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) Ser 38 constructs recruited ␤-arrestins-1 or -2 in response to trypsin stimulation, whereas both ␤-arrestins were recruited to these mutants by SLIGRL-NH 2 . The lack of trypsin-triggered ␤-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsinstimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of G␣ i (pertussis toxin),, or the epidermal growth factor (EGF) receptor [4-(3Ј-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rhokinase inhibitor (R)-(ϩ)-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.Proteinase-activated receptors (PARs) are unique members of the G-protein-coupled superfamily of receptors (GPCRs), modeled as seven transmembrane domain cell-surface receptors that mediate diverse signaling events in response to proteolytic exposure of an N-terminal tethered ligand (TL) sequence. PAR 2 , the second member of this family to be cloned (Nystedt et al., 1994;Bohm et al., 1996) ABBREVIATIONS: PAR, proteinase-activated receptor; A23187, calcimycin; AG1478, 4-(3Ј-chloroanilino)-6,7-dimethoxy-quinazoline; BRET, bioluminescence resonance energy transfer; DKO, double-knockout ␤-arrestin-deficient mouse embryo-derived fibroblasts; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GP2A,pyrazole-3-carboxamide; GPCR, G-protein-coupled receptor; H1152, (S)-(ϩ)-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; PAR 2 or wt-rPAR 2 , wild-type rat proteinase-activated receptor-2 having the trypsin-revealed tethered ligand sequence SLIGRL-; PAR 2 -Ala 37-38 , mutated rat PAR 2 with a trypsin-revealed tethered ligand sequence AAIGRL-; PAR 2 -Ala 39 -42 , mutated rat PAR 2 with a trypsin-revealed tethered ligand sequence SLAAAA-; PAR 2 -Leu 37 Ser 38 , mutated rat PAR 2 with a trypsin-revealed tethered ligand sequence LSIGRL...

Beta-arrestins-1 and 2 are known to play important roles in desensitization of membrane receptors and facilitation of signal transduction pathways. It has been previously shown that beta-arrestins are required for signal termination, internalization, and ERK1/2 activation downstream of protease-activated-receptor-2 (PAR-2), but it is unclear whether they are functionally redundant or mediate specific events. Here, we demonstrate that in mouse embryonic fibroblasts (MEFs) from beta-arrestin-1/2 knockout mice, G alpha q signaling by PAR-2, as measured by mobilization of intracellular Ca(2+), is prolonged. Only expression of beta-arrestin-1 shortened the signal duration, whereas either beta-arrestin-1 or 2 was able to restore PKC-induced receptor desensitization. Beta-arrestin-1 also mediated early, while beta-arrestin-2 mediated delayed, receptor internalization and membrane-associated ERK1/2 activation. While beta-arrestin-1 colocalized with a lysosomal marker (LAMP-1), beta-arrestin-2 did not, suggesting a specific role for beta-arrestin-1 in lysosomal receptor degradation. Together, these data suggest distinct temporal and functional roles for beta-arrestins in PAR-2 signaling, desensitization, and internalization.

Background: Many GPCRs that utilize ␤-arrestins differ with respect to downstream signaling and cellular consequences. Results: Exchanging the C termini of two GPCRs switches the ␤-arrestin responses and relative affinities for the two receptors. Conclusion: Sequences within the C termini of different GPCRs are important for determining the nature of ␤-arrestin recruitment and signaling. Significance: These studies provide new insight regarding receptor-specific ␤-arrestin signals.

Protease Activated Receptor‐2 (PAR‐2) is a G Protein Coupled Receptor (GPCR), whose ligand is tethered to its N‐terminus and is activated only when it's N‐terminus is removed by an extracellular protease. Upon activation, PAR‐2 couples to Gq to mobilize intracellular calcium, leading to activation of both Phophatidyl Inositol 3 Kinase (PI3K) and Extracellular signal Regulated Kinase (ERK1/2). Paradoxically, PAR‐2‐evoked ERK1/2 activation requires beta‐arrestins 1 and 2, which are traditionally known to play a role in the desensitization and the internalization of receptors. Recent studies have demonstrated that beta‐arrestins can also mediate & generate independent signaling, but a distinct role of each individual beta‐arrestin in PAR‐2 desensitization, internalization and signaling has not been elucidated. Here we show that each isoform of beta‐arrestin can mediate the internalization, desensitization & the downstream signaling response following PAR‐2 activation. However, while beta‐arrestin‐1 is required for early, beta‐arrestin‐2 is required for prolonged receptor internalization and ERK1/2 activation. Furthermore, beta‐arrestin‐1 is sufficient to target a pool of PAR‐2 to lysosomes, while beta‐arrestin‐2 does not. These studies suggest that beta‐arrestins are not redundant in PAR‐2 signaling but work cooperatively to elicit specific downstream responses. α

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