Cellular Localization of Membrane-type Serine Protease 1 and Identification of Protease-activated Receptor-2 and Single-chain Urokinase-type Plasminogen Activator as Substrates

• Published in: The Journal of biological chemistry Vol. 275; no. 34; pp. 26333 - 26342
• Main Authors: Takeuchi, T, Harris, J L, Huang, W, Yan, K W, Coughlin, S R, Craik, C S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25.08.2000
• Subjects: Animals; Binding Sites; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular - metabolism; HeLa Cells; Humans; Models, Molecular; Rabbits; Receptor, PAR-2; Receptors, Thrombin - metabolism; Serine Endopeptidases - metabolism; Structure-Activity Relationship; Substrate Specificity; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator - metabolism; Xenopus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002941200

Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-( X )P2-(Ser)P1-(Arg)P1′-(Ala)) and (P4-( X )P3-(Arg/Lys)P2-(Ser)P1(Arg) P1′(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 n m but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 n m . Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 n m ), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.