News and Publications

Structure and Function of Integrins

The development and functioning of multicellular animals depend on integrins. These adhesion receptors link to the actin cytoskeleton, resulting in transmission of biochemical signals and of force during cell migration and interactions with the extracellular matrix. We found that a simple, reversible, posttranslational modification of an integrin, when appropriately regulated in time and space, gives directionality to a migrating cell.

We have been studying an apparent paradox of cellular migration: the effect of α₄ integrin phosphorylation. The cytoplasmic side of α₄ mediates migration by binding to paxillin, a signaling protein in the focal adhesion complex, which in turn interacts with cytoskeletal and signaling proteins. Previously, we found that phosphorylation of α₄ integrin inhibits paxillin binding. Thus, it follows that constitutive phosphorylation of the integrin blocks cell movement. However, versions of α₄ that cannot be phosphorylated also block migration, indicating that the phospho group is not simply an on-off switch for movement.

We have now discovered that α₄ phosphorylation is restricted to the leading edge, where paxillin and integrin do not associate. Forced association of α₄ and paxillin throughout the cell, obtained either by fusing the 2 proteins or by using a mutant α₄ version that cannot be phosphorylated, inhibited migration by destabilizing lamellipodia. Thus, the spatial regulation of α₄ integrin phosphorylation by protein kinase A means that interaction of α₄ with paxillin inhibits misplaced lamellipodia from forming along the sides and rear of the cell. At the leading edge, where α₄ and paxillin do not interact, the lamellipodium remains stable, and the cell can move in its chosen direction.

Most integrin ß cytoplasmic tails contain 1 or 2 NPXY/F motifs that can form ß turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding domains, protein modules present in a wide variety of signaling and cytoskeletal proteins. Indeed, the signaling proteins talin and integrin cytoplasmic domain-associated protein-1 α bind to integrin ß tails via an interaction between a phosphotyrosine-binding domain and an NPXY ligand. To assess the generality of this interaction, we examined the binding of a series of recombinant phosphotyrosine-binding domains to a panel of short integrin ß tails.

In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated phosphotyrosine-binding domains bound to integrin tails. Furthermore, Dok-1 physically associated with integrin α_IIbß₃. Mutations of the integrin ß tails confirmed that these interactions are canonical interactions between phosphotyrosine-binding domains and ligands. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions occurred with the phosphotyrosine-binding domains of proteins such as Dab, EPS8, and tensin. We used this specificity and a molecular model of interaction between an integrin ß tail and a phosphotyrosine-binding domain to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in ß₇ and ß₃ tails. These data establish that short integrin ß tails interact with a large number of proteins containing phosphotyrosine-binding domains through a structurally conserved mechanism.

Publications

Ambroise, Y., Yaspan, B., Ginsberg, M.H., Boger, D.L. Inhibitors of cell migration that inhibit intracellular paxillin/α₄ binding: a well-documented use of positional scanning libraries. Chem. Biol. 9:1219, 2002.

Baker, S.E., Lorenzen, J.A., Miller, S.W., Bunch, T.A., Jannuzi, A.L., Ginsberg, M.H., Perkins, L.A., Brower, D.L. Genetic interaction between integrins and moleskin, a gene encoding a Drosophila homolog of importin-7. Genetics 162:285, 2002.

Calderwood, D.A., Fujioka, Y., de Pereda, J.M., Garcia-Alvarez, B., Nakamoto, T., Margolis, B., McGlade, C.J., Liddington, R.C., Ginsberg, M.H. Integrin ß cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling. Proc. Natl. Acad. Sci. U. S. A. 100:2272, 2003.

Calderwood, D.A., Yan, B., de Pereda, J.M., Garcia-Alvarez, B., Fujioka, Y., Liddington, R.C., Ginsberg, M.H. The phosphotyrosine binding-like domain of talin activates integrins. J. Biol. Chem. 277:21749, 2002.

Garcia-Alvarez, B., de Pereda, J.M., Calderwood, D.A., Ulmer, T.S., Critchley, D., Campbell, I.D., Ginsberg, M.H., Liddington, R.C. Structural determinants of integrin recognition by talin. Mol. Cell 11:49, 2003.

Hansen, M., Rusyn, E.V., Hughes, P.E., Ginsberg, M.H., Cox, A.D., Willumsen, B.M. R-Ras C-terminal sequences are sufficient to confer R-Ras specificity to H-Ras. Oncogene 21:4448, 2002.

Hill, J.M., Vaidyanathan, V., Ramos. J.W., Ginsberg, M.H., Werner, M.H. Recognition of ERK MAP kinase by PEA-15 reveals a common docking site within the death domain and death effector domain. EMBO J. 21:6494, 2002.

Hughes, P.E., Oertli, B., Hansen, M., Chou, F.-L., Willumsen, B.M., Ginsberg, M.H. Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase. Mol. Biol. Cell 13:2256, 2002.

Liddington, R.C., Ginsberg, M.H. Integrin activation takes shape. J. Cell Biol. 158:833, 2002.

Lin, J.Y., Pollack, J.R., Chou, F.-L., Rees, C.A., Christian, A.T., Bedford, J.S., Brown, P.O., Ginsberg, M.H. Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays. Genome Biol. 3:RESEARCH0026, 2002.

Liu, S., Kiosses, W.B., Rose, D.M., Slepak, M., Salgia, R., Griffin, J.D., Turner, C.E., Schwartz, M.A., Ginsberg, M.H. A fragment of paxillin binds the α₄ integrin cytoplasmic domain (tail) and selectively inhibits α₄-mediated cell migration. J. Biol. Chem. 277:20887, 2002.

Riederer, M.A., Ginsberg, M.H., Steiner, B. Blockade of platelet GPIIB-IIIA (integrin α_IIbß₃) in flowing human blood leads to passivation of prothrombotic surfaces. Thromb. Haemost. 88:858, 2002.

Rose, D.M., Han J., Ginsberg, M.H. Alpha 4 integrins and the immune response. Immunol. Rev. 186:118, 2002.

Schwartz, M.A., Ginsberg, M.H. Networks and crosstalk: integrin signaling spreads. Nat. Cell Biol. 4:E65, 2002.

Woodside, D.G., Obergfell, A., Talapatra, A., Calderwood, D.A., Shattil, S.J., Ginsberg, M.H. The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin ß cytoplasmic domains. J. Biol. Chem. 277:39401, 2002.