IDENTIFICATION OF A FUNCTIONALLY IMPORTANT SEQUENCE IN THE CYTOPLASMIC TAIL OF INTEGRIN BETA(3) BY USING CELL-PERMEABLE PEPTIDE ANALOGS

Integrins are major two-way signaling receptors responsible for the at tachment of cells to the extracellular matrix and for cell-cell intera ctions that underlie immune responses, tumor metastasis, and progressi on of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta(3) (glycoprotein ma ) based on the cellular import of synthetic peptide analogs of this re gion. Among the four overlapping cell-permeable peptides, only the pep tide carrying residues 747-762 of the carboxyl-terminal segment of int egrin beta(3) inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen med iated by integrin beta(3) heterodimers, alpha(IIb)beta(3), and alpha(v ) beta(3), respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta(3) peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta(1) het erodimers. Conversely, a cell-permeable peptide representing homologou s portion of the integrin beta(1) cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta (3) peptide (residues 747-762) carrying a known loss-of-function mutat ion (Ser(752)Pro) responsible for the genetic disorder Glanzmann throm basthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cell s, whereas the beta(3) peptide carrying a Ser(752)Ala mutation was inh ibitory. Although Ser(752) is not essential, Tyr(747) and Tyr(759) for m a functionally active tandem because conservative mutations Tyr(747) Phe or Tyr(759)Phe resulted in a nonfunctional cell permeable integrin beta(3) peptide. We propose that the carboxyl-terminal segment of the integrin pg cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modula tes the interaction of integrin beta(3)-expressing cells with immobili zed fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition ''from within'' of the adhesive function of the se integrins.