News and Publications

Structure and Function of Integrins

D. Calderwood, M. Cong, C. Fenczik, J. Han, P. Hughes, J. Lin, S. Liu, B. Oertli, J. Ramos, M. Roesel, D. Rose, D. Woodside, R. Zent, M. Ginsberg

The cytoplasmic domains of integrins connect these receptors to the cytoskeleton. Furthermore, interactions between integrins and the cytoskeleton involve binding of ligand (occupancy) to the integrin extracellular domain and clustering of the integrin. To construct mimics of the cytoplasmic face of an occupied and clustered integrin, we fused the cytoplasmic domains of integrin ß subunits to an N-terminal sequence containing 4 heptad repeat sequences. The heptad repeats form coiled-coil dimers in which the cytoplasmic domains are parallel, dimerized, and held in an appropriate vertical stagger.

In these mimics, we found that both conformation and protein-binding properties are altered by insertion of glycine spacers C-terminal to the heptad repeat sequences and that the cytoskeletal proteins talin and filamin are among the polypeptides that bind to the integrin ß_1A tail. Binding of filamin, but not talin, is enhanced by the insertion of glycine spacers. Binding of both cytoskeletal proteins to ß_1A is direct and specific, because it occurs with purified talin and filamin and is inhibited in a point mutant and in splice variants known to disrupt cytoskeletal associations of ß₁ integrins. In addition, the muscle-specific splice variant, ß_1D, binds talin more tightly than does ß_1A and is therefore predicted to form more stable cytoskeletal associations.The ß₇ cytoplasmic domain binds filamin better than does ß_1A. The structural specificity of these associations suggests that these mimics offer a useful approach for analysis of the interactions and structure of the integrin cytoplasmic face.

We also devised a genetic strategy to dissect integrin signaling pathways. Overexpression of isolated integrin ß₁ cytoplasmic domains blocks integrin activation (dominant suppression). Proteins involved in integrin signaling were detected by their capacity to complement dominant suppression in a cDNA expression cloning scheme. CD98, an early T-cell activation antigen that specifically associates with functional integrins, was cloned as a regulator of integrin activation. This unbiased approach should facilitate analysis of integrin signaling in cells or in multicellular organisms.

PUBLICATIONS

Du, X., Ginsberg, M.H. Integrin _IIbß₃ and platelet function. Thromb. Haemost. 78:96, 1997.

Du, X., Ginsberg, M.H. Signaling and platelet adhesion. In: Adhesive Interactions of Cells. Garrod, D.R., North, A., Chidgey, M.A.J. (Eds.). JAI Press, Greenwich, CT, in press.

Felding-Habermann, B., Silletti, S., Mei, F., Siu, C.H., Yip, P.M., Brooks, P.C., Cheresh, D.A., O'Toole, T.E., Ginsberg, M.H., Montgomery, A.M.P. A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins. J. Cell Biol. 139:1567, 1997.

Fenczik, C.A., Sethi, T., Ramos, J.W., Hughes, P.E., Ginsberg, M.H. Complementation of dominant suppression implicates CD98 in integrin activation. Nature 370:81, 1997.

Gao, J., Zoller, K.E., Ginsberg, M.H., Brugge, J.S., Shattil, S.J. Regulation of the pp72syk protein tyrosine kinase by platelet integrin _IIbß₃. EMBO J. 16:6414, 1997.

Huttenlocher, A., Palecek, S.P., Lu, Q., Zhang, W., Mellgren, R.L., Lauffenburger, D.A., Ginsberg, M.H., Horwitz, A.F. Regulation of cell migration by the calcium-dependent protease calpain. J. Biol. Chem. 272:32719, 1997.

Indig, F.E., Diaz-Gonzalez, F., Stuiver, I., Ginsberg, M.H. Analysis of the tetraspanin CD9-integrin _IIbß₃ (GPIIb-IIIa) complex in platelet membranes and transfected cells. Biochem. J. 327:291, 1997.

Pfaff, M., Liu, S., Erle, D.J., Ginsberg, M.H. Integrin beta cytoplasmic domains differentially bind to cytoskeletal proteins. J. Biol. Chem. 273:6104, 1998.

Ross, R., Vu, H., Fenczik, C., Glembotski, C.C., Ginsberg, M.H., Loftus, J.C. Integrins participate in the hypertrophic response of rat ventricular myocytes. Circ. Res., in press.

Shattil, S.J., Ginsberg, M.H. Integrin signaling in vascular biology. J. Clin. Invest. 100:1, 1997.