Olivier Déry

Proteinase-activated receptors: novel mechanisms of signaling by serine proteases

Although serine proteases are usually considered to act principally as degradative enzymes, certain proteases are signaling molecules that specifically regulate cells by cleaving and triggering members of a new family of proteinase-activated receptors (PARs). There are three members of this family, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, a receptor for trypsin and mast cell tryptase. Proteases cleave within the extracellular NH2-terminus of their receptors to expose a new NH2-terminus. Specific residues within this tethered ligand domain interact with extracellular domains of the cleaved receptor, resulting in activation. In common with many G protein-coupled receptors, PARs couple to multiple G proteins and thereby activate many parallel mechanisms of signal transduction. PARs are expressed in multiple tissues by a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation. Because the cleaved receptor is physically coupled to its agonist, efficient mechanisms exist to terminate signaling and prevent uncontrolled stimulation. These include cleavage of the tethered ligand, receptor phosphorylation and uncoupling from G proteins, and endocytosis and lysosomal degradation of activated receptors.

Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation.

Thrombin and mast cell tryptase regulate guinea‐pig myenteric neurons through proteinase‐activated receptors‐1 and −2

1 Proteases regulate cells by cleaving proteinase‐activated receptors (PARs). Thrombin and trypsin cleave PAR‐1 and PAR‐2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR‐1 and PAR‐2. 2 We detected immunoreactive PAR‐1 and PAR‐2 in > 60 % of neurons from the myenteric plexus of guinea‐pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR‐1 and PAR‐2. We confirmed expression of PAR‐1 and PAR‐2 in the myenteric plexus by RT‐PCR using primers based on sequences of cloned guinea‐pig receptors. 3 Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR‐1 and PAR‐2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR‐1 agonists responded to PAR‐2 agonists, and > 90 % of PAR‐1 and PAR‐2 responsive neurons responded to ATP. 4 These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR‐1 and PAR‐2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Trafficking of Proteinase-activated Receptor-2 and β-Arrestin-1 Tagged with Green Fluorescent Protein β-ARRESTIN-DEPENDENT ENDOCYTOSIS OF A PROTEINASE RECEPTOR

Proteases cleave proteinase-activated receptors (PARs) to expose N-terminal tethered ligands that bind and activate the cleaved receptors. The tethered ligand, once exposed, is always available to interact with its binding site. Thus, efficient mechanisms must prevent continuous activation, including receptor phosphorylation and uncoupling from G-proteins, receptor endocytosis, and lysosomal degradation. β-Arrestins mediate uncoupling and endocytosis of certain neurotransmitter receptors, which are activated in a reversible manner. However, the role of β-arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subcellular distribution of β-arrestins have not been examined. We studied trafficking of PAR2 and β-arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of β-arrestin1 from the cytosol to the plasma membrane, where it co-localized with PAR2; (b) internalization of β-arrestin1 and PAR2 into the same early endosomes; (c) redistribution of β-arrestin1 to the cytosol concurrent with PAR2 translocation to lysosomes; and (d) mobilization of PAR2 from the Golgi apparatus to the plasma membrane. Overexpression of a C-terminal fragment of β-arrestin-319–418, which interacts constitutively with clathrin but does not bind receptors, inhibited agonist-induced endocytosis of PAR2. Our results show that β-arrestins mediate endocytosis of PAR2 and support a role for β-arrestins in uncoupling of PARs.

Mast cell tryptase and proteinase‐activated receptor 2 induce hyperexcitability of guinea‐pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long‐term hyperexcitability in submucosal neurons from the guinea‐pig ileum by activating proteinase‐activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT‐PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL‐NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min–hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR‐NH2) had no effect on neuronal membrane potential or long‐term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5‐HT, prostaglandins) also caused depolarization, and following washout of antigen, long‐term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long‐term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and β-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of β-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or β-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of β-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and β-arrestin1 into the same endosomes containing SP and the transferrin receptor (2–10 min); (c) prolonged colocalization of the NK1R and β-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and β-arrestin1 in the cytosol (4–6 h). SP stimulated a similar redistribution of immunoreactive β-arrestin1 and β-arrestin2. In contrast, SP did not affect Gαq/11distribution, which remained at the plasma membrane. Expression of the dominant negative β-arrestin319–418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of β-arrestins to the plasma membrane, where they participate in NK1R endocytosis. β-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and β-arrestins return to the cytosol.

Dynamin and Rab5a-dependent Trafficking and Signaling of the Neurokinin 1 Receptor

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca2+ mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

Heterologous regulation of trafficking and signaling of G protein-coupled receptors: β-Arrestin-dependent interactions between neurokinin receptors

Cells express multiple G protein-coupled receptors that are simultaneously or sequentially activated by agonists. The consequences of activating one receptor on signaling and trafficking of another receptor are unknown. We examined the effects of selective activation of the neurokinin 1 receptor (NK1R) on signaling and trafficking of the NK3R and vice versa. Selective agonists of NK1R and NK3R induced membrane translocation of β-arrestins (β-ARRs). Dominant negative β-ARR319–418 inhibited endocytosis of NK1R and NK3R. Whereas an NK1R agonist caused sequestration of NK1R with β-ARR in the same endosomes, thereby depleting them from the cytosol, β-ARRs did not prominently sequester with the activated NK3R and rapidly returned to the cytosol. In cells coexpressing both receptors, prior activation of the NK1R inhibited endocytosis and homologous desensitization of the NK3R, which was dose-dependently reversed by overexpression of β-ARR1. Similar results were obtained in enteric neurons that naturally coexpress the NK1R and NK3R. In contrast, activation of the NK3R did not affect NK1R endocytosis or desensitization. Thus, the high-affinity and prolonged interaction of the NK1R with β-ARRs depletes β-ARRs from the cytosol and limits their role in desensitization and endocytosis of the NK3R. Because β-ARRs are critical for desensitization, endocytosis, and mitogenic signaling of many receptors, this sequestration is likely to have important and widespread implications.