Integrin alpha 4 cysteines 278 and 717 modulate VLA-4 ligand binding and also contribute to alpha 4/180 formation.
Here we describe experiments in which we mutated four of the six integrin alpha 4 subunit cysteine residues that are not present in most other integrin alpha subunits that lack an I domain. In four different types of ligand binding assay we found that optimal integrin alpha 4 beta 1 and/or to CS1 peptide required the presence of both alpha 4 Cys 278 and Cys 717. In addition, optimal ligand binding required divalent cations and reduced cysteines, as evidenced by EDTA and N-ethylmaleimide inhibition results. In a control experiment, an alpha 4 mutation that completely eliminated the alpha 4 80/70 proteolytic cleavage site had no effect on ligand binding. Notably, although Cys 278 an Cys 717 mutations markedly altered ligand binding, they had no adverse effect on cell adhesion. Thus, compared with cell adhesion, ligand binding is a distinct and apparently more stringent test of VLA-4 integrin-ligand interactions. In addition, we have established that the formation of the previously described alpha 4/180 [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028-2035] also requires Cys 278 and Cys 717, divalent cations and reduced cysteines. thus alpha 4/180 appears to be more functionally relevant than alpha 4/150.
Pujades
C
C
Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Teixidó
J
J
Bazzoni
G
G
Hemler
M E
ME
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D039441:Integrin alpha4; D039041:Integrin alpha4beta1; D016023:Integrins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D051379:Mice; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D015703:Antigens, CD; D039041:Integrin alpha4beta1; D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D010455:Peptides; D016201:Receptors, Lymphocyte Homing; D019010:Vascular Cell Adhesion Molecule-1; C076687:CS1 peptide; D039441:Integrin alpha4; D003545:Cysteine