ABSTRACT
The serpin plasminogen activator inhibitor type-1 (PAI-1), as the primary physiological inhibitor of both urokinase-type (uPA) and tissue-type (tPA) plasminogen activator, plays an important role in the regulation of the fibrinolytic system as well as in extracellular remodeling in both physiological and pathophysiological processes. In plasma as well as in the extracellular matrix PAI-1 binds to vitronectin (Vn), an interaction that affects the function of both proteins. As PAl-1/Vn interaction has a significant regulatory function in fibrinolysis, thrombolysis, and cell adhesion in cancer spread, there is a strong interest in defining the binding sites on PAI-1 and Vn as the basis of a rational design of novel drugs that may modulate PAI-1/Vn-mediated effects. In this minireview, we give an overview on the approaches to define the Vn binding site of PAI-1 and vice versa. Although in the case of PAI-1 the region around alpha-helix E and alpha-helix F of PAI-1 has been demonstrated to be important for its interaction with Vn, the precise location of the Vn-binding region has not completely been resolved. The major high-affinity PAI-1 binding region of Vn is localized within the N-terminal somatomedin B (SMB) domain of Vn. There are indications for at least one other low-affinity PAI-1 binding site in the C-terminal region of Vn, which seems to be involved in the formation of larger PAI-1/Vn complexes.
Subject(s)
Plasminogen Activator Inhibitor 1/metabolism , Vitronectin/metabolism , Binding Sites , Humans , Plasminogen Activator Inhibitor 1/chemistry , Protein Binding , Vitronectin/chemistryABSTRACT
The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.
