The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin beta subunits to the cytoskeleton.
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 alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 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.
Moes
Michèle
M
Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), University of Luxembourg, L-1511 Luxembourg, Grand Duchy of Luxembourg, Luxembourg.
Rodius
Sophie
S
Coleman
Stacey J
SJ
Monkley
Susan J
SJ
Goormaghtigh
Erik
E
Tremuth
Laurent
L
Kox
Corinne
C
van der Holst
Patrick P G
PP
Critchley
David R
DR
Kieffer
Nelly
N
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003599:Cytoskeleton / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006868:Hydrolysis; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D013055:Spectrophotometry, Infrared; D016608:Talin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016023:Integrins; D016608:Talin