Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29)
Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].
Kamata
T
T
Department of Vascular Biology, Scripps Research Intitute, La Jolla, CA 92037, USA.
Puzon
W
W
Takada
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells / Q000378:metabolism; D002412:Cations; D015815:Cell Adhesion Molecules / Q000378:metabolism; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D005353:Fibronectins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1
D000911:Antibodies, Monoclonal; D002412:Cations; D015815:Cell Adhesion Molecules; D000939:Epitopes; D005353:Fibronectins; D039041:Integrin alpha4beta1; D016023:Integrins; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1; D001224:Aspartic Acid