β1 Integrin Cytoplasmic Domain Residues Selectively Modulate Fibronectin Matrix Assembly and Cell Spreading through Talin and Akt-1

J.Angelo Green,Allison L Berrier,Roumen Pankov,Kenneth M Yamada

doi:10.1074/jbc.m805934200

Abstract

The integrin beta(1) cytoplasmic domain (tail) serves as a scaffold for numerous intracellular proteins. The mechanisms by which the tail coordinates these proteins to facilitate extracellular matrix assembly and cell spreading are not clear. This study demonstrates that the beta(1) cytoplasmic domain can regulate cell spreading on fibronectin and fibronectin matrix assembly through Akt- and talin-dependent mechanisms, respectively. To identify these mechanisms, we characterized GD25 cells expressing the beta(1) integrin cytoplasmic domain mutants W775A and R760A. Although cell spreading appears normal in R760A mutant-integrin cells compared with wild type, it is inhibited in W775A mutant cells. In contrast, both mutant cell lines show defective fibronectin matrix assembly. Inhibition of cell spreading, but not matrix assembly, in the W775A mutant cells is due to a specific defect in Akt-1 activation. In addition, we find that both W775A and R760A mutant integrins have reduced surface expression of the 9EG7 epitope that correlates with reduced recruitment of talin to beta(1) integrin cytoplasmic complexes. Down-regulation of talin with small interfering RNA or expression of green fluorescent protein-talin head domain inhibits matrix assembly in beta(1) wild-type cells, mimicking the defect seen with the W775A and R760A mutant cells. These results demonstrate distinct mechanisms by which integrins regulate cell spreading and matrix assembly through the beta(1) integrin cytoplasmic tail.

Highlights

A peptide containing both this tryptophan residue and an NPXY motif inhibited talin binding to a recombinant ␤1A integrin cytoplasmic domain (6)
It has been recently demonstrated that the W775A ␤1 integrin mutation directly inhibits binding of talin to the ␤1 integrin cytoplasmic domain and inhibits integrin activation (35)
To our knowledge, the R760 ␤1 integrin cytoplasmic residue itself has not been implicated until now in regulating talin recruitment to the cytoplasmic domain, the membrane proximal region containing the homologous residue in the ␤3 cytoplasmic tail (Arg-724) interacts with talin (34)

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Antibodies against ␤1 integrin were rat 9EG7 (BD Pharmingen), mouse K20 (Immunotech), mouse 12G10 (22), mouse 4B4 (Beckman Coulter), and rabbit antibody Rab 4080 (23). 5 ϫ 105 cells were incubated with an anti-integrin antibody or control IgG at 10 ␮g/ml final concentration for 40 min on ice, washed three times with BSA buffer, and stained with fluorescein isothiocyanate-conjugated secondary antibodies. 5 ϫ 106 cells were incubated with fluorescein isothiocyanate-conjugated K20 (anti-total ␤1 integrin antibody) for 30 min on ice. After the antibody incubation, the cells were washed with PBSϪ/1% BSA and resuspended in complete DMEM. The bead complexes were subsequently washed five times with cold CSK buffer containing 0.5% Triton X-100 and 300 mM NaCl. To elute the proteins from the beads, 2ϫ SDS sample buffer (Invitrogen) was added, and the samples were probe-sonicated with a Microson XL ultrasonic cell disrupter at setting 2–3 for three 5-s periods to reduce the viscosity of the samples. One-way analysis of variance with pairwise multiple comparisons made using the Tukey-Kramer test

RESULTS

We therefore tested whether expression of constitutive active

Protein Depletion of Talin Inhibits Fibronectin Matrix

Cell spreading