Specific interaction of SPARC with endothelial cells is mediated through a carboxyl-terminal sequence containing a calcium-binding EF hand.
SPARC is a secreted, Ca(2+)-binding protein that modulates cell shape and gene expression (Sage, E.H., and Bornstein, P. (1991) J. Biol. Chem. 266, 14831-14834). In the present study we questioned whether SPARC interacted with an endothelial cell surface receptor. The binding of 125I-SPARC to bovine aortic endothelial cells was dependent on Ca2+ and was sensitive to small changes in extracellular pH; maximal binding occurred at pH 7.1. Scatchard analysis indicated approximately 2.3 x 10(7) binding sites/cell with an apparent KI of 1.1 nM. The interaction was diminished specifically by competition with synthetic peptides corresponding to amino acids 54-73 (SPARC 54-73) and 254-273 (SPARC254-273). The binding of 125I-SPARC254-273, a sequence containing a Ca(2+)-binding EF-hand, was saturated within 45 min at a concentration of 5 microM; Scatchard analysis indicated 4.2 x 10(7) sites/cell and a KI of 2.4 nM. Iodinated proteins from plasma membranes were affinity-chromatographed on SPARC254-273; several proteins with apparent masses ranging from 153 to 100 kDa (unreduced) or from 153 to 122 kDa (reduced) were eluted with the soluble peptide. These proteins represent candidates for a SPARC receptor(s) that mediates the biological activity of this protein on endothelial cells.
Yost
J C
JC
Department of Biological Structure, University of Washington, Seattle 98195.
Sage
E H
EH
D000818:Animals; D001011:Aorta; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D015676:Osteonectin / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism
D015676:Osteonectin; D010446:Peptide Fragments; D002118:Calcium