Critical amino acid residues of the alpha 4 subunit for alpha 4 beta 7 integrin function

A characteristic feature of integrin-ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified i n the alpha and beta subunit of the alpha4 integrins, alpha4 beta1 and alph a4 beta7, and within their ligands which display the tripeptide LDV in fibr onectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular dom ain of the murine and human alpha4-subunit contains three conserved LDV mot ifs, designated LDV-1 to -3. Using site directed mutagenesis and transfecti on studies, we now examined the functional relevance of the LDV motifs for alpha4 beta7 integrins. We present evidence that LDV-1 mutants (D489N) beha ve like alpha4 wt cells, but LDV-3 mutants (D881N) are impaired in alpha4 b eta7 integrin-triggered homotypic cell aggregation and in adhesion and spre ading on a4 specific ligands. Further characterization of LDV-3 mutants rev ealed a defect in mAb-induced alpha4 beta7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of alpha4 beta7 integrin ce ll surface expression. Our results indicate: (i) that LDV-3, located proxim al to the cell membrane, is important for alpha4 beta7 integrin-triggered f unctions and for lateral clustering and (ii) that LDV-2 affects alpha4 beta 7 heterodimer stability. (C) 2001 Wiley-Liss, Inc.