A fragment of paxillin binds the α4 integrin cytoplasmic domain (tail) and selectively inhibits α4-mediated cell migration

Abstract

The α4 integrins play important roles in embryogenesis, hematopoiesis, cardiac development, and the immune responses. The α4 integrin subunit is indispensable for these biological processes, possibly because the α4 subunit regulates cellular functions differently from other integrin a subunits. We have previously reported that the a4 cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule, and this interaction accounts for some of the unusual functional responses to α4 integrin-mediated cell adhesion. We also have identified a conserved 9-amino acid region (Glu983-Tyr991) in the α4 cytoplasmic domain that is sufficient for paxillin binding, and an alanine substitution at either Glu983 or Tyr991 within this region disrupted the α4-paxillin interaction and reversed the effects of the α4 cytoplasmic domain on cell spreading and migration. In the current study, we have mapped the α4-binding site within paxillin using mutational analysis, and examined its effects on the α4 tail-mediated functional responses. Here we report that sequences between residues Ala176 and Asp275 of paxillin are sufficient for binding to the α4 tail. We found that the α4 tail, paxillin, and FAT, the focal adhesion targeting domain of pp125FAK, could form a ternary complex and that the α4-binding paxillin fragment, P(Ala176-Asp275), specifically blocked paxillin binding to the α4 tail more efficiently than it blocked binding to FAT. Furthermore, when expressed in cells, this α4-binding paxillin fragment specifically inhibited the α4 tail-stimulated cell migration. Thus, paxillin binding to the α4 tail leads to enhanced cell migration and inhibition of the α4-paxillin interaction selectively blocks the α4-dependent cellular responses.