Integrin αIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of β3-subunits in actin cytoskeletal organization

Using truncated or mutated alpha(IIb) integrin cytoplasmic domains fused to the alpha(V) extracellular domain and expressed with the beta(3) integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alpha(IIb) integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta(3) integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alpha(IIb)-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alpha(IIb) did not affect beta(3) tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta(3) phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta(3) integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta(3) integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta(3) phosphorylation is demonstrated without mutation of the beta(3) subunit. In support of our previous hypothesis of a role for beta(3) phosphorylation in adhesion, these studies demonstrate a strong correlation between beta(3) tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.