Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries

Background and Purpose: Calcitonin gene‐related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor‐like receptor (CLR) and receptor activity‐modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR•RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin‐converting enzyme‐1 (ECE‐1). However, it is not known if ECE‐1 regulates the resensitization of CGRP‐induced responses in functional arterial tissue. Experimental Approach: CLR, ECE‐1a‐d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA‐SMCs) and mesenteric arteries was analyzed by RT‐PCR and by immunofluorescence and confocal microscopy. CGRP‐induced signaling in cells was examined by measuring cAMP production and ERK activation. CGRP‐induced relaxation of arteries was measured by isometric wire myography. ECE‐1 was inhibited using the specific inhibitor, SM‐19712. Key Results: RMA‐SMCs and arteries contained mRNA for CLR, ECE‐1a‐d and RAMP1. ECE‐1 was present in early endosomes of RMA‐SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium‐independent relaxation of arteries. ECE‐1 inhibition had no effect on initial CGRP‐induced responses but reduced cAMP generation in RMA‐SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions and Implications: ECE‐1 regulates the resensitization of responses to CGRP in RMA‐SMCs and mesenteric arteries. CGRP‐induced relaxation does not involve endothelium‐derived pathways. This is the first report of ECE‐1 regulating CGRP responses in SMCs and arteries. ECE‐1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.

Multimodel estimates of intercontinental source-receptor relationships for ozone pollution

Understanding the surface O3 response over a “receptor” region to emission changes over a foreign “source” region is key to evaluating the potential gains from an international approach to abate ozone (O3) pollution. We apply an ensemble of 21 global and hemispheric chemical transport models to estimate the spatial average surface O3 response over east Asia (EA), Europe (EU), North America (NA), and south Asia (SA) to 20% decreases in anthropogenic emissions of the O3 precursors, NOx, NMVOC, and CO (individually and combined), from each of these regions. We find that the ensemble mean surface O3 concentrations in the base case (year 2001) simulation matches available observations throughout the year over EU but overestimates them by >10 ppb during summer and early fall over the eastern United States and Japan. The sum of the O3 responses to NOx, CO, and NMVOC decreases separately is approximately equal to that from a simultaneous reduction of all precursors. We define a continental-scale “import sensitivity” as the ratio of the O3 response to the 20% reductions in foreign versus “domestic” (i.e., over the source region itself) emissions. For example, the combined reduction of emissions from the three foreign regions produces an ensemble spatial mean decrease of 0.6 ppb over EU (0.4 ppb from NA), less than the 0.8 ppb from the reduction of EU emissions, leading to an import sensitivity ratio of 0.7. The ensemble mean surface O3 response to foreign emissions is largest in spring and late fall (0.7–0.9 ppb decrease in all regions from the combined precursor reductions in the three foreign regions), with import sensitivities ranging from 0.5 to 1.1 (responses to domestic emission reductions are 0.8–1.6 ppb). High O3 values are much more sensitive to domestic emissions than to foreign emissions, as indicated by lower import sensitivities of 0.2 to 0.3 during July in EA, EU, and NA when O3 levels are typically highest and by the weaker relative response of annual incidences of daily maximum 8-h average O3 above 60 ppb to emission reductions in a foreign region (<10–20% of that to domestic) as compared to the annual mean response (up to 50% of that to domestic). Applying the ensemble annual mean results to changes in anthropogenic emissions from 1996 to 2002, we estimate a Northern Hemispheric increase in background surface O3 of about 0.1 ppb a−1, at the low end of the 0.1–0.5 ppb a−1 derived from observations. From an additional simulation in which global atmospheric methane was reduced, we infer that 20% reductions in anthropogenic methane emissions from a foreign source region would yield an O3 response in a receptor region that roughly equals that produced by combined 20% reductions of anthropogenic NOx, NMVOC, and CO emissions from the foreign source region.

Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR(2)), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR(2) deubiquitination and its importance in trafficking and signaling of endocytosed PAR(2) are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR(2). Trapping PAR(2) in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR(2) with beta-arrestin2 or the duration of PAR(2)-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR(2) but not for regulating PAR(2) dissociation from beta-arrestin2 or PAR(2)-mediated ERK2 activation.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous alpha,beta-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.

Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor-mediated signalling

Key point summary • Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells. • Mutant mice with deficits in Ca2+ channel auxiliary α2δ-2 subunits are used as models of cerebellar ataxia. • Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing. • We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter-matched control mice. • These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ-2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias. Abstract Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’ du2J mouse model of ataxia and absence epilepsy represents a clean knock-out of the auxiliary Ca2+ channel subunit, α2δ-2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell (GC) layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone-Purkinje cell(IN-PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN-PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)-mediated modulation of spontaneous neuronal spike firing and CB1Rmediated presynaptic inhibition of synaptic transmission at IN-PC synapses in both +/du2J and du2J/du2J mutants; effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid, DCA, taurolithocholic acid, TLCA) and the selective agonists oleanolic acid (OA) and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide (CCDC) stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, assessed by confocal microscopy. DCA, TLCA and OA did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, determined by bioluminescence resonance energy transfer. CCDC stimulated a low level of TGR5 interaction with β-arrestin2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of extracellular signal regulated kinase (ERK1/2). BRET analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

Staphylococcus aureus lipoteichoic acid inhibits platelet activation and thrombus formation via the Paf receptor

Impaired healing is common in wounds infected with the major human pathogen Staphylococcus aureus, although the underlying mechanisms are poorly understood. Here, we show that S.aureus lipoteichoic acid (LTA) inhibits platelet aggregation caused by physiological agonists and S. aureus and reduced platelet thrombus formation in vitro. The presence of D-alanine on LTA is necessary for the full inhibitory effect. Inhibition of aggregation was blocked using a monoclonal anti-platelet activating factor receptor (PafR) antibody and Ginkgolide B, a well-defined PafR antagonist, demonstrating that the LTA inhibitory signal occurs via PafR. Using a cyclic AMP (cAMP) assay and a western blot for phosphorylated VASP, we determined that cAMP levels increase upon platelet incubation with LTA, an effect which inhibits platelet activation. This was blocked when platelets were preincubated with Ginkgolide B. Furthermore, LTA reduced haemostasis in a mouse tail-bleed assay.

Tackling drought stress: RECEPTOR-LIKE KINASES present new approaches

Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops.