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-Ala^37–38, and rPAR₂-Ala^39–42 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-Ala^39–42 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-Ala^37–38 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 Gα_i (pertussis toxin), Gα_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 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.