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Teleocidin A2 inhibits human proteinase-activated receptor 2 signaling in tumor cells.

ABSTRACT: Enhanced expression of the proteinase-activated receptor 2 (PAR2) is linked to cell proliferation and migration in many cancer cell types. The role of PAR2 in cancer progression strongly illustrates the need for PAR2-inhibiting compounds. However, to date, potent and selective PAR2 antagonists have not been reported. The natural product teleocidin A2 was characterized against PAR2-activating peptide SLIGKV-NH ₂, and trypsin-induced PAR2-dependent intracellular Ca²⁺ mobilization in tumor and in primary endothelial or epithelial cells. Further biochemical and cell-based studies were conducted to evaluate teleocidin specificity. The antagonizing effect of teleocidin A2 was confirmed in PAR2-dependent cell migration and rearrangement of actin cytoskeleton of human breast adenocarcinoma cell line (MDA-MB 231) breast cancer cells. Teleocidin A2 antagonizes PAR2-dependent intracellular Ca²⁺ mobilization induced by either SLIGKV-NH ₂ or trypsin with IC ₅₀ values from 15 to 25 nmol/L in MDA-MB 231, lung carcinoma cell line, and human umbilical vein endothelial cell. Half maximal inhibition of either PAR1 or P2Y receptor-dependent Ca²⁺ release is only achieved with 10- to 20-fold higher concentrations of teleocidin A2. In low nanomolar concentrations, teleocidin A2 reverses both SLIGKV-NH ₂ and trypsin-mediated PAR2-dependent migration of MDA-MB 231 cells, and has no effect itself on cell migration and no effect on cell viability. Teleocidin A2 further controls PAR2-induced actin cytoskeleton rearrangement of MDA-MB 231 cells. Thus, for the first time, the small molecule natural product teleocidin A2 exhibiting PAR2 antagonism in the low nanomolar range with potent antimigratory activity is described.

SUBMITTER: Stahn S

PROVIDER: S-EPMC5242168 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Teleocidin A2 inhibits human proteinase-activated receptor 2 signaling in tumor cells.

Stahn Sonja S Thelen Lisa L Albrecht Ina-Maria IM Bitzer Jens J Henkel Thomas T Teusch Nicole Elisabeth NE

Pharmacology research & perspectives 20160610 4

Enhanced expression of the proteinase-activated receptor 2 (PAR2) is linked to cell proliferation and migration in many cancer cell types. The role of PAR2 in cancer progression strongly illustrates the need for PAR2-inhibiting compounds. However, to date, potent and selective PAR2 antagonists have not been reported. The natural product teleocidin A2 was characterized against PAR2-activating peptide SLIGKV-NH <sub>2</sub>, and trypsin-induced PAR2-dependent intracellular Ca<sup>2+</sup> mobiliza ...[more]

PMID: 28116092

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Project description:We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR(2)) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR(2) and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR(2)-Leu(37)Ser(38), rPAR(2)-Ala(37-38), and rPAR(2)-Ala(39-42) were compared with the trypsin-revealed wild-type rPAR(2) TL sequence, S(37)LIGRL(42)-. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR(2) and rPAR(2)-Ala(39-42) triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR(2)-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR(2)-Ala(37-38) nor rPAR(2)-Leu(37)Ser(38) constructs recruited beta-arrestins-1 or -2 in response to trypsin stimulation, whereas both beta-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered beta-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Galpha(i) (pertussis toxin), Galpha(q) [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the proteolytically revealed TL sequence(s) and the mode of its presentation to the receptor (tethered versus soluble) can confer biased signaling by PAR(2), its arrestin recruitment, and its internalization. Thus, PAR(2) can signal to multiple pathways that are differentially triggered by distinct proteinase-revealed TLs or by synthetic signal-selective activating peptides.

Project description:The C-terminal tail of G-protein-coupled receptors (GPCR) contain important regulatory sites that enable interaction with intracellular signaling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383-385, Ser387-Thr392) and the helix-8 palmitoylation site (Cys361) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined Gαq/11-coupled calcium signaling, β-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background with both peptide stimulation of the receptor (SLIGRL-NH2) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser383-385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated β-arrestin recruitment. In contrast, alanine substitutions in the Ser387-Thr392 cluster resulted in a large (∼50%) decrease in β-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated β-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a large decrease in both Gαq/11 coupling and β-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gαq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of β-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of β-arrestins also enhanced Gαq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased β-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate β-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.

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Teleocidin A2 inhibits human proteinase-activated receptor 2 signaling in tumor cells.

Publications

Teleocidin A2 inhibits human proteinase-activated receptor 2 signaling in tumor cells.

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