A deletion mutant of vitronectin lacking the somatomedin B domain exhibits residual plasminogen activator inhibitor-1-binding activity.
Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (rDeltasBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.
Schar
Christine R
CR
Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Blouse
Grant E
GE
Minor
Kenneth H
KH
Peterson
Cynthia B
CB
D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D017353:Gene Deletion; D006493:Heparin / Q000737:chemistry; D006801:Humans; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013002:Somatomedins / Q000378:metabolism*; D018411:Spodoptera / Q000378:metabolism; D020298:U937 Cells; D014461:Ultracentrifugation; D019096:Vitronectin / Q000235:genetics* / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D006493:Heparin