Abstract
Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade 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 phosphorylation are not understood. Previous studies have shown that the integrin beta1, beta3, and beta5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, beta3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin beta3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the beta3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the beta3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the beta3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the beta3 cytoplasmic domain was found to 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 beta3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the beta3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling.
Highlights
Cellular environment [2,3,4,5]
To identify the amino acids within the 3 cytoplasmic domain required to signal increases in FAK phosphorylation, we made a series of amino acid deletions and substitutions in the 3 cytoplasmic domain (Fig. 1) based on three criteria: (a) the identity of amino acid residues important for FAK binding in vitro [41], (b) the identity of the amino acids replaced by alternative splicing of 1B and 3B [25], and (c) the regions of homology in the C-terminal regions of the 1, 3, and 5 cytoplasmic domains known to contain sufficient information to trigger FAK phosphorylation [25, 34]
Integrin-triggered FAK Phosphorylation ing cells and triggered FAK phosphorylation in a manner dependent upon the presence of the integrin  cytoplasmic domain [34]. Elements in Both the N-terminal and C-terminal Segments of the 3 Cytoplasmic Domain Are Required to Trigger FAK Phosphorylation—Differences in the abilities of the 3 and 3B cytoplasmic domains to trigger FAK phosphorylation suggested either that the C-terminal amino acids of the 3 cytoplasmic domain are required for signaling FAK phosphorylation or that the C-terminal amino acids of the 3B cytoplasmic domain inhibit this signaling event
Summary
Cellular environment [2,3,4,5]. Integrin engagement triggers intracellular signals that direct cell adhesion and regulate other aspects of cell behavior including cell proliferation, differentiation, and survival. The 3 deletion mutant, 3-d742–762, which lacked the C-terminal amino acids that differ between the 3 and 3B cytoplasmic domains, consistently did not signal FAK phosphorylation above background levels
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