Abstract
We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR2) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR2 and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR2-Leu37Ser38, rPAR2-Ala37–38, and rPAR2-Ala39–42 were compared with the trypsin-revealed wild-type rPAR2 TL sequence, S37LIGRL42—. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR2 and rPAR2-Ala39–42 triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR2-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 rPAR2-Ala37–38 nor rPAR2-Leu37Ser38 constructs recruited β-arrestins-1 or -2 in response to trypsin stimulation, whereas both β-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered β-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 Gαi (pertussis toxin), Gαq [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 PAR2, its arrestin recruitment, and its internalization. Thus, PAR2 can signal to multiple pathways that are differentially triggered by distinct proteinase-revealed TLs or by synthetic signal-selective activating peptides.
- PAR, proteinase-activated receptor
- A23187, calcimycin
- AG1478, 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline
- BRET, bioluminescence resonance energy transfer
- DKO, double-knockout β-arrestin-deficient mouse embryo-derived fibroblasts
- EGF, epidermal growth factor
- EGFR, epidermal growth factor receptor
- ERK, extracellular signal-regulated kinase
- GP2A, N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide
- GPCR, G-protein-coupled receptor
- H1152, (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride
- HEK, human embryonic kidney
- KNRK, normal rat kidney cell line transformed by Kirsten murine sarcoma virus
- MAP, mitogen-activated protein
- MAPK, mitogen-activated protein kinase
- MEF, mouse embryo-derived fibroblasts
- MMP, matrix metalloproteinase
- PAR2 or wt-rPAR2, wild-type rat proteinase-activated receptor-2 having the trypsin-revealed tethered ligand sequence SLIGRL—
- PAR2-Ala37–38, mutated rat PAR2 with a trypsin-revealed tethered ligand sequence AAIGRL—
- PAR2-Ala39–42, mutated rat PAR2 with a trypsin-revealed tethered ligand sequence SLAAAA—
- PAR2-Leu37Ser38, mutated rat PAR2 with a trypsin-revealed tethered ligand sequence LSIGRL—
- PBS, phosphate-buffered saline
- PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine
- PTX, pertussis toxin
- r, rat
- TBST, Tris-buffered saline/Tween 20
- TL, tethered ligand
- wt, wild type
- YFP, yellow fluorescent protein
- Y27362, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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These studies were supported in part the Canadian Institutes of Health Research [Grants MT-13759, MOP-53289] (to M.D.H.), [Grant FRN11215] (to M.B.); the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM066151] (to K.D.); and by a Canadian Association of Gastroenterology/Canadian Institutes of Health Research/Ortho-Jensen postdoctoral fellowship.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- Received February 27, 2009.
- Accepted July 14, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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