Proteinase-Activated Receptor-2 and Human Lung Epithelial Cells

Abstract

Proteinase-activated receptor (PAR)-2 is cleaved within its aminoterminal extracellular domain by serine proteinases such as trypsin, unmasking a new aminoterminus starting with the sequence SLIGKV, which binds intramolecularly and activates the receptor. PAR-2 has been reported to be involved in inflammation within the lungs. We show that PAR-2 is expressed not only by human alveolar (A549), but also by bronchial (16HBE) epithelial cell lines, using RT-PCR and flow cytometry with a PAR-2 antibody whose epitope maps over the trypsin cleavage site. PAR-2 activation by trypsin and by the activating peptide SLIGKV-NH(2) leads to intracellular calcium mobilization in both lung epithelial cells. During lung inflammation, airspaces are burdened by neutrophils that release elastase and cathepsin G, two serine proteinases. We demonstrate that these proteinases do not activate PAR-2, but rather disarm the receptor, preventing activation by trypsin but not by SLIGKV-NH(2). Preincubation of a PAR-2-transfected cell line, as well as 16HBE and A549 cells, with either proteinase led to the disappearance of the cleavage/activation epitope recognized by the PAR-2 antibody. We hypothesize that elastase and cathepsin G disarm PAR-2 by proteolysis of the extracellular domain downstream from the trypsin cleavage/activation site, while leaving unmodified the SLIGKV-NH(2)-binding site. These findings suggest that the neutrophil serine proteinases may play a role in PAR-2-mediated lung inflammation.

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

“…Because elastase cleaves PAR 2 distal to the trypsin cleavage site, elastase pretreatment can prevent subsequent activation by trypsin, and thereby "disarm" the receptor (49,56). We found that pretreatment with elastase prevented trypsin signals in oocytes, which confirms this mechanism.…”

“…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.…”

Activity of recombinant trypsin isoforms on human proteinase‐activated receptors (PAR): mesotrypsin cannot activate epithelial PAR‐1, ‐2, but weakly activates brain PAR‐1

“…Such cleavage may prevent receptor activation by amputating or destroying the tethered ligand, depending upon the site where the additional cleavage occurs. Chymotrypsin is known to disable PAR-1 (Vouret-Craviari et al, 1995), cathepsin G can disable PAR-1 and PAR-2 (Molino et al, 1995;Dulon et al, 2003;Sokolova et al, 2005), and bacterial metalloprotease thermolysin was shown by us to disable PAR-2 in HBE and A549 cells (Ubl et al, 2002). We have tested the possibility for mesotrypsin to act as a disabling protease.…”

The contribution of PARs to inflammation and immunity to fungi

“…24 Fungi control secretory responses of PMNs, including protease activity, through TLR signaling. 22,23 Therefore, we hypothesized that fungi, through TLR activation, stimulate the release of proteases from PMNs that cleave PAR 1 and PAR 2 and modulate their activity.…”