THE TYPE-III CONNECTING SEGMENT OF FIBRONECTIN CONTAINS AN ASPARTIC-ACID RESIDUE THAT REGULATES THE RATE OF BINDING TO INTEGRIN ALPHA(4)BETA(1)

The type III connecting segment (IIICS) within fibronectin is the majo r binding site for the integrin alpha(4) beta(1). Most integrin ligand s have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at posi tion 21. Alanine scanning mutagenesis was used to determine the amino acid residues within the intact IIICS domain required for interaction with alpha(4) beta(1). IIICS was cloned and expressed as a fusion prot ein with glutathione S-transferase. This recombinant form of IIICS sup ports the adhesion of CHO cells that express human alpha(4) beta(1) in a cation dependent manner. Alanine scanning mutagenesis of the EILDVP sequence in recombinant IIICS demonstrated that only two of these res idues are critical for adhesion of alpha(4) beta(1) expressing cells. Mutations of leucine at position 20 and aspartic acid at position 21 t o alanine significantly reduced cell adhesion. Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also re duced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacem ent. Most importantly, we show that although the mutation of asp 21 im pairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion throu gh alpha(4) beta(1). In comparison to wild type IIICS, the asp 21 to a la mutant supported minimal adhesion at early time points (10-30 min.) , but was equivalent to wild type IIICS in supporting adhesion over on e hour.