title: Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin.
abstract: Osteopontin (OPN) is a ligand for the α4ß1 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of post-translational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines and compared OPN interaction with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn(2+): the EC50 of adhesion was not affected by phosphorylation or glycosylation status. Thrombin cleavage of OPN at the C-terminus of the α4 integrin-binding site also did not affect binding affinity. THP-1 cells express a low-affinity conformation of the integrin and adhered to OPN only in the presence of Mn(2+) plus PMA or an activating antibody. This was in contrast to VCAM and fibronectin: THP-1 cells adhered to these ligands without integrin activation. Studies with ligand-induced binding site antibodies demonstrated that the SVVYGLR peptide of OPN bound to the α4 integrin with a similar affinity as the LDV peptide of fibronectin, suggesting that a high off-rate is responsible for the reduced binding of OPN to the low-affinity forms of this integrin. Together, the results suggest OPN has very low affinity for the α4 integrin on human leukocytes under physiological conditions.
authors.affiliation: Department of Immunology and Infectious Disease, The Forsyth Institute, Cambridge, MA, USA.
authors.forename: Tommy
authors.initials: T
authors.lastname: Hui
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
authors.affiliation: Department of Immunology and Infectious Disease, The Forsyth Institute, Cambridge, MA, USA; Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA, USA. Electronic address: srittling@forsyth.org.
authors.forename: Susan R
authors.initials: SR
authors.lastname: Rittling
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D006801:Humans; D039441:Integrin alpha4 / Q000737:chemistry / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000378:metabolism; D019169:Jurkat Cells; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D053495:Osteopontin / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D013917:Thrombin / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D039081:Integrin alpha5beta1; C506895:SPP1 protein, human; D019010:Vascular Cell Adhesion Molecule-1; D053495:Osteopontin; D039441:Integrin alpha4; D013917:Thrombin
keywords: Adhesion; Integrin Alpha4; Osteopontin; Phosphorylation; VCAM; VLA-4