Mechanisms of Desensitization and Resensitization of Proteinase-activated Receptor-2

Abstract

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.

Presence of functionally active protease‐activated receptors 1 and 2 in myenteric glia

“…The intracellular origin of the initial calcium increase has been demonstrated in epithelial cell and myenteric neurons, where activation of these receptors induces increased [Ca 2+ ] i 22 in absence of extracellular calcium (Bohm et al 1996;Corvera et al 1999).…”

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

“…No immunofluorescence was seen when only the second antibody was applied or when the primary antibody was pre-incubated with its immunizing peptide (not shown). Since PAR-2 has been shown to be internalized upon activation by trypsin or activating peptide AP2 (Bohm et al, 1996a), we also performed immunofluorescence studies following incubation of HT-29 cells with either activator. It appeared that cell surface labelling dramatically decreased (Figure 3), further assessing for the relevance of the immunofluorescence.…”

Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway

“…injection of OVA and alum (10 g in 0.2 ml Al(OH) 3 ) on days 0 and 14 (23). To examine infiltration of immune cells into BAL, mice were exposed to a single challenge by OVA aerosol (5%, 20 min) or PBS aerosol (100 mM, 20 min, control) on day 21 and were studied 24, 72, or 96 h later.…”

“…Proteolytic activation of PARs is irreversible and cleaved receptors internalize and are degraded in lysosomes (3,4). Therefore, PARs are single-use receptors that probably mediate signaling during pathophysiological or "emergency" conditions when PAR agonists are generated or released.…”