TY - JOUR
T1 - The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin β subunits to the cytoskeleton
AU - Moes, Michèle
AU - Rodius, Sophie
AU - Coleman, Stacey J.
AU - Monkley, Susan J.
AU - Goormaghtigh, Erik
AU - Tremuth, Laurent
AU - Kox, Corinne
AU - Van Der Holst, Patrick P.G.
AU - Critchley, David R.
AU - Kieffer, Nelly
PY - 2007/6/8
Y1 - 2007/6/8
N2 - Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in α helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this α helix, which disrupted the α-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to αIIbβ3 integrin in focal adhesions and to inhibit in vitro this association as shown by an αIIbβ3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1-/- cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.
AB - Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in α helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this α helix, which disrupted the α-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to αIIbβ3 integrin in focal adhesions and to inhibit in vitro this association as shown by an αIIbβ3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1-/- cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.
UR - http://www.scopus.com/inward/record.url?scp=34447116981&partnerID=8YFLogxK
U2 - 10.1074/jbc.M611846200
DO - 10.1074/jbc.M611846200
M3 - Article
C2 - 17430904
AN - SCOPUS:34447116981
SN - 0021-9258
VL - 282
SP - 17280
EP - 17288
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 23
ER -