Introduction of an RRHR motif into chicken urokinase-type plasminogen activator (ch-uPA) confers sensitivity to plasminogen activator inhibitor (PAI)-1 and PAI-2 and allows ch-uPA-mediated extracellular matrix degradation to be controlled by PAI-1
Abstract
Comparison of the amino acid sequence of the chicken and human urokinase-type plasminogen activators (uPAs) revealed that the putative PAI-binding site found in the variable region 1 (VR1) loop of mammalian PAs is absent in the homologous region of ch-uPA. ch-uPA, unlike mammalian PAs, also appears to be refractory to inhibition by human PAIs and as a naturally occurring PAI-resistant variant, constitutes a unique model system for assessing the functional relevance of the PAI-binding site. Therefore, we molecularly constructed a ch-uPA, ch-uPARRHR, which contains the putative PAI-binding motif RRHR (residues 192-195) in its VR1 loop. As a result of this substitution, the second-order rate constant of inhibition of PAI-1 increased ≈700-fold from 4.50 × 104 M-1.s-1 for wild-type ch-uPA to 3.02 × 107 M-1.s-1 for ch-uPARRHR, and the ability to form SDS-stable, uPA-PAI-1 complexes increased ≈1000-fold. Furthermore, the interaction of ch-uPARRHR with PAI-2 was also substantially enhanced, while the interaction with other members of the serine proteinase inhibitor superfamily, protein nexin 1, α1-PI, and C1-inhibitor, was unaffected indicating that the RRHR motif is not a general serine proteinase inhibitor binding site. Finally, we show that extracellular matrix degradation by cells expressing ch-uPARRHR is inhibited by PAI-1 in a dose-dependent manner, while matrix breakdown by cells expressing wild-type ch-uPA is unaffected by PAI-1. Thus acquisition of sensitivity to PAI-1 through a structural motif that enhances the specificity of the protease-inhibitor interaction confers to ch-uPA an added level of regulation in the context of the degradative cellular phenotype.
- Publication:
-
Proceedings of the National Academy of Science
- Pub Date:
- April 1997
- DOI:
- 10.1073/pnas.94.7.2933
- Bibcode:
- 1997PNAS...94.2933S