Two Synergistic Activation Mechanisms of α2β1 Integrin-mediated Collagen Binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, α2β1 integrin. Concomitantly, TPA increases the avidity of α2β1 for collagen and the number of conformationally activated α2β1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the “open” (active) conformation of the inserted domain in the α2 subunit (α2I). Amino acid residue Glu-336 in α2 subunit is proposed to mediate the interaction between α2I domain and β1 subunit. Glu-336 seems to regulate a switch between open and “closed” conformations, since the mutation α2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or α2β1 aggregation. Thus, α2β1 integrin avidity is regulated by two synergistic mechanisms, first an α2E336-dependent switch to the open α2I conformation, and second an α2E336-independent mechanism temporally associated with receptor aggregation. Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, α2β1 integrin. Concomitantly, TPA increases the avidity of α2β1 for collagen and the number of conformationally activated α2β1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the “open” (active) conformation of the inserted domain in the α2 subunit (α2I). Amino acid residue Glu-336 in α2 subunit is proposed to mediate the interaction between α2I domain and β1 subunit. Glu-336 seems to regulate a switch between open and “closed” conformations, since the mutation α2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or α2β1 aggregation. Thus, α2β1 integrin avidity is regulated by two synergistic mechanisms, first an α2E336-dependent switch to the open α2I conformation, and second an α2E336-independent mechanism temporally associated with receptor aggregation. Integrin-type cell adhesion receptors undergo functional regulation via different conformational states (1Springer T.A. Wang J.H. Adv. Protein Chem. 2004; 68: 29-63Crossref PubMed Scopus (138) Google Scholar). In an inactive integrin α/β heterodimer, the ligand binding headpiece is bent toward the cell surface (2Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar), while an active integrin stands tall but may still have multiple activation states. Nine α subunits out of 24 human integrin heterodimers have an inserted αI (or αA) ligand binding domain, which can exist in multiple configurations of differing affinity. The conformation of αI is regulated by a spring-like mechanism formed by a conserved glutamate residue in the α7 helix acting as an intrinsic ligand of the I-like domain of the β subunit (βI; Refs. 3Alonso J.L. Essafi M. Xiong J.P. Stehle T. Arnaout M.A. Curr. Biol. (Lond.). 2002; 12: 340-342Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and 4Yang W. Shimaoka M. Salas A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2906-2911Crossref PubMed Scopus (78) Google Scholar). Avidity, the overall strength of integrin mediated interactions between cells and extracellular ligands, is multideterminant, involving the affinity of individual receptors and variations in the spatial arrangement of the integrins. The total number of receptors participating in the interaction, or valency, is expected to be larger if integrins form clusters. The relationship between conformational regulation and cluster formation has been thoroughly studied in αLβ2 integrin, a leukocyte receptor for intercellular adhesion molecules (5Jin M. Andricioaei I. Springer T.A. Structure (Lond.). 2004; 12: 2137-2147Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 6Salas A. Shimaoka M. Kogan A.N. Harwood C. von Andrian U.H. Springer T.A. Immunity. 2004; 20: 393-406Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 7Sarantos M.R. Raychaudhuri S. Lum A.F. Staunton D.E. Simon S.I J. Biol. Chem. 2005; 280: 28290-28298Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Recent studies suggest that αLβ2 clustering takes place only after binding a multivalent ligand and that clustering strengthens adhesion after ligand binding (8Kim M. Carman C.V. Yang W. Salas A. Springer T.A. J. Cell Biol. 2004; 167: 1241-1253Crossref PubMed Scopus (203) Google Scholar). The requirement of a ligand for receptor clustering has also been reported for αVβ3 (9Cluzel C. Saltel F. Lussi J. Paulhe F. Imhof B.A. Wehrle-Haller B. J. Cell Biol. 2005; 171: 383-392Crossref PubMed Scopus (278) Google Scholar) and αIIbβ3 integrins (10Loftus J.C. Albrecht R.M. J. Cell Biol. 1984; 99: 822-829Crossref PubMed Scopus (90) Google Scholar). Four collagen receptors, α1β1, α2β1, α10β1, and α11β1, form a subgroup of αI domain containing integrins. Integrin α2β1 is expressed on platelets, epithelial cells, and many mesenchymal cell types (11White D.J. Puranen S. Johnson M.S. Heino J. Int. J. Biochem. Cell Biol. 2004; 36: 1405-1410Crossref PubMed Scopus (145) Google Scholar). The α2I domain, like other integrin I domains, can exist in both an “open” and a “closed” conformation (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar), as well as a less frequent, intermediate form (13Shimaoka M. Xiao T. Liu J-H. Yang Y. Dong Y. Jun C.-D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J.-H. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Thus, α2β1 can also be found in more than one conformation (14Cruz M.A. Chen J. Whitelock J.L. Morales L.D. Lopez J.A. Blood. 2005; 105: 1986-1991Crossref PubMed Scopus (57) Google Scholar), likely regulated similarly to other αI domain integrins. The unique functions of collagen receptor integrins may also be governed by the regulation of the receptor distribution and local density. For example, α2β1 and α11β1 participate in the formation and rearrangement of collagen fibrils (15Velling T. Risteli J. Wennerberg K. Mosher D.F. Johansson S. J. Biol. Chem. 2002; 277: 37377-37381Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 16Li S. Van Den Diepstraten C. D'Souza S.J. Chan B.M. Pickering J.G. Am. J. Pathol. 2003; 163: 1045-1056Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and echovirus-1-dependent clustering leads to rapid relocation and internalization of α2β1. In this process, α2β1 behaves differently from other integrin family members, such as αV integrins (17Upla P. Marjomaöki V. Kankaanpaöaö P. Ivaska J. Hyypiaö T. Van Der Goot F.G. Heino J. Mol. Biol. Cell. 2004; 15: 625-636Crossref PubMed Scopus (147) Google Scholar). The phorbol ester TPA 2The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; CHO, Chinese hamster ovary; col I, collagen I; BSA, bovine serum albumin; mAb, monoclonal antibody; BIS, bisindolylmaleimide. can increase the avidity of α2β1 binding to collagen (18Polanowska-Grabowska R. Gear A.R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 3044-3054Crossref PubMed Scopus (15) Google Scholar). We report here that while TPA induces rapid (5-15 min) clustering of cell surface α2β1inthe absence of ligand and increases α2β1 avidity for collagen, it also activates the integrin via a conformational mechanism involving α2E336 and intermolecular cross-talk between the α2I and β1I domains. Aggregation of α2β1 is independent of E336 and temporally associated with increased avidity of adhesion. We conclude that the avidity of α2β1 for collagen is regulated by two synergistic mechanisms. Cell Culture—Chinese hamster ovary (CHO) and Saos (ATCC) cells were stably transfected with human integrin α2 (19Riikonen T. Westermarck J. Koivisto A. Heino J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. Ivaska J. J. T. Heino J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), subunit J. J. M. J. T. P. Marjomaöki V. P. Farndale R.W. D.E. Heino J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) in or only as The cell has been by Ivaska J. Westermarck J. Koivisto Heino J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). Integrin α2 and E336A were by a and cell were by using α2 positive cells were first with positive cells were with integrin α2 12F1 and and by Cell and also and from from and F. of stably human integrin α2 subunit and β1 or β1 in have been K. M. Johansson S. R. J. Cell Biol. PubMed Scopus Google Scholar, K. A. T. M. R. Mosher D.F. Johansson S. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar, Mosher D.F. T. K. Cell 2003; PubMed Scopus Google Scholar). Cell were with collagen I I, or were to in with for cells were by as J. P. J. D.J. Ivaska J. P. Heino J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). TPA was to cells in serum cells were and in with and were with or and and by cells were first with or α2 integrin antibody for col or Heino J. Biochem. 2005; PubMed Scopus Google TPA was cells were and and by Heino J. Biochem. 2005; PubMed Scopus Google Scholar). Integrin and on in were with and with TPA, or were for a total of 24 were in containing bovine serum and with or TPA for or cells were with for by of TPA for cells were cells were and with α2 integrin a from of and In cells were on were with TPA for or and cells were with and in were by and and and were with of human α2β1 integrin headpiece were on the of the αVβ3 integrin J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Protein and the α2 domain J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (845) Google Protein were from the Protein J. 2000; PubMed Scopus Google Scholar) and from the A. R. B. S. Lopez R. M. M.J. C. 2005; PubMed Scopus Google Scholar). were in J. M. A. J. T. T.A. M. Johnson M.S. J. Mol. 2004; PubMed Scopus Google Scholar) and using M.A. A. A. R. F. A. 2000; PubMed Scopus Google Scholar). were using The 2002; Google Scholar). clustering and internalization of α2β1 integrin in The was using TPA a but Aggregation and of α2β1 and α2β1 clustering to (17Upla P. Marjomaöki V. Kankaanpaöaö P. Ivaska J. Hyypiaö T. Van Der Goot F.G. Heino J. Mol. Biol. Cell. 2004; 15: 625-636Crossref PubMed Scopus (147) Google Scholar). the of the phorbol ester TPA, a on integrin clustering and internalization was using Saos cells transfected to α2β1 integrin J. Westermarck J. Koivisto Heino J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). were with TPA, and the were in the absence of collagen In both and internalization of α2β1 integrin by TPA were and The of TPA was but not as that by which was used as a positive shown in the for the of after TPA was and the for internalization was For were and clustering or internalization was in cells that were not in BIS, a of the formation of integrin and internalization Thus, between activation and integrin receptor clustering activates (17Upla P. Marjomaöki V. Kankaanpaöaö P. Ivaska J. Hyypiaö T. Van Der Goot F.G. Heino J. Mol. Biol. Cell. 2004; 15: 625-636Crossref PubMed Scopus (147) Google Scholar) and the aggregation. TPA the of to in a have reported a and cell adhesion on the of and Heino J. Biochem. 2005; PubMed Scopus Google Scholar). studies of the of TPA on the integrin avidity for collagen. cells transfected to α2β1, α10β1, or α2β1 integrin as only collagen receptor Refs. P. Ivaska J. J. T. Heino J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. M. J. T. P. Marjomaöki V. P. Farndale R.W. D.E. Heino J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Westermarck J. Koivisto Heino J. J. Cell Biol. 1999; PubMed Scopus Google Scholar and J. Heino J. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar) were The binding of cells to collagen I was by Cell adhesion to collagen I to increase for to However, cells were with TPA, a which in was TPA a increase in cell adhesion with cells adhesion was the the of activation it has been shown that the of the α2 subunit is for mediated by α2β1 integrin J. Westermarck J. Koivisto Heino J. J. Cell Biol. 1999; PubMed Scopus Google Scholar, J. Heino J. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). In a α2 integrin subunit an to be to mediate the of the α2 in cells were in of both and cell adhesion to collagen I the adhesion of However, the TPA mediated increase in cell adhesion was in cells than in cells Thus, the mutation did not the of the receptor to cells integrin were similarly In this the adhesion in both TPA and cells than in cells the TPA increased cell adhesion to collagen I more than in or cells an overall the that the of integrin α subunits may not participate in regulation by TPA and that the is not to the α2β1 that the increases in adhesion were mediated by α2β1 integrin, a of were using and cell types an of interaction with which were or with cells, cells, did not with collagen However, and cells to collagen a both and was by cells with the α2 that adhesion in via the α2β1 integrin that the increase in α2β1 integrin avidity to collagen I a the BIS, was in the and absence of and cells were with BIS, the cell adhesion to collagen However, did not the adhesion of cells to collagen I. In the reduced adhesion in cells Integrin β1 for TPA-related in Cell to is a protein and a well for Biochem. PubMed Scopus Google Scholar). binding to the of the integrin β subunit is for the of integrin activation S. S.J. K. V. Liddington R.C. Science. 2003; PubMed Scopus Google Scholar). the of the β1 subunit in integrin avidity used cells of α2β1 or α2β1 with in the of the β1 subunit Mosher D.F. T. K. Cell 2003; PubMed Scopus Google Scholar). in conserved of the β has shown to be to binding S. S.J. K. V. Liddington R.C. Science. 2003; PubMed Scopus Google Scholar, A. C. R. J. Cell Biol. PubMed Scopus (88) Google Scholar). in with studies Mosher D.F. T. K. Cell 2003; PubMed Scopus Google Scholar), both α2β1 and integrin heterodimers mediate cell to collagen by TPA increased the collagen binding in cells as In TPA did not the activation of cells The suggest that integrin β1 subunit is for activation of α2β1 by TPA the of on the Cell have monoclonal that to activation states of the α2 integrin subunit (14Cruz M.A. Chen J. Whitelock J.L. Morales L.D. Lopez J.A. Blood. 2005; 105: 1986-1991Crossref PubMed Scopus (57) Google Scholar, K. J. I. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). the that with TPA α2 integrin conformational activation in cells was were different with TPA, and of and open α2 integrin subunit were by α2 antibody is the ligand binding of the α2 was used for of total α2 conformation was using which has been shown to an activated conformation of α2 subunit on (14Cruz M.A. Chen J. Whitelock J.L. Morales L.D. Lopez J.A. Blood. 2005; 105: 1986-1991Crossref PubMed Scopus (57) Google Scholar). The of integrin α2 on the cell surface in the of TPA between the first two and min) in independent as shown in was not that internalization of α2β1 integrin was by in TPA Saos cells the the of α2 integrin on the cell surface after in In a increase in the number of 12F1 to the active conformation of α2 integrin was in between and after with The increase was in from to to the conformational activation of the α2 integrin to than the increase in avidity to but the TPA-related leukocyte in and αLβ2 ligand binding is shown first to a change in the conformation of the αI domain, a of the α7 is by a change in the domain, to of the of the α and β subunits (1Springer T.A. Wang J.H. Adv. Protein Chem. 2004; 68: 29-63Crossref PubMed Scopus (138) Google Scholar, 3Alonso J.L. Essafi M. Xiong J.P. Stehle T. Arnaout M.A. Curr. Biol. (Lond.). 2002; 12: 340-342Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 4Yang W. Shimaoka M. Salas A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2906-2911Crossref PubMed Scopus (78) Google Scholar, M. Xiao T. Liu J-H. Yang Y. Dong Y. Jun C.-D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J.-H. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, M.A. B. Xiong J.P. Cell Biol. 2005; PubMed Scopus (424) Google Scholar). conserved glutamate residue on the α7 helix of the αI domain was to as an intrinsic ligand for the β1I The structural between the αI and seems to be in integrin and regulation of affinity. The glutamate in the α2I domain, the conserved may in a with the β1I domain and participate in the switch between open and α2I domain We also to the that the conformational regulation of α2β1 takes place in a different than the regulation of αLβ2 and Thus, acid and is to the C of the α7 or helix of the α2I domain and with the β1I domain binding residue was to to the of the The mutation was to the of the interaction between the α2 and the β1I surface is between two and to be a interaction the of the α2 of this interaction, on the αVβ3 (2Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar), be one of the in a interaction with of this interaction to in conformational cells transfected to integrin α2 subunits or E336A and were by for to to collagen that the adhesion of to collagen I was with cells also α2 integrin binding to collagen I, mutation cell binding to collagen I is in with studies that and integrins to after the mutation of the residue to E336 in α2 in and in Refs. 3Alonso J.L. Essafi M. Xiong J.P. Stehle T. Arnaout M.A. Curr. Biol. (Lond.). 2002; 12: 340-342Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and R. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). cells a of avidity to collagen and were to on collagen in However, cells, as well as and cells, more on collagen The conformational activation of the α2 integrin with the E336A mutation was studied in a by using cells and total or open α2 integrins on the cell In cells, the of total α2 was the of the in cells, as to cells the α2 subunit the TPA did not an increase in the of the active α2 integrin conformation by 12F1 α2E336A the TPA-related in for α2β1 cells with or were with TPA, avidity to collagen increased Thus, α2β1 integrin avidity is also regulated by an mechanism to the in integrin The cluster formation of α2β1 integrin was also studied in and cells using TPA the formation of on cells which is in with using cells clustering was in both and cells The clustering was more to in cells than in Saos cells of the of the integrin However, clustering is in from the of the cells in which it to by antibody while it is in cells in to conformational TPA also induces other integrin We that ligand-independent α2β1 integrin clustering is one of the mechanisms, which increases The interaction of α2β1 integrin and has been in a number of and such as and The regulation of integrin α2β1 activation is for for Thromb. PubMed Scopus Google Scholar). In integrin activation is an mechanism for the formation of adhesion Thus, the mechanism of α2β1 activation thoroughly a of report that α2β1 integrin activation is regulated by two synergistic receptor clustering and conformational The structural of in leukocyte integrins has been in (1Springer T.A. Wang J.H. Adv. Protein Chem. 2004; 68: 29-63Crossref PubMed Scopus (138) Google Scholar, M.A. B. Xiong J.P. Cell Biol. 2005; PubMed Scopus (424) Google Scholar). The integrins to have different with to In inactive the ligand binding is bent toward the in a that the binding of However, such as binding to the of the β subunits S. S.J. K. V. Liddington R.C. Science. 2003; PubMed Scopus Google Scholar) can a in the integrin the integrin The avidity of integrin for ligand can still be increased by conformational In αI domain integrins the avidity conformation has been as the open conformation of the αI have studied a in which α2β1 integrins were activated by was that the TPA on integrin avidity be by the of TPA to switch α2I from the to the open conformational change was studied using 12F1 binding to the cell In a the monoclonal antibody 12F1 was shown to the open conformation of the α2 subunit on (14Cruz M.A. Chen J. Whitelock J.L. Morales L.D. Lopez J.A. Blood. 2005; 105: 1986-1991Crossref PubMed Scopus (57) Google Scholar). antibody which recognizes an the ligand binding of the α2 was used to the total α2 on the cell studies on αLβ2 and integrins have proposed that a glutamate residue in the α7 helix of the αI domain in and in can as an intrinsic ligand for the β subunit and mediate conformational regulation between the αI domain and the β subunit in two J.L. Essafi M. Xiong J.P. Stehle T. Arnaout M.A. Curr. Biol. (Lond.). 2002; 12: 340-342Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 4Yang W. Shimaoka M. Salas A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2906-2911Crossref PubMed Scopus (78) Google Scholar). of α2β1 integrin the that Glu-336 in the α2 subunit have a was using cells transfected with the α2 subunit the E336A E336A increases in the number of 12F1 on the cell surface the of Glu-336 in conformational the that integrin conformation was shown to participate in the regulation of it that likely to was on two the conformational change in α2β1 integrins was only after while the avidity to increase a increase in the number of 12F1 after with E336A mutation not the of In the mutation in which is an to E336A in the of TPA on avidity (8Kim M. Carman C.V. Yang W. Salas A. Springer T.A. J. Cell Biol. 2004; 167: 1241-1253Crossref PubMed Scopus (203) Google Scholar). In cell adhesion was in than in cells, which is in with that have the of ligand binding by and integrins after mutation in the residue J.L. Essafi M. Xiong J.P. Stehle T. Arnaout M.A. Curr. Biol. (Lond.). 2002; 12: 340-342Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The clustering of αLβ2 is after ligand and it is an mechanism in the of adhesion (8Kim M. Carman C.V. Yang W. Salas A. Springer T.A. J. Cell Biol. 2004; 167: 1241-1253Crossref PubMed Scopus (203) Google Scholar). binding to α2β1 integrin is to clustering and activation of (17Upla P. Marjomaöki V. Kankaanpaöaö P. Ivaska J. Hyypiaö T. Van Der Goot F.G. Heino J. Mol. Biol. Cell. 2004; 15: 625-636Crossref PubMed Scopus (147) Google Scholar). Thus, receptor clustering is a well However, the of ligand-independent integrin clustering in the regulation of avidity has been a M. Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar, C.V. Springer T.A. Curr. Cell Biol. 2003; 15: PubMed Scopus Google Scholar). using TPA was to the of α2β1 integrins in α2β1 cells and The of integrin aggregation was by two clustering place in less than after TPA similarly to the increase in the E336A mutation not prevent integrin and it not prevent the increase in The of TPA on α2β1 avidity and clustering likely mediated by activation of since a the of have shown that α2β1 clustering can the α of and of prevent integrin internalization (17Upla P. Marjomaöki V. Kankaanpaöaö P. Ivaska J. Hyypiaö T. Van Der Goot F.G. Heino J. Mol. Biol. Cell. 2004; 15: 625-636Crossref PubMed Scopus (147) Google Scholar). Thus, that α2β1 integrin clustering and may regulate other in both the structural of α2β1 integrin were also studies have shown that an integrin the α2 extracellular domain, and the domain of the subunit mediate J. Westermarck J. Koivisto Heino J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). In this this mutation the of the receptor to activated by TPA the of In both and collagen I the increase was the suggest that the of integrin α subunits may not participate in regulation by In collagen binding by cells transfected to integrin was also activated by that with β1 subunit T. A. S. M.J. J. 1999; PubMed Scopus Google Scholar) or integrin activation J. A. B. W. S.J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). For αIIbβ3 integrin has shown to be activated by TPA only and J. A. B. W. S.J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In cells α2 and β1 subunit were not by The suggest that of the α the β subunit is for We conclude that α2β1 integrin TPA activation and that receptor may to the increase in the avidity of adhesion. a has that ligand of αLβ2 integrin may A. B. M. F. I. R. J.A. M. C.G. Mol. Biol. Cell. PubMed Scopus Google Scholar), while in other receptor clustering has only ligand binding (8Kim M. Carman C.V. Yang W. Salas A. Springer T.A. J. Cell Biol. 2004; 167: 1241-1253Crossref PubMed Scopus (203) Google Scholar, C. Saltel F. Lussi J. Paulhe F. Imhof B.A. Wehrle-Haller B. J. Cell Biol. 2005; 171: 383-392Crossref PubMed Scopus (278) Google Scholar). In studies have that clustering regulate integrin avidity conformational activation of individual receptors Y. C.G. Curr. Cell Biol. 2000; 12: PubMed Scopus Google Scholar, R. B. A. J. P. 2002; PubMed Scopus (145) Google Scholar). report and suggest that the that activated by ligand mediated clustering may also structural activation seems to be for the avidity of the α2β1 integrin. Thus, a in which α2β1 clustering and conformational activation place in a synergistic We to from and F.