P2X1 receptors and the endothelium

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, a, b methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.

The effect of combined polymorphisms in chemokines and chemokine receptors on the clinical course of HIV-1 infection in a Brazilian population

Polymorphisms in genes that encode chemokines or their receptors can modulate susceptibility to human immunodeficiency virus (HIV) infection and disease progression. The objective of this study was to assess the frequency of polymorphisms CCR5-Δ32, CCR2-64I, CCR5-59029A and SDF1-3'A and their role in the course of HIV infection in a Southern Brazilian population. Clinical data were obtained from 249 patients for an average period of 6.4 years and genotypes were determined by standard polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Survival analyses were conducted for three outcomes: CD4+ T-cell counts below 200 cells/µL, acquired immune deficiency syndrome (AIDS) or death. The frequency of the polymorphisms CCR5-Δ32, CCR2-64I, CCR5-59029A and SDF1-3'A were 0.024, 0.113, 0.487 and 0.207, respectively. CCR5-Δ32 was associated with a reduction in the risk for CD4+ T-cell depletion and with an increased risk for death after AIDS diagnosis. CCR2-64I was associated with a reduction in the risk for developing AIDS. SDF1-3'A was also associated with decreased risk for AIDS, but its effect was only evident when CCR2-64I was present as well. These results highlight the possibility of using these markers as indicators for the prognosis of disease progression and provide evidence for the importance of analysing the effects of gene polymorphisms in a combined fashion.

Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.

The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1.

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

The α1-adrenergic receptors: diversity of signaling networks and regulation.

The α(1)-adrenergic receptor (AR) subtypes (α(1a), α(1b), and α(1d)) mediate several physiological effects of epinephrine and norepinephrine. Despite several studies in recombinant systems and insight from genetically modified mice, our understanding of the physiological relevance and specificity of the α(1)-AR subtypes is still limited. Constitutive activity and receptor oligomerization have emerged as potential features regulating receptor function. Another recent paradigm is that β arrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. The aim of this review is to summarize our current knowledge on some recently identified functional paradigms and signaling networks that might help to elucidate the functional diversity of the α(1)-AR subtypes in various organs.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

Characterization of Tollip in the Interleulkin-1 Receptor/Toll Like Receptors signaling pathways

SUMMARY IL-1R and TLRs are key players in innate immunity and inflammation. Tollip was identified as a component of IL-1RI, TLR2 and TLR4 signaling complexes that activate NF-κB and MAP kinase pathways. Tollip was previously shown as a negative regulator of NF-κB and MAP Kinase activation. We have characterized the role of Tollip in IL-R/TLRs induced signaling by the analysis of the Tollip deficient mice. We showed that NF-κB and MAPK (p38, JNK, or ERK1/2) signaling appeared normal in Tollip deficient cells following stimulation with IL-1β, lipopolysaccharide (LPS), and other TLR ligands. Also IL-1β and TLRs ligands induced activation of immune cells was indistinguishable from wild-type cells. Strikingly, in Tollip deficient mice the production of the inflammatory cytokines, IL-6 or TNF-α was significantly reduced relative to control mice after treatment with physiological doses of IL-1β or LPS, whereas no difference was observed at high doses of stimulation with LPS or in LPS induced septic shock. Therefore, Tollip could be critical for regulation of optimal responses to IL-1β and LPS, in addition to its role as negative regulator of the signaling. We also studied the role of Tollip as an endocytic adaptor for IL-1R endocytosis. We could show that Il-1R is ubiquitinated after IL-1β stimulation, and that Tollip's CUE domain binds IL-1RI in an ubiquitin-dependent manner. We followed IL-1R internalization and Tollip localization by confocal microscopy. Consistent with a role for Tollip in sorting of ubiquitinated IL-1RI, a significant amount of Tollip was also localized at the late endosomal compartment. We could show that Tollip is required for efficient lysosomal targeting of ubiquitinated IL-1R1, In the absence of Tollip or in Tollip deficient cells reconstituted with a Tollip mutant (defective in ubiquitin binding) IL-1RI accumulates in enlarged late endosomes. In addition, Tollip was shown to interact with, another endocytic adapter, Toml, and both interact with IL-1RI. In conclusion, we showed that Tollip is required for IL-1β and LPS signaling for cytokine production. In addition we showed and that Tollip has a role as an endocytic adapter, necessary for efficient trafficking and lysosomal degradation of IL-1RI. Resumé Le récepteur à l'interleukine-1 (IL-1R) et les récepteurs "Toll-like" (TLRs) sont des acteurs cruciaux de la réponse immunitaire innée et de l'inflammation. La proteine Tollip a été identifiée comme étant un élément des complexes de signalisation, induits par les récepteurs IL-1RI, TLR-2 et TLR-4, qui mènent à l'activation de la voie des MAP kinases et de NF-κB. Dans de précédentes études, il a été montré que Tollip pouvait inhiber ces deux voies de signalisation. Nous avons voulu caractériser plus précisément le rôle de Tollip dans l'activation des voies de signalisation mitées par IL-1R/TLRs en utilisant une lignée murine déficiente pour la protéine Tollip. Ainsi, en absence de Tollip, les cascades d'activation de NF-κB et MAPK (p38, JNK, or ERK1/2) ne semblent pas affectées après stimulation avec IL-1β, lipopolysaccharide (LPS) ou d' autres ligands des TLR. La réponse des cellules du système immunitaire induite par la stimulation avec IL-1β et les ligands des TLR est également comparable entre les souris sauvages et les souris deficientes pour Tollip. Par contre, dans cette lignée murine, la production de cytokines proinflammatoires IL-6 et TNFα induite par la stimulation à dose physiologique de IL-1β or LPS, est réduite. Cependant, lors de stimulation à plus hautes doses de LPS ou pendant un choc septique induit par de LPS, cette réduction n'est pas observée. Ces résultats montrent que Tollip pourrait avoir un rôle déterminant dans l'activation optimale en réponse à l' IL-1β et au LPS qui s'ajoute à sa fonction inhibitrice des mêmes voies de signalisation. Nous avons aussi étudié le rôle de Tollip comme molécule adaptatatrice du mécanisme endocytique d'internalisation de l' IL-1RI. Ainsi, l' IL-1R est ubiquitiné après stimulation par l' IL-1β , permettant à Tollip de se lier au récepteur. Cette interaction est réalisée entre le domaine CUE de Tollip et l'IL-1R via l'ubiquitine. L'internalisation et la localisation intracellulaire de l'IL-1RI et de Tollip ont été observés par microscopie confocale. En accord avec le rôle de Tollip dans le triage et la recirculation des IL-1R ubiquitiné, une quantité importante de Tollip été détectée dans l' endosome tardif. Nous avons pu démontrer que Tollip était nécessaire pour diriger efficacement ubiquitiné vers les lysosomes. Dans des cellules déficientes pour Tollip, ou reconstituées avec un mutant de Tollip (MF/AA) incapable de lier l'ubiquitine, IL-1RI s'accumule dans des vesicules anormales de l'endosome tardif. Dans ce travail, nous avons pu confirmer et préciser la fonction de la protéine Tollip dans l' activation de la production de cytokines induites par l' IL-1p and le LPS lors de l'inflammation et découvrir son rôle d'adaptateur dans l' internalisation et l'endocytose de l' IL-1RI.

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

Fibroblast Growth Factor 8 and its Receptors in The Growth and Angiogenesis of Experimental Breast Cancer . With Special Reference to Thrombospondin-1 as a Novel Target Gene for FGF-8

The growth of breast cancer is regulated by hormones and growth factors. Recently, aberrant fibroblast growth factor (FGF) signalling has been strongly implicated in promoting the progression of breast cancer and is thought to have a role in the development of endocrine resistant disease. FGFs mediate their auto- and paracrine signals through binding to FGF receptors 1-4 (FGFR1-4) and their isoforms. Specific targets of FGFs in breast cancer cells and the differential role of FGFRs, however, are poorly described. FGF-8 is expressed at elevated levels in breast cancer, and it has been shown to act as an angiogenic, growth promoting factor in experimental models of breast cancer. Furthermore, it plays an important role in mediating androgen effects in prostate cancer and in some breast cancer cell lines. We aimed to study testosterone (Te) and FGF-8 regulated genes in Shionogi 115 (S115) breast cancer cells, characterise FGF-8 activated intracellular signalling pathways and clarify the role of FGFR1, -2 and -3 in these cells. Thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis, was recognised as a Te and FGF-8 regulated gene. Te repression of TSP-1 was androgen receptor (AR)-dependent. It required de novo protein synthesis, but it was independent of FGF-8 expression. FGF-8, in turn, downregulated TSP-1 transcription by activating the ERK and PI3K pathways, and the effect could be reversed by specific kinase inhibitors. Differential FGFR1-3 action was studied by silencing each receptor by shRNA expression in S115 cells. FGFR1 expression was a prerequisite for the growth of S115 tumours, whereas FGFR2 expression alone was not able to promote tumour growth. High FGFR1 expression led to a growth advantage that was associated with strong ERK activation, increased angiogenesis and reduced apoptosis, and all of these effects could be reversed by an FGFR inhibitor. Taken together, the results of this thesis show that FGF-8 and FGFRs contribute strongly to the regulation of the growth and angiogenesis of experimental breast cancer and support the evidence for FGF-FGFR signalling as one of the major players in breast cancers.

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4,-6 and-7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed RMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K-d 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.