CB1 receptor allosteric modulators display both agonist and signaling pathway specificity

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) which display a contradictory pharmacological profile: increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signalling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor agonist-induced [35S]GTPγS binding, inhibition and stimulation of forskolin stimulated cAMP production, phosphorylation of ERK, and β arrestin recruitment. We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signalling as compared to WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced [35S]GTPγS binding, simulation (Gαs mediated) and inhibition (Gαi mediated) of cAMP production and β arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphoryation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high affinity CB1 agonist binding sites. The receptor conformation stabilised by the allosterics appears to induce signalling and also selectively traffics orthosteric agonist signalling via the ERK phosphorylation pathway.

Trafficking and signaling of G protein-coupled receptors in the nervous system: implications for disease and therapy

G protein-coupled receptors (GPCRs) are expressed throughout the nervous system where they regulate multiple physiological processes, participate in neurological diseases, and are major targets for therapy. Given that many GPCRs respond to neurotransmitters and hormones that are present in the extracellular fluid and which do not readily cross the plasma membrane, receptor trafficking to and from the plasma membrane is a critically important determinant of cellular responsiveness. Moreover, trafficking of GPCRs throughout the endosomal system can initiate signaling events that are mechanistically and functionally distinct from those operating at the plasma membrane. This review discusses recent advances in the relationship between signaling and trafficking of GPCRs in the nervous system. It summarizes how receptor modifications influence trafficking, discusses mechanisms that regulate GPCR trafficking to and from the plasma membrane, reviews the relationship between trafficking and signaling, and considers the implications of GPCR trafficking to drug development.

Endosomes: a legitimate platform for the signaling train

Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).

Alpha-synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of alpha-synuclein (alpha-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the alpha-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type alpha-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of alpha-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the alpha-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+](i) in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of alpha-synuclein. However, only WT alpha-syn transfected cells displayed significantly impaired viability. Our findings suggest that alpha-syn regulates Ca2+ entry pathways and, consequently, that abnormal alpha-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Ion channels as effectors in carbon monoxide signaling

A wealth of recent studies has highlighted the diverse and important influences of carbon monoxide (CO) on cellular signaling pathways. Such studies have implicated CO, and the enzymes from which it is derived (heme oxygenases) as potential therapeutic targets, particularly (although not exclusively) in inflammation, immunity and cardiovascular disease.1 In a recent study,2 we demonstrated that CO inhibited cardiac L-type Ca(2+) channels. This effect arose due to the ability of CO to bind to mitochondria (presumably at complex IV of the electron transport chain) and so cause electron leak, which resulted in increased production of reactive oxygen species. These modulated the channel's activity through interactions with three cysteine residues in the cytosolic C-terminus of the channel's major, pore-forming subunit. Our study provided a potential mechanism for the cardioprotective effects of CO and also highlighted ion channels as a major potential target group for this gasotransmitter.

Hypoxic modulation of ca(2+) signaling in human venous and arterial endothelial cells

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca(2+) homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O(2), 24 h). Basal [Ca(2+)]( i ) and store depletion-mediated Ca(2+) entry were significantly different between the two cell types, yet agonist (ATP)-mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca(2+) entry only in venous cells. Clearly, Ca(2+) signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.

Reading is now 'cool': a study of English teachers' perspectives on e-reading devices as a challenge and an opportunity

E-reading devices such as The Kindle have rapidly secured a significant place in a number of societies as at least one major platform for reading. To some extent they are part of the overarching move towards a fully digitised world but they have a distinctiveness in being deliberately “book-like”. Teachers generally have some suspicion towards “New Media”, especially when it challenges their established practice and nothing dominates the school more than the physical book. What may be the challenges but also the benefits of e-readers to teachers and students? What may be the particular challenges to those teachers who are, traditionally, the guardians of the book, that is the teachers of mother tongue literature? This article reports on a survey of English teachers in England to gauge their reactions to e-readers, both personally and professionally and describes their speculations about the place of e-readers in schools in the future. There is a mixed reaction with some teachers concerned about the demise of the book and the potential negative impact on reading. However, the majority welcome e-readers as a dynamic element within the reading environment with particular potential to enthuse reluctant readers and those with special or linguistic needs. They also, some grudgingly, view the fact that reading using this form of technology appeals to the “e-generation” and may succeed in making reading “cool”. This form of technology is, ironically (given that it appears to threaten traditional books) likely to be rapidly adopted in classrooms.

MICAL-1 is a negative regulator of MST-NDR kinase signaling and apoptosis.

MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.

Infotainment across access devices: the perceptual impact of multimedia QoS

The introduction of multimedia on pervasive and mobile communication devices raises a number of perceptual quality issues. However, limited work has been done examining the 3-way interaction between use of equipment, user perceptual quality and quality of service. Our work measures user perceptual quality with the quality of perception (QoP) metrics which comprises levels of informational transfer (objective) and user satisfaction (subjective) when users are presented with multimedia video clips at three different frame rates, using four different display devices. Finally, our results will show that variation in frame-rate does not impact a user’s level of information assimilation (IA), however, does impact a users’ perception of multimedia video ‘quality’.

A functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA and secreted protein level (75 and 94%, respectively) and elicited a 77% reduction in CYP17A1 mRNA level and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 mRNA level (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ~2-fold. The CYP17 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling.

A conceptual valuation framework (VF) for home telecare system devices

Purpose – The aim of this paper is to present a conceptual valuation framework to allow telecare service stakeholders to assess telecare devices in the home in terms of their social, psychological and practical effects. The framework enables telecare service operators to more effectively engage with the social and psychological issues resulting from telecare technology deployment in the home and to design and develop appropriate responses as a result. Design/methodology/approach – The paper provides a contextual background for the need for sociologically pitched tools that engage with the social and cultural feelings of telecare service users before presenting the valuation framework and how it could be used. Findings – A conceptual valuation framework is presented for potential development/use. Research limitations/implications – The valuation framework has yet to be extensively tested or verified. Practical implications – The valuation framework needs to be tested and deployed by a telecare service operator but the core messages of the paper are valid and interesting for readership. Social implications – In addressing the social and cultural perspectives of telecare service stakeholders, the paper makes a link between the technologies in the home, the feelings and orientations of service users (e.g. residents, emergency services, wardens, etc.) and the telecare service operator. Originality/value – The paper is an original contribution to the field as it details how the sociological orientations of telecare technology service users should be valued and addressed by service operators. It has a value through the conceptual arguments made and through valuation framework presented.

Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets.

Phosphatidylinositol 3-kinase (PI3K) isoforms PI3Kbeta and PI3Kgamma are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3Kbeta or PI3Kgamma. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein-coupled receptors for adenosine 5'-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3Kbeta, but not PI3Kgamma, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor glycoprotein VI (GPVI). Moreover, PI3Kbeta was a major essential regulator of platelet adhesion to fibrinogen and of integrin alpha(IIb)beta(3)-mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3Kbeta in signaling through GPVI and integrin alpha(IIb)beta(3).

C-slow retimed parallel histogram architectures for consumer imaging devices

A parallel pipelined array of cells suitable for real-time computation of histograms is proposed. The cell architecture builds on previous work obtained via C-slow retiming techniques and can be clocked at 65 percent faster frequency than previous arrays. The new arrays can be exploited for higher throughput particularly when dual data rate sampling techniques are used to operate on single streams of data from image sensors. In this way, the new cell operates on a p-bit data bus which is more convenient for interfacing to camera sensors or to microprocessors in consumer digital cameras.