Critical amino acid residues of the α4 subunit for α4β7 integrin function

A characteristic feature of integrin–ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the α and β subunit of the α4 integrins, α4β1 and α4β7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human α4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for α4β7 integrins. We present evidence that LDV-1 mutants (D489N) behave like α4 wt cells, but LDV-3 mutants (D811N) are impaired in α4β7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on α4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced α4β7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of α4β7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for α4β7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects α4β7 heterodimer stability. J. Cell. Biochem. 83: 304–319, 2001. © 2001 Wiley-Liss, Inc.