We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.

The large and functionally diverse group of G-protein-coupled receptors includes receptors for many different signalling molecules, including peptide and non-peptide hormones and neurotransmitters, chemokines, prostanoids and proteinases. Their principal function is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins, and they thereby participate in many aspects of regulation. Cellular responses to agonists of these receptors are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis and downregulation. Agonists are removed by dilution, uptake by transporters and enzymic degradation. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second-messenger kinases, interaction of phosphorylated receptors with arrestins and receptor uncoupling from

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E 2 and F 1␣ from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E 2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

Proteinase-activated receptor-2 (PAR-2) is a G-protein-coupled receptor that is expressed by intestinal epithelial cells, which are episodically exposed to pancreatic trypsin in the intestinal lumen. Trypsin cleaves PAR-2 to expose a tethered ligand, which irreversibly activates the receptor. Thus, PAR-2 may desensitize and resensitize by novel mechanisms. We examined these mechanisms in kidney epithelial cells, stably expressing human PAR-2, and intestinal epithelial cells, which naturally express PAR-2. Trypsin stimulated a prompt increase in [Ca 2؉ ] i , due to mobilization of intracellular Ca 2؉, followed by a sustained plateau, due to influx of extracellular Ca 2؉ . Repeated application of trypsin caused marked desensitization of this response, which is due in part to (a) irreversible cleavage of the receptor by trypsin and (b) protein kinase C-mediated termination of signaling. Trypsin exposure resulted in internalization of PAR-2 into early endosomes and then lysosomes; but endocytosis was not the mechanism of rapid desensitization. Thus, activated PAR-2 is endocytosed and degraded. The Ca 2؉ response to trypsin resensitized by 60 -90 min. Brefeldin A, which disrupted Golgi stores of PAR-2, and cycloheximide, which inhibited protein synthesis, markedly attenuated resensitization. Thus, PAR-2-mediated Ca 2؉ mobilization desensitizes by irreversible receptor cleavage, protein kinase C-mediated termination of signaling, and PAR-2 targeting to lysosomes. It resensitizes by mobilization of large Golgi stores and synthesis of new receptors.Cellular responses to agonists of G-protein-coupled receptors are rapidly attenuated in the continuous presence of agonist and desensitize to repeated application of agonist (1, 2). With time between challenges, responses recover and cells resensitize. These are important processes, since they determine the ability of cells to respond to agonists; desensitization prevents the uncontrolled stimulation of cells, whereas resensitization allows cells to recover or maintain their responsiveness. Desensitization and resensitization are controlled at the level of the receptors and at more distal steps in the signaling pathway, but regulation of the receptors is of critical importance. The mechanisms may be distinct for different classes of G-proteincoupled receptors. Receptors for hormones and neurotransmitters, exemplified by the ␤ 2 AR 1 and the NK1-R, bind agonists reversibly and desensitize in a reversible manner.