Neutrophil Elastase Acts as a Biased Agonist for Proteinase-activated Receptor-2 (PAR2)

Abstract

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR 2 ). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G q -coupled PAR 2 calcium signaling; but all of these proteinases can disarm PAR 2 , releasing the N-terminal TL sequence, thereby preventing G q -coupled PAR 2 signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR 2 -mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ␤-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR 2 , downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR 2 signaling by preventing/disarming the G q /calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR 2 signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

“…6D). These results suggest that elastase stimulates ERK1/2 activation and cAMP accumulation in DRG by a PAR 2 -dependent process, consistent with our current observations and with published studies showing similar stimulator actions of elastase in KNRK-PAR 2 but not KNRK-VC cells (32).…”

“…Previous studies using immunofluorescence and confocal microscopy suggest that whereas trypsin-activated PAR 2 undergoes endocytosis, elastase-activated PAR 2 remains at the plasma membrane (32,49). To quantitatively assess PAR 2 trafficking at the plasma membrane, we measured the BRET signal between PAR 2 -RLuc8 and two proteins that reside at the plasma membrane: KRas-Venus, which is present in cholesterol-independent microdomains (50), and RIT-Venus, which is uniformly distributed.…”

“…Because elastase cleaves PAR 2 distal from the trypsin site, elastase can remove the trypsin-exposed tethered ligand and disarm the receptor to subsequent activation by trypsin (32,49). To determine whether elastase also disarms PAR 2 in oocytes, we expressed PAR 2 and examined trypsin-evoked PAR 2 activation of whole cell currents.…”

“…biased agonism). Such proteases include cathepsin S, which cleaves at Gly 41 2Lys 42 (30) (32). However, the precise molecular mechanisms and the physiological consequences of biased protease signaling are poorly defined.…”

“…Elastase cleaves PAR 1 at Leu 45 2Arg 46 , distal to the thrombin cleavage site, which reveals a tethered ligand domain (RNPND-KYEPF-NH 2 ) that activates G␣ i/o -mediated ERK signaling (34). Elastase cleaves PAR 2 at Ser 67 2Val 68 , distal to the trypsin cleavage site, which activates PAR 2 by a mechanism that does not involve exposure of a tethered ligand domain (32). However, the functional importance of the elastase-biased agonism of PAR 2 is uncertain.…”