A biochemical characterization of the binding of osteopontin to integrins alpha v beta 1 and alpha v beta 5.
Osteopontin (OPN) is an extracellular matrix protein that binds to integrin alpha v beta 3. Here we demonstrate that two other integrins, alpha v beta 1 and alpha v beta 5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin alpha v beta 1. When the 293 cells are transfected with the beta 5 subunit, they can also adhere to GST-OPN using integrin alpha v beta 5. Divalent cations regulate the binding of GST-OPN to both alpha v beta 1 and alpha v beta 5. Mg2+ and Mn2+ support the binding of GST-OPN to these integrins but Ca2+ does not. The highest affinity is observed in Mn2+. In the presence of this ion, the affinity of GST-OPN for alpha v beta 1 is 18 nM and the affinity for alpha v beta 5 is 48 nM. The antibody 8A2, which is an agonist for beta 1, promotes the adhesion of 293 cells to GST-OPN even when Ca2+ is present. This observation suggests that cellular events could modulate the affinity of alpha v beta 1 for OPN. Collectively, these findings prove that integrins alpha v beta 1, alpha v beta 3, and alpha v beta 5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.
Hu
D D
DD
Cancer Research Center, La Jolla Cancer Research Foundation, California 92037, USA.
Lin
E C
EC
Kovach
N L
NL
Hoyer
J R
JR
Smith
J W
JW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002448:Cell Adhesion; D002460:Cell Line; D003001:Cloning, Molecular; D005434:Flow Cytometry; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D016023:Integrins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007680:Kidney Neoplasms; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D053495:Osteopontin; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D013724:Teratoma; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016023:Integrins; D009842:Oligopeptides; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C064660:integrin alphaVbeta5; C094340:integrin alphavbeta1; D053495:Osteopontin; D008345:Manganese; D005982:Glutathione Transferase; D008274:Magnesium; D002118:Calcium