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 major binding site for the integrin alpha 4 beta 1. Most integrin ligands have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at position 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 protein with glutathione S-transferase. This recombinant form of IIICS supports 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 residues are critical for adhesion of alpha 4 beta 1 expressing cells. Mutations of leucine at position 20 and aspartic acid at position 21 to alanine significantly reduced cell adhesion. Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also reduced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacement. Most importantly, we show that although the mutation of asp 21 impairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion through alpha 4 beta 1. In comparison to wild type IIICS, the asp 21 to ala mutant supported minimal adhesion at early time points (10-30 min.), but was equivalent to wild type IIICS in supporting adhesion over one hour.
Jongewaard
I N
IN
Program on Cell Adhesion and Extracellular Matrix, La Jolla Cancer Research Foundation, CA 92037, USA.
Tsai
P M
PM
Smith
J W
JW
D000596:Amino Acids / Q000502:physiology; D000818:Animals; D001224:Aspartic Acid / Q000502:physiology*; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017469:Receptors, Fibronectin / Q000378:metabolism*; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D005353:Fibronectins; D039041:Integrin alpha4beta1; D016023:Integrins; D017469:Receptors, Fibronectin; D016201:Receptors, Lymphocyte Homing; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D008345:Manganese; D005982:Glutathione Transferase; D008274:Magnesium