 subunit, which inactivated α2β1 as a collagen receptor, enhanced α2β1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of α2β1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in α2β1. Furthermore, the mutation E336A did neither prevent EV1 induced and α2β1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on α2β1 clustering, but do not require the conformational regulation of the receptor. Introduction Adhesion receptors of the integrin family are known to anchor most cell types to the surrounding matrix. Several intracellular pathogens also bind to integrins to gain entry to the cell. Integrins are optimal virus receptors for several reasons. They are abundantly expressed on the cell surface and they have relatively low affinity for their natural ligands. In addition, integrins are connected to signalling proteins that may trigger endocytotic pathways. Activation of integrin-mediated signalling is considered to be an essential mechanism for the internalization of viruses. In this process they may mimic the natural ligands. The initial step in virus infection is binding of the virus particle to a specific receptor on the cell surface. Many adenoviruses (Wickham et al, 1993), coxsackievirus A9 (Chang et al, 1989; Roivainen et al, 1991, 1994; Williams et al, 2004), human parechovirus 1 (Hyypiä et al, 1992; Stanway et al, 1994; Joki-Korpela et al, 2001), foot-and-mouth disease virus (Fox et al, 1989; Jackson et al, 1997) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8; Akula et al, 2002; Veettil et al, 2008) have surface proteins harbouring an arginine–glycine–aspartic acid (RGD) motif, a well-known recognition sequence for a subset of integrins (Ruoslahti and Pierschbacher, 1987). Integrin α2β1, a collagen receptor, binds to human echovirus 1 (EV1; Bergelson et al, 1992) and rotavirus (Zárate et al, 2000) in an RGD-independent manner. Recent investigations have unveiled many essential facts concerning the structural basis of integrin signalling (for reviews, see Springer and Wang, 2004; Arnaout et al, 2005). Inactivated integrins are proposed to take a bent conformation. Activating 'inside-out' signals, such as talin or kindlin binding to β-integrin cytoplasmic domain, can trigger a conformational change leading to the extension of integrin ectodomain (for review, see Moser et al, 2009). Natural ligand binding to a site formed by the inserted domain of the β-subunit (βI domain) and the β-propeller domain of the α-subunit triggers a conformational change in the βI domain, leading to the separation of the α- and β-leg regions (Xiao et al, 2004). This results further in the dissociation of α- and β-cytoplasmic domains, allowing activation of intracellular signalling protein binding to the integrins. Multivalent ligands can, in addition to conformational changes, induce integrin cluster formation. Integrin-binding viruses have been thought to act in a manner similar to natural multivalent ligands. However, the results reported here indicate that EV1 diverges from all previously studied α2β1 integrin ligands. Integrin α2β1, as well as the three other human collagen receptor integrins (α1β1, α10β1 and α11β1) and the five leukocyte integrins (αLβ2, αMβ2, αXβ2, αDβ2 and αEβ7), belong to a structurally distinct subgroup of integrins. These nine α-subunits have a ligand-binding αI domain, homologous to the βI domain found in all the integrin β-subunits. The 'I' domains or 'inserted' domains are also called 'A' domains on the basis of their structural similarity to von Willebrand factor A domains (Arnaout et al, 2005). The αL and αM integrin αI domains can assume a closed, an intermediate or an open conformation (Shimaoka et al, 2003a), whereas in the α1I and α2I domains the intermediate form may not exist (Jin et al, 2004), suggesting that ligand binding to the latter domains triggers a change from the closed to the open conformation (Emsley et al, 2000). The open conformation is also detected in activated integrins before ligand binding, and it may represent a high avidity state of the αI domain. In a recombinant α2I domain, the alteration from the closed to the open conformation can be induced by the gain-of-function mutation E318W (Aquilina et al, 2002). All natural ligands, including different collagen and laminin subtypes, have shown better binding to the open α2I domain when compared with the closed domain form (Aquilina et al, 2002; Tulla et al, 2008). The key mechanism involved in signalling by the αMβ2 and αLβ2 integrins seems to act through a glutamate residue, located close to the C-terminus of the α7 helix in the αI domain (E310 in αL and E320 in αM), which acts as an intrinsic ligand for the β2I domain and participates in the conformational activation of the integrin receptor (Alonso et al, 2002; Shimaoka et al, 2003a; Yang et al, 2004). In αLβ2 integrin mutation E310A has been reported to push the equilibrium between the bent and extended conformations towards the bent conformation (Salas et al, 2004). In the α2 integrin, glutamate 336 (E336) in the α7 helix of the αI domain seems to have a similar role (Connors et al, 2007), as mutation E336A affects the activation of α2β1 and the regulation of α2I domain conformation (Connors et al, 2007). Thus α2β1 integrin that harbours E336A mutation is in the bent rather than extended conformation. We report here that EV1, unlike any currently known extracellular matrix ligand, favours binding to the closed α2I domain and inactive α2β1 integrin. Furthermore, the activation of protein kinase Cα (PKCα) and the EV1 entry pathway are independent of E336. We have also studied structural requirements of α2β1 signalling through p38 mitogen-activated protein kinase (MAPK) pathway, a signalling pathway strongly linked to α2β1 integrin (Ivaska et al, 1999; Ravanti et al, 1999; Xu et al, 2001; Bix et al, 2004; Mazharian et al, 2005). Our results indicate that clustering, mediated either by collagen or antibodies, leads to rapid and transient activation of p38 MAPK. We also demonstrate that E336 in the α2I domain is a key determinant in the α2β1-mediated activation of p38. Thus, the activation of p38 pathway can be considered as an indicator of E336-dependent conformational change in α2β1 integrin. However, the clustering of α2β1 integrin by EV1 did not significantly activate the p38 pathway during the early stage of infection. Thus, our results demonstrate that there seem to be fundamental differences in the mechanisms of EV1 action when compared with the natural ligands of α2β1 integrin. EV1 seems to gain its entry by activation of signalling pathways that are dependent on α2β1 clustering, but do not require conformational activation of the integrin. Results EV1 binds to the closed conformation of the α2I domain To compare the possible interactions of the open and closed conformations of α2I with EV1, we constructed a model of the α2I (open)–EV1 complex by superimposing the open-form crystal structure on our previously published model of the α2I (closed)–EV1 complex. The model is based on a cryoelectron microscopy (cryo-EM) structure of the complex, into which the high-resolution crystal structures of EV1 and the closed form of α2I have been fitted (Xing et al, 2004). EV1 is known to bind in a metal ion-independent manner (Bergelson et al, 1993) at a binding site different from the MIDAS (King et al, 1997). The site has also been mapped by mutagenesis (King et al, 1997; Dickeson et al, 1999) to the side-face of the αI domain as opposed to the MIDAS location on top of the domain (Figure 1A). The comparison of the open and closed conformations shows that most of the region participating in virus binding on the αI domain surface is not involved in the closed–open conformational alteration and remains essentially unchanged (Figure 1A). The α7 helix that undergoes a large movement is located on the opposite side of the αI domain, compared with the virus-binding surface. Most of the loop between the β7 strand and the α6 helix, including the αC helix in the closed conformation, is also not in close contact with EV1. Thus, the structural model can be considered to be compatible with EV1 binding to either the closed or the open conformation of the α2I domain. Figure 1.EV1 prefers the closed conformation of the α2I domain. (A) The crystal structure of the α2I domain in the closed (left) and the open conformations (right). Both conformations are also shown docked onto the surface of EV1 on the basis of the structure of the α2I–EV1 complex determined by cryo-EM (bottom). The surfaces of two protomers of the EV1 capsid are shown, one coloured light blue and the other light grey. The fivefold symmetry axis of the icosahedral capsid is labelled '5'. The regions in α2I that undergo the most extensive conformational changes (grey arrows) are the α7 helix (green), the βE-α6 loop (orange; includes the αC helix in the closed conformation) and the MIDAS (the metal ion in yellow). The position of amino-acid E318 is indicated. Residues that have been shown by mutagenesis to affect EV1 binding are coloured red (residues 199–201, 212–216 and 289), and the residues that are within 4.0 Å of the virus structure in the model of the complex are coloured blue. Most of the regions of the α2I involved in the conformational changes are not in close contact with the virus. Microtitre plates were coated with (B) collagen I (Col I) or (C) EV1. Delfia® Diluent II (BSA; Δ) was used as a background control. The GST fusion proteins, containing wild-type α2I (▪) or high affinity mutant α2I E318W (O) domains, were allowed to react with the ligand for 1 h in the presence of 2 mM MgCl2. Bound αI domains were detected with Eu3+-labelled GST antibody, and the signal was measured using time-resolved fluorescence. Data are presented as mean values±s.d. of triplicate measurements. Approximate Kd values for αI domain binding were obtained by fitting the binding data for αI domain concentrations series to a Michaelis–Menten form equation. (D) BIAcore analyses of the interactions between 1 μM α2I WT (solid line) or α2I E318W (dashed line) with immobilized EV1 are presented as overlaid sensograms. After a short (120 s) association phase, the measurement of the dissociation was continued for 25 min. (E, F) Binding of α2I WT (▪) and α2I E318W (O) to immobilized EV1 was competed with increasing concentrations of soluble (E) EV1 (0.04–0.7 nM) or (F) triple helical GFOGER peptide (0.01–1000 μM) that mimic a high-affinity integrin-binding site on collagen by using Eu3+-based solid phase binding assay described above (B, C). Results were fitted to the model representing a dose–response curve with variable Hill slope. Download figure Download PowerPoint Previously, residue Asn289 in the αC helix of α2I has been shown by mutagenesis to be required for EV1 binding (Dickeson et al, 1999). Here, in the modelled complexes (Figure 1A), this amino acid is in contact with the virus, but it is located at the periphery of the α2I–EV1 interface and part of the residue is exposed to solvent. Asn289 is in the middle of a segment that undergoes an extensive structural rearrangement upon the α2I change from the closed to open form, and therefore, it is possible to hypothesize that EV1 binding to Asn289 may affect the avidity or may even activate a conformational effect in the αI domain (Xing et al, 2004). The introduction of the mutation E318W into α2I has previously been shown to lead to a shift from the closed to the high affinity state open conformation (Aquilina et al, 2002; Tulla et al, 2008). Glu318 is located in helix α7, and it is not in contact with EV1 (Figure 1A). Here, the binding of human recombinant α2I domains (α2I WT and α2I E318W) to EV1 was tested in a solid phase binding assay using microtitre plates coated with either the virus or collagen I as a control. The results were fitted to a Michaelis–Menten form equation and approximate Kd values were determined to quantify α2I domain binding (Figures 1B and C). As expected, the mutation E318W increased α2I domain binding to collagen I from Kd≈39±3.5 nM (α2I WT) to Kd≈7±0.5 nM (α2I E318W; Figure 1B). Wild-type α2I bound tighter to EV1 than to collagen I in accordance with our previously published results (Xing et al, 2004). Surprisingly, the approximate Kd for α2IE318W domain binding to EV1 seemed to be weaker (Kd≈3±0.2 nM) than the binding of wild-type α2I (Kd≈0.8±0.2 nM; Figure 1C). Thus, the requirements for EV1 binding seem to differ from all previously tested α2β1 integrin ligands that have been shown to favour the open α2I conformation. To further study the virus–receptor interaction additional experiments were performed. For BIAcore surface plasmon resonance measurements, EV1 was covalently coupled on the surface of the sensor chip. The measurements indicated that the association of both α2I WT and α2I E318W to immobilized EV1 was very fast (Figure 1D). Importantly, the dissociation of α2I WT was remarkably slower when compared with α2E318W (Figure 1D), which partially explains the tighter binding of α2I WT to EV1 seen in solid phase binding assays. We also used BIAcore to perform kinetic titration series, in which samples were injected sequentially without a regeneration step. The results failed to fit 1:1 binding model (Karlsson et al, 2006), indicating that more than one kind of binding site exist in EV1 (data not shown). To make further estimates of the stoichiometry of the virus binding to α2I, increasing concentrations (0.04–0.7 nM) of soluble EV1 were allowed to compete with 1.5 nM α2I WT in binding to immobilized EV1 in a solid phase binding assay (Figure 1E). In two independent experiments, 0.17–0.34 nM EV1 seemed to block α2I binding to immobilized EV1. On the basis of calculations using estimated Mr of virion (5.65 × 106) and α2I domain–GST fusion (49 500), the results indicated that in these conditions 7–15% of 60 putative integrin-binding sites per virion were occupied. On the basis of molecular modelling, we have previously proposed that collagen and EV1 cannot concomitantly bind to α2I domain (Xing et al, 2004). To test this experimentally, a soluble triple helical GFOGER peptide, which mimics a high-affinity integrin-binding site in collagen (Knight et al, 1998), was used in the competition study. Interestingly, the collagenous peptide (0.01–1000 μM) effectively inhibited the binding of open α2I (E318W) to EV1 (Figure 1F), but could not compete with the binding of closed α2I (WT) to EV1 within the concentration range used. The result suggests that in the presence of collagen, EV1 significantly benefits from its preference for inactive α2β1. To test the phenomenon at the cellular level, Chinese hamster ovary (CHO) cells expressing either full-length α2WT or α2E318W were allowed to attach to immobilized collagen I, EV1 or BSA for 15 min. CHO cells do not naturally express integrin-type collagen receptors (Nykvist et al, 2000). Adherent cells were detected using WST-1 reagent. In agreement with the experiments performed at the α2I domain level, integrin activation significantly (P<0.001) increased cell adhesion to collagen I (Figure 2). At the same time the activation decreased cell adhesion to EV1 (P<0.05; Figure 2). Thus, EV1 seemed to bind better to the closed than open α2I conformation and not only at the α2I domain level but also when the tests are performed using full-length α2 integrins expressed on cell surface. Figure 2.Integrin activation decreased EV1 binding to cellular α2β1. CHO-α2 and CHO-α2E318W cells were allowed to adhere to immobilized EV1 or collagen I for 15 min. Adhered cells were detected using WST-1 reagent. Integrin activation significantly (P<0.001) increased cell adhesion to collagen I. At the same time, EV1 seemed to favour CHO-α2WT cells (P<0.05). Mean values±s.d. of eight parallel measurements are shown. Statistical significances were determined by two-tailed Student's t-test. Download figure Download PowerPoint EV1 favours inactive α2β1 integrin To study further the structural requirements of EV1 binding to α2β1 integrin, we constructed a conformationally inactive integrin and expressed it on cell surface. We have used molecular modelling to assess, whether the α2I domain could interact with the β1 subunit in the same manner as αLI and αMI interact with the β2 subunit (Alonso et al, 2002; Shimaoka et al, 2003b; Yang et al, 2004; Arnaout et al, 2005). Our model suggests that when the α2I domain is in the open conformation, E336 could interact with the metal ion of MIDAS of the β1I domain (Connors et al, 2007; Figure 3A). The model predicts that collagen binding to the α2I domain will most probably induce a conformational change in the β1I domain that leads to the separation of the integrin leg regions, as has been described for other integrins (Alonso et al, 2002; Kim et al, 2003; Yang et al, 2004). In addition to E336 at the C-terminal end of helix α7, E309 in the loop preceding helix α7 was chosen as a target for mutagenesis (Figure 3A). Figure 3.EV1 favours inactive α2β1 integrin. (A) A structural model of the human α2β1 integrin head region was built based on the crystal structures of the αVβ3 integrin and the α2I domain. (A; bottom, left) Modelling indicates that the closed conformation of the α2I domain does not form specific contacts with the β1I domain (blue). (A; bottom, right) However, when the α2I domain adopts an open conformation, E336 at the C-terminal end of helix α7 is positioned in such a way that it could coordinate to the metal ion (yellow) of the MIDAS in the β1I domain and act as an intrinsic ligand. E309 does not seem to participate in the process. (B) In a cell spreading assay, CHO-α2 and CHO-α2E309A cells attached and spread on a collagen I (Col I) matrix in 120 min, whereas α2E336A mutation caused a dramatic decrease in the spreading. Cells were not able to attach to or spread on BSA, which was used as a negative control. (C) When CHO-α2 and CHO-α2E336A cells were allowed to adhere to immobilized EV1 or collagen I for 15 min, the E336A mutation seemed to decrease α2-mediated cell adhesion to collagen I. Interestingly, EV1 seemed to favour CHO-α2E336A cells. Mean values±s.d. of four parallel measurements are shown (B, C). (D) However, at 15 min, CHO-α2 and CHO-α2E336A cell adhesion to both collagen I and EV1 was significantly increased in the presence of integrin cluster-inducing TPA (100 nM; black columns). Mean values±s.d. of four parallel measurements are shown. Download figure Download PowerPoint Wild-type α2 and mutant α2 integrins, containing either E336A or E309A, were expressed in CHO cells. Equal expression levels of the mutant α2 integrins were confirmed by flow cytometry (Supplementary Figure S1). In addition, cell lines were metabolically labelled with 35S-methionine/cysteine, and the α2β1 complex was immunoprecipitated with α2 antibodies under conditions that maintained the subunit interactions. Analysis by SDS–PAGE confirmed that the expression levels of the mutant integrins were practically equal. Furthermore, the mutations seemed to have no effect on the stability of the α2β1 heterodimer (data not shown). When tested by a spreading assay on collagen I, CHO-α2E309A cells did not significantly differ from CHO-α2 cells expressing the wild-type α2 subunit (Figure 3B). Cells carrying the point mutation, E336A, in their α2 subunit were able to attach to collagen I but their spreading was delayed. Similar results were obtained when the CHO-α2 and CHO-α2E336A cells were allowed to attach to an immobilized collagen I for 15 min and adherent cells were detected with WST-1 reagent (Figure 3C). The results suggest that the mechanism of α2β1 action would closely resemble that of αLβ2 integrin (Yang et al, 2004): the communication of the conserved residue E336 in α2 with the metal ion at MIDAS of the β1I domain may induce the high-affinity conformation of the α2I domain. Importantly, when CHO-α2 and CHO-α2E336A binding to EV1 was analysed in the adhesion assay, EV1 seemed to favour the inactive state of α2β1 (Figure 3C). Thus, the results with the E336A variant confirmed the idea that EV1 binds to inactive rather than active integrins. Furthermore, the corresponding mutation in αL integrin has been reported to push the equilibrium between the bent and extended conformations towards the bent conformation (Salas et al, 2004). Therefore, the data propose that EV1 may bind to bent rather than extended integrins. We have previously shown that activation of PKC by TPA induces both ligand-independent macroaggregation of α2β1 integrins and conformational activation of α2I, whereas the E336A mutation prevents the change in conformation but not receptor clustering (Connors et al, 2007). When TPA-treated (100 nM) cells were allowed to attach to collagen I for 15 min, both CHO-α2 and CHO-α2E336A cell adhesions were increased (Figure 3D). Similarly, TPA increased binding of EV1 to the α2WT and the α2E336A mutant cells. A previous study has also shown that TPA can increase EV1 binding to α2β1 integrin (Bergelson et al, 1993). Our observations indicate that the formation of α2β1 clusters before ligand binding, rather than the conformational activation of the integrin, may explain the increased integrin avidity to EV1. Activation of p38 after α2β1 integrin clustering by collagen I requires E336-dependent conformational changes in the integrin Next we tested the structural requirements of α2β1 signalling after integrin clustering. The formation of α2β1 integrin clusters after treatment with α2 subunit-specific primary antibodies and clustering secondary antibodies was imaged using confocal microscopy. At 15 min, clear integrin clusters appeared when primary and secondary antibodies were used together (Figure 4A). Collagen binding to α2β1 has been reported to lead to specific activation of the p38α MAPK signalling pathway (Ivaska et al, 1999; Ravanti et al, 1999; Xu et al, 2001; Bix et al, 2004; Mazharian et al, 2005). To analyse the effect of α2β1 cluster formation on p38 activation, Saos-α2 and CHO-α2 cells were treated with the antibodies and p38 phosphorylation was analysed by immunoblotting. Antibody-induced clustering caused a rapid and transient phosphorylation of p38 at 15 min in both cell lines (Figures 4B and C). The transient nature of p38 activation (Figure 4B) is most probably because of the fact that antibody-generated α2β1 clusters are rapidly internalized (Upla et al, 2004). To confirm the observation, we also analysed the phosphorylation of p38 by a flow cytometry-based method and a similar activation of p38 in CHO-α2 cells was detected (Figure 4D). It was also obvious that neither the α2 integrin-specific antibody, nor the secondary antibody alone affected p38 phosphorylation (Figure 4D). Figure 4.Clustering of α2β1 integrins induces a rapid transient phosphorylation of p38. (A, bottom) Volume renderings of confocal image data show that secondary antibodies (goat anti-mouse IgG) were able to induce integrin clustering in Saos-α2 cells treated with α2 primary antibody (Alexa Fluor 555-conjugated 16B4). (A, top) Without secondary antibodies, no clustering occurs. In both cases, the same living cell was imaged at 0- and 15-min time points. (B) When p38 phosphorylation (P-p38) induced by the antibody (16B4 and anti-mouse IgG)-mediated α2β1 clustering was analysed in Saos-α2 cells at successive time points, a rapid and transient p38 phosphorylation, peaking at 15 min, was obvious. (C) Similarly, antibody-mediated clustering caused p38 activation even in CHO-α2 cells in 15 min. Representative immunoblots of one experiment (B, C) and statistical analyses of scanned blot images of one (C) or five (B) independent experiments are shown. Mean levels of phosphorylated p38 (P-p38)±s.d. relative to total p38 or β-actin levels are shown. (D)
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