Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates.
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 nm but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nm. Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nm), 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.
Takeuchi
T
T
Department of Pharmaceutical Chemistry and Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA.
Harris
J L
JL
Huang
W
W
Yan
K W
KW
Coughlin
S R
SR
Craik
C S
CS
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D011817:Rabbits; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D014981:Xenopus
D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; C101392:MTSP-1 serine protease; D012697:Serine Endopeptidases; D014568:Urokinase-Type Plasminogen Activator