The 41 integrin binds osteopontin
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abstract
Osteopontin (OPN) is an extracellular matrix protein whose expression is upregulated at sites of inflammation and cardiovascular injury. Here, we present data showing that OPN can promote leukocyte adhesion through the 41 integrin. Using cell adhesion assays, OPN promoted dose-dependent adhesion of a model leukocyte cell line, HL-60 in the presence of physiologic concentrations of Ca2+ and Mg2+. Adhesion of HL-60 cells was inhibited by anti-integrin antibodies directed at either the 4 or 1 integrin subunits but not by control antibodies directed to other HL-60 cell integrins. Further adhesion experiments revealed that HL-60 and Ramos (a second leukocyte cell line) cell binding to OPN was completely inhibited by the 41-binding peptide Leu-Asp-Val (LDV), while the control, non-binding peptide, Leu-Glu-Val (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the 41 integrin bound specifically to OPN. Immunoprecipitation of EDTA eluted fractions from this column positively identified the 41 integrin. In order to identify potential 41-binding sites within OPN, the protein was proteolytically cleaved with thrombin. Using fast protein liquid chromatography, a major 30 kDa N-terminal fragment was isolated and found to promote 41 dependent leukocyte adhesion in a manner similar to that of the intact protein. These data clearly demonstrate that the 41 integrin is a new adhesion receptor for OPN and that an 41 binding site exists in the NH2-terminal thrombin fragment of OPN.
