Identification of the ligand binding site for the integrin α9β1 in the third fibronectin type III repeat of tenascin-C

Abstract

The integrin α9 subunit forms a single heterodimer, α9β1 that mediates cell adhesion to a site within the third fibronectin type III repeat of tenascin-C (TNfn3). In contrast to at least 3 other integrins that bind to this region of tenascin-C, α9β1 does not recognize the common integrin recognition motif, Arg-Gly-Asp (RGD). In this report, we have used substitution mutagenesis to identify a unique ligand recognition sequence in TNfn3. We introduced mutations substituting alanine for each of the acidic residues in or adjacent to each of the exposed loops predicted from the solved crystal structure. Most of these mutations had little or no effect on adhesion of α9-transfected SW480 colon carcinoma cells, but mutations of either of two acidic residues in the B-C loop region markedly reduced attachment of these cells. In contrast, cells expressing the integrin α(v)β3, previously reported to bind to the RGD sequence in the adjacent F- G loop, attached to all mutant fragments except one in which the RGD site was mutated to RAA. The peptide, AEIDGIEL, based on the sequence of human tenascin-C in this region blocked the binding of α9-transfected cells, but not β3-transfected cells to wild type TNfn3. This sequence contains a tripeptide, IDG, homologous to the sequences LDV, IDA, and LDA in fibronectin and IDS in VCAM-1 recognized by the closely related integrin α4β1. These findings support the idea that this tripeptide motif serves as a ligand binding site for the α4/α9 subfamily of integrins.