THE ROLE OF CONSERVED AMINO-ACID MOTIFS WITHIN THE INTEGRIN BETA(3) CYTOPLASMIC DOMAIN IN TRIGGERING FOCAL ADHESION KINASE PHOSPHORYLATION

Integrin-mediated adhesion of cells to extracellular matrix proteins t riggers a variety of intracellular signaling pathways including a casc ade of tyrosine phosphorylations, In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however , the molecular mechanisms regulating integrin-triggered FAK phosphory lation are not understood, Previous studies have shown that the integr in beta(1), beta(3), and beta(5) subunit cytoplasmic domains all conta in sufficient information to trigger FAK phosphorylation when expresse d in single-subunit chimeric receptors connected to an extracellular r eporter, In the present study, beta(3) cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin beta(3) cytoplasmic domain that regulate its ability to t rigger FAK phosphorylation, Cells transiently expressing chimeric rece ptors containing these mutant cytoplasmic domains were magnetically so rted and assayed for the tyrosine phosphorylation of FAK, Analysis of these mutants indicated that structural information in both the membra ne proximal and C-terminal segments of the beta(3) cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal seg ment of the beta(3) cytoplasmic domain, the highly conserved NPXY moti f was found to be required for the beta(3) cytoplasmic domain to trigg er FAK phosphorylation. However, the putative FAK binding domain withi n the N-terminal segment of the beta(3) cytoplasmic domain was found t o be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the be ta(2) cytoplasmic domain to signal FAK phosphorylation, suggesting tha t a single mutation in the beta(3) cytoplasmic domain can inhibit both ''inside-out'' and ''outside-in'' integrin signaling.