Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases.
The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.
Mirza
H
H
Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8151, USA.
Schmidt
V A
VA
Derian
C K
CK
Jesty
J
J
Bahou
W F
WF
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D053818:Chymases; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis*; D015398:Signal Transduction; D053802:Tryptases
D007377:Interleukin-3; D009842:Oligopeptides; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C509190:Tpsb2 protein, mouse; C099570:seryl-leucyl-isoleucyl-glycyl-arginyl-leucine; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509222:Tpsab1 protein, mouse; D053802:Tryptases