Constitutive Receptor Activity Research Articles

Tumor promotion and inhibition by phenobarbital in livers of conditional Apc-deficient mice.

Activation of Wnt/β-catenin signaling is important for human and rodent hepatocarcinogenesis. In mice, the tumor promoter phenobarbital (PB) selects for hepatocellular tumors with activating β-catenin mutations via constitutive androstane receptor activation. PB-dependent tumor promotion was studied in mice with genetic inactivation of Apc, a negative regulator of β-catenin, to circumvent the problem of randomly induced mutations by chemical initiators and to allow monitoring of PB- and Wnt/β-catenin-dependent tumorigenesis in the absence of unknown genomic alterations. Moreover, the study was designed to investigate PB-induced proliferation of liver cells with activated β-catenin. PB treatment provided Apc-deficient hepatocytes with only a minor proliferative advantage, and additional connexin 32 deficiency did not affect the proliferative response. PB significantly promoted the outgrowth of Apc-deficient hepatocellular adenoma (HCA), but simultaneously inhibited the formation of Apc-deficient hepatocellular carcinoma (HCC). The probability of tumor promotion by PB was calculated to be much lower for hepatocytes with loss of Apc, as compared to mutational β-catenin activation. Comprehensive transcriptomic and phosphoproteomic characterization of HCA and HCC revealed molecular details of the two tumor types. HCC were characterized by a loss of differentiated hepatocellular gene expression, enhanced proliferative signaling, and massive over-activation of Wnt/β-catenin signaling. In conclusion, PB exerts a dual role in liver tumor formation by promoting the growth of HCA but inhibiting the growth of HCC. Data demonstrate that one and the same compound can produce opposite effects on hepatocarcinogenesis, depending on context, highlighting the necessity to develop a more differentiated view on the tumorigenicity of this model compound.

Ligand-Independent Adenosine A2B Receptor Constitutive Activity as a Promoter of Prostate Cancer Cell Proliferation.

Aberrant ligand-independent G protein-coupled receptor constitutive activity has been implicated in the pathophysiology of a number of cancers. The adenosine A2B receptor (A2BAR) is dynamically upregulated under pathologic conditions associated with a hypoxic microenvironment, including solid tumors. This, in turn, may amplify ligand-independent A2BAR signal transduction. The contribution of A2BAR constitutive activity to disease progression is currently unknown yet of fundamental importance, as the preferred therapeutic modality for drugs designed to reduce A2BAR constitutive activity would be inverse agonism as opposed to neutral antagonism. The current study investigated A2BAR constitutive activity in a heterologous expression system and a native 22Rv1 human prostate cancer cell line exposed to hypoxic conditions (2% O2). The A2BAR inverse agonists, ZM241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol] or PSB-603 (8-(4-(4-(4-chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine), mediated a concentration-dependent decrease in baseline cAMP levels in both cellular systems. Proliferation of multiple prostate cancer cell lines was also attenuated in the presence of PSB-603. Importantly, both the decrease in baseline cAMP accumulation and the reduction of proliferation were not influenced by the addition of adenosine deaminase, demonstrating that these effects are not dependent on stimulation of A2BARs by the endogenous agonist adenosine. Our study is the first to reveal that wild-type human A2BARs have high constitutive activity in both model and native cells. Furthermore, our findings demonstrate that this ligand-independent A2BAR constitutive activity is sufficient to promote prostate cancer cell proliferation in vitro. More broadly, A2BAR constitutive activity may have wider, currently unappreciated implications in pathologic conditions associated with a hypoxic microenvironment.

Versatile Signaling Activity of Adhesion GPCRs.

The adhesion G protein-coupled receptors (aGPCRs) are a family of 33 receptors in humans that are widely expressed in various tissues and involved in many diverse biological processes. These receptors possess extremely large N-termini (NT) containing a variety of adhesion domains. A distinguishing feature of these receptors is the presence within the NT of a highly conserved GPCR autoproteolysis-inducing (GAIN) domain, which mediates autoproteolysis of the receptors into N-terminal and C-terminal fragments that stay non-covalently associated. The downstream signaling pathways and G protein-coupling preferences of many aGPCRs have recently been elucidated, and putative endogenous ligands for some aGPCRs have also been discovered and characterized in recent years. A pivotal observation for aGPCRs has been that deletion or removal of the NT up the point of GAIN cleavage results in constitutive receptor activation. For at least some aGPCRs, this activation is dependent on the unmasking of specific agonistic peptide sequences within the N-terminal stalk region (i.e., the region between the site of GAIN domain cleavage and the first transmembrane domain). However, the specific peptide sequences involved and the overall importance of the stalk region for activation can vary greatly from receptor to receptor. An emerging theme of work in this area is that aGPCRs are capable of versatile signaling activity that may be fine-tuned to suit the specific physiological roles played by the various members of this family.

Role of P2Y12 Receptor in Thrombosis.

P2Y12 receptor is a 342 amino acid Gi-coupled receptor predominantly expressed on platelets. P2Y12 receptor is physiologically activated by ADP and inhibits adenyl cyclase (AC) to decrease cyclic AMP (cAMP) level, resulting in platelet aggregation. It also activates PI3 kinase (PI3K) pathway leading to fibrinogen receptor activation, and may protect platelets from apoptosis. Abnormalities of P2Y12 receptor include congenital deficiencies or high activity in diseases like diabetes mellitus (DM) and chronic kidney disease (CKD), exposing such patients to a prothrombotic condition. A series of clinical antiplatelet drugs, such as clopidogrel and ticagrelor, are designed as indirect or direct antagonists of P2Y12 receptor to reduce incidence of thrombosis mainly for patients of acute coronary syndrome (ACS) who are at high risk of thrombotic events. Studies on novel dual-/multi-target antiplatelet agents consider P2Y12 receptor as a promising part in combined targets. However, the clinical practical phenomena, such as "clopidogrel resistance" due to gene variations of cytochrome P450 or P2Y12 receptor constitutive activation, call for better antiplatelet agents. Researches also showed inverse agonist of P2Y12 receptor could play a better role over neutral antagonists. Personalized antiplatelet therapy is the most ideal destination for antiplatelet therapy in ACS patients with or without other underlying diseases like DM or CKD, however, there is still a long way to go.

Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule development.

KNOX transcription factors (TFs) regulate different aspects of plant development essentially through their effects on phytohormone metabolism. In particular, KNOX TF SHOOTMERISTEMLESS activates the cytokinin biosynthesis ISOPENTENYL TRANSFERASE (IPT) genes in the shoot apical meristem. However, the role of KNOX TFs in symbiotic nodule development and their possible effects on phytohormone metabolism during nodulation have not been studied to date. Cytokinin is a well-known regulator of nodule development, playing the key role in the regulation of cell division during nodule primordium formation. Recently, the activation of IPT genes was shown to take place during nodulation. Therefore, it was hypothesized that KNOX TFs may regulate nodule development and activate cytokinin biosynthesis upon nodulation. This study analysed the expression of different KNOX genes in Medicago truncatula Gaertn. and Pisum sativum L. Among them, the KNOX3 gene was upregulated in response to rhizobial inoculation in both species. pKNOX3::GUS activity was observed in developing nodule primordium. KNOX3 ectopic expression caused the formation of nodule-like structures on transgenic root without bacterial inoculation, a phenotype similar to one described previously for legumes with constitutive activation of the cytokinin receptor. Furthermore, in transgenic roots with MtKNOX3 knockdown, downregulation of A-type cytokinin response genes was found, as well as the MtIPT3 and LONELYGUY2 (MtLOG2) gene being involved in cytokinin activation. Taken together, these findings suggest that KNOX3 gene is involved in symbiotic nodule development and may regulate cytokinin biosynthesis/activation upon nodule development in legume plants.

Atypical antipsychotics and inverse agonism at 5-HT2 receptors.

It is now well accepted that receptors can regulate cellular signaling pathways in the absence of a stimulating ligand, and inverse agonists can reduce this ligand-independent or "constitutive" receptor activity. Both the serotonin 5-HT2A and 5-HT2C receptors have demonstrated constitutive receptor activity in vitro and in vivo. Each has been identified as a target for treatment of schizophrenia. Further, most, if not all, atypical antipsychotic drugs have inverse agonist properties at both 5-HT2A and 5-HT2C receptors. This paper describes our current knowledge of inverse agonism of atypical antipsychotics at 5-HT2A/2C receptor subtypes in vitro and in vivo. Exploiting inverse agonist properties of APDs may provide new avenues for drug development.

Inhibition of ligand-independent constitutive activation of the Met oncogenic receptor by the engineered chemically-modified antibody DN30

An awesome number of experimental and clinical evidences indicate that constitutive activation of the Met oncogenic receptor plays a critical role in the progression of cancer toward metastasis and/or resistance to targeted therapies. While mutations are rare, the common mechanism of Met activation is overexpression, either by gene amplification (‘addiction’) or transcriptional activation (‘expedience’). In the first instance ligand-independent kinase activation plays a major role in sustaining the transformed phenotype. Anti-Met antibodies directed against the receptor binding site behave essentially as ligand (Hepatocyte Growth Factor, HGF) antagonists and are ineffective to counteract ligand-independent activation. The monovalent chimeric MvDN30 antibody fragment, PEGylated to extend its half-life, binds the fourth IPT domain and induces ‘shedding’ of the Met extracellular domain, dramatically reducing both the number of receptors on the surface and their phosphorylation. Downstream signaling is thus inhibited, both in the absence or in the presence of the ligand. In vitro, MvDN30 is a strong inhibitor not only of ligand-dependent invasive growth, sustained by both paracrine and autocrine HGF, but notably, also of ligand-independent growth of ‘Met-addicted’ cells. In immunocompromised mice, lacking expression of Hepatocyte Growth Factor cross-reacting with the human receptor – thus providing, by definition, a model of ‘ligand-independent’ Met activation – PEGylated MvDN30 impairs growth of Met ‘addicted’ human gastric carcinoma cells. In a Met-amplified patient-derived colo-rectal tumor (xenopatient) MvDN30-PEG overcomes the resistance to EGFR targeted therapy (Cetuximab). The PEGylated MvDN30 is thus a strong candidate for targeting tumors sustained by ligand-independent Met oncogenic activation.

Latent sensitization: a model for stress-sensitive chronic pain.

Latent sensitization is a rodent model of chronic pain that reproduces both its episodic nature and its sensitivity to stress. It is triggered by a wide variety of injuries ranging from injection of inflammatory agents to nerve damage. It follows a characteristic time course in which a hyperalgesic phase is followed by a phase of remission. The hyperalgesic phase lasts between a few days to several months, depending on the triggering injury. Injection of μ-opioid receptor inverse agonists (e.g., naloxone or naltrexone) during the remission phase induces reinstatement of hyperalgesia. This indicates that the remission phase does not represent a return to the normal state, but rather an altered state in which hyperalgesia is masked by constitutive activity of opioid receptors. Importantly, stress also triggers reinstatement. Here we describe in detail procedures for inducing and following latent sensitization in its different phases in rats and mice.

ProKinO: a unified resource for mining the cancer kinome.

Protein kinases represent a large and diverse family of evolutionarily related proteins that are abnormally regulated in human cancers. Although genome sequencing studies have revealed thousands of variants in protein kinases, translating “big” genomic data into biological knowledge remains a challenge. Here, we describe an ontological framework for integrating and conceptualizing diverse forms of information related to kinase activation and regulatory mechanisms in a machine readable, human understandable form. We demonstrate the utility of this framework in analyzing the cancer kinome, and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify a novel mutational hotspot in the αC-β4 loop of the kinase domain and demonstrate the functional impact of the identified variants in epidermal growth factor receptor (EGFR) constitutive activity and inhibitor sensitivity. We provide a unified resource for the kinase and cancer community, ProKinO, housed at http://vulcan.cs.uga.edu/prokino.

Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.

Hunting for the function of orphan GPCRs - beyond the search for the endogenous ligand.

Seven transmembrane-spanning proteins (7TM), also called GPCRs, are among the most versatile and evolutionary successful protein families. Out of the 400 non-odourant members identified in the human genome, approximately 100 remain orphans that have not been matched with an endogenous ligand. Apart from the classical deorphanization strategies, several alternative strategies provided recent new insights into the function of these proteins, which hold promise for high therapeutic potential. These alternative strategies consist of the phenotypical characterization of organisms silenced or overexpressing orphan 7TM proteins, the search for constitutive receptor activity and formation of protein complexes including 7TM proteins as well as the development of synthetic, surrogate ligands. Taken together, a variety of ligand-independent functions can be attributed to orphan 7TM proteins that range from constitutive activity to complex formation with other proteins and include 'true' orphans for which no ligand exist and 'conditional' orphans that behave like orphans in the absence of ligand and as non-orphans in the presence of ligand.

Cross-species transcriptomic analysis elucidates constitutive aryl hydrocarbon receptor activity.

BackgroundResearch on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances.ResultsWe found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements.ConclusionsThese findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1053) contains supplementary material, which is available to authorized users.

Noninvasive saliva-based EGFR gene mutation detection in patients with lung cancer.

Constitutive activation of the epidermal growth factor receptor (EGFR) is prevalent in epithelial cancers, particularly in non-small cell lung carcinoma (NSCLC). Mutations identified in EGFR predict the sensitivity to EGFR-targeted therapy. Detection of these mutations is mainly based on tissue biopsy, which is invasive, expensive, and time consuming. Noninvasive, real-time, inexpensive detection and monitoring of EGFR mutations in patients with NSCLC is highly desirable. We developed a novel core technology, electric field-induced release and measurement (EFIRM), which relies on a multiplexible electrochemical sensor that can detect EGFR mutations directly in bodily fluids. We established EFIRM for the detection of the EGFR mutations in vitro and correlated the results with tumor size from xenografted mice. In clinical application, we demonstrated that EFIRM could detect EGFR mutations in the saliva and plasma of 22 patients with NSCLC. Finally, a blinded test was performed on saliva samples from 40 patients with NSCLC. The receiver operating characteristic analysis indicated that EFIRM detected the exon 19 deletion with an area under the curve of 0.94 and the L858R mutation with an area under the curve of 0.96. Our data indicate that EFIRM is effective, accurate, rapid, user-friendly, and cost effective for the detection of EGFR mutations in the saliva of patients with NSCLC. We termed this saliva-based EGFR mutation detection (SABER).

The TLR4 D299G and T399I SNPs are constitutively active to up-regulate expression of Trif-dependent genes.

Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and ∼12 fold higher IFN-β gene expression levels compared to wild-type subjects (P<0.05; MWU test) and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.

Dowstream events in the NIK-mediated defense associated with resistance to begomovirus

The NSP-interacting kinase (NIK) receptor-mediated antiviral signaling has been identified as a virulence target of the begomovirus nuclear shuttle protein (NSP) [1,2]. NSP suppresses the activity of the NIK receptor through specific binding to the kinase domain and hence enhances begomovirus pathogenicity [1,3]. NIK receptors belong to the plant defense group of the leucine-rich repeat (LRR) receptor-like kinase (RLK) subfamily, designated LRR-RLK II. This subfamily of RLKs from tomato and Arabidopsis is constituted by 14 proteins harboring four complete LRRs (with 24 residues) and a fifth incomplete LRR (with 16 residues) arranged in a single continuous block within the extracellular domain [4]. Based on sequence conservation and structural features, the members of the LRR-RLK II subfamily are clustered into three distinct branches: (i) antiviral defense proteins (ii) developmental and defense proteins, such as the somatic embryogenesis receptor-like kinases (SERK-like) including SERK1 and SERK3/BAK1 and (iii) functionally unassigned proteins. The Arabidopsis NSP-interacting kinase 1, NIK1 (At5g16000), NIK2 (At3g25560) and NIK3 (At1g60800) are in the defense group I of the LRR-RLKII subfamily and are virulence targets of the bipartite geminivirus nuclear shuttle protein, NSP [1,4]. NSP from CaLCuV (Cabbage leaf curl virus interacts with all three NIKs from Arabidopsis to suppress their kinase activity [4]. The NSP-NIK interaction is also conserved among geminivirus NSPs and NIK homologs from different hosts [2]. Tomato and soybean NIK homologs also interact stably with NSP from CaLCuV [2] and with NSPs from the tomato-infecting geminiviruses TGMV (Tomato golden mosaic virus), TCrLYV (Tomato crinkle leaf yellows virus) and ToYSV (Tomato yellow spot virus) [1,2]. Several lines of evidence indicate that NIK functions in defense. NSP from CaLCuV acts as a virulence factor to suppress the kinase activity of transmembrane receptor NIKs [1]. Second, loss of NIK1, NIK2 or NIK3 function in Arabidopsis is also linked to an enhanced susceptibility phenotype to CaLCuV infection [1,3]. In addition, overexpression of NIK1 from Arabidopsis in tomato plants attenuates symptom development and delays ToYSV infection [5]. NIK exhibits trans-autophosphorylation activity in vitro and substrate phosphorylation activity in vitro and in vivo, and interacts with the ribosomal proteins L10 (rpL10) and L18 (rpL18) [5]. NIK-mediated phosphorylation of rpL10 promotes translocation of the ribosomal protein to the nucleus where it may function to mount a defense response that negatively impacts virus infection. This is consistent with the notion that the regulated nucleocytoplasmic shuttling of rpL10 links the antiviral response to receptor activation. We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues [6]. The conserved Thr-474 and Thr-469 were found to be phosphorylated in vitro and mutations at Thr-474 impaired autophosphorylation and were defective for kinase activation in vitro and in vivo. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. Our results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response [6,7]. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation. To identify novel regulators of NIK-mediated defense response, we screened a two-hybrid library for partners of rpL10. We discovered a novel transcriptional factor, which interacts with rpL10 in the nucleus of plant cells to down-regulate the expression of ribosomal genes. These data are consistent with the observation that constitutive activation of the NIK receptor by replacing Thr-474 with aspartate impairs global translation in Arabidopsis and tomato transgenic lines and confers broad-spectrum tolerance to begomovirus infection. Our data also indicate that the NIK immune receptor-mediated antiviral signaling operates through a bipartite module. One defense signal-transducing branch is mediated by the regulated nucleocytoplasmic transport of ribosomal protein to impair translation and the second branch of the antiviral signaling transduces a typical defense signal through induction of the plant immune system. We will discuss novel insights into the regulation of the NIK-mediated antiviral signaling and an efficient-acquired defense against begomovirus by modulating the activity of the immune defense receptor NIK in tomato plants.

Discovery of Biomarkers Predictive of GSI Response in Triple-Negative Breast Cancer and Adenoid Cystic Carcinoma

Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.

Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

Background and purposeDespite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants.

Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4 R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4 R) and rat H4 receptor (rH4 R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4 R-F169V, mH4 R-V171F, hH4 R-S179A, hH4 R-S179M; double mutants: hH4 R-F169V+S179A, hH4 R-F169V+S179M and mH4 R-V171F+M181S. Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1 γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([(3) H]-histamine), and in functional [(35) S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. Constitutive activity decreased from the hH4 receptor via the hH4 R-F169V mutant to the hH4 R-F169V+S179A and hH4 R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4 R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc.

In vitro combined treatment with cetuximab and trastuzumab inhibits growth of colon cancer cells.

Overexpression or constitutive activation of epidermal growth factor receptors (EGFR) is involved in growth of human cancers. We investigated effects of EGFR and HER-2 blockade in colon cancer cell lines using cetuximab and trastuzumab, with the aim of developing novel approaches to cancer therapy. We studied effects of treatment on cell growth, cell cycle distribution, induction of apoptosis, changes in EGFR and HER-2 mRNA-protein expression and EGFR and HER-2 gene copy number in Caco-2, HT-29 and HCT-116 cells. Treatment of cells resulted in no effect in one of the three cell lines and in inhibition of cell proliferation in a time- and dose-dependent manner in the other two, with modulation of EGFR and HER-2 mRNA and protein levels. Differences in sensitivity to cetuximab and trastuzumab were observed. Treatment induced specific changes in cell cycle distribution in both cell lines affected, while apoptosis was not increased. Fluorescence in situ hybridization analysis revealed abnormal copy number of two genes resulting from aneuploidy; this was not responsible for different sensitivity to combination between the two cell lines. Targeting EGFR and HER-2 simultaneously could have useful applications in colorectal cancer treatment. To improve pharmacological efficacy of cetuximab and trastuzumab combination, molecular mechanisms involved in their activity need to be elucidated.