Identification of Amino Acid Sequences in the Integrin/31 Cytoplasmic Domain Implicated in Cytoskeletal Association

Abs t rac t . Wild-type and mutant chicken integrin/31 subunit (/31o) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-/3L: antibodies. Mutant/31c integrins containing internal deletions of 13 amino acids adjacent to the membrane, A759-771, and 20 centrally located amino acids, A771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (79%800). The l 1-residue cyto-1 signal is only found on integrin/3 subunit sequences, except/34. Four residues within this region, 13764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an tx helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conforrnation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (79%800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.