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.
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-uPA(RRHR), 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 approximately 700-fold from 4.50 x 10(4) M(-1) x s(-1) for wild-type ch-uPA to 3.02 x 10(7) M(-1) x s(-1) for ch-uPA(RRHR), and the ability to form SDS-stable, uPA-PAI-1 complexes increased approximately 1000-fold. Furthermore, the interaction of ch-uPA(RRHR) with PAI-2 was also substantially enhanced, while the interaction with other members of the serine proteinase inhibitor superfamily, protein nexin 1, alpha1-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-uPA(RRHR) 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.
Sipley
J D
JD
Department of Pathology, State University of New York, Stony Brook 11794, USA.
Alexander
D S
DS
Testa
J E
JE
Quigley
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology*; D017396:Plasminogen Activator Inhibitor 2 / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D017396:Plasminogen Activator Inhibitor 2; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator