The cationic region of Rhes mediates its interactions with specific Gβ subunits

Ras homologue enriched in striatum (Rhes) is a small monomeric G protein which functions in a variety of cellular processes, including attenuation of G protein-coupled receptor (GPCR)signalling. There have been many studies into the effects of Rhes, but there is no molecular information about how Rhes might bring about these effects. Rhes shares striking sequence homology to AGS1 (activator of G protein signalling 1) and we considered whether the two proteins function in similar ways. AGS1 binds to the Gβ1 subunit of heterotrimeric G proteins and we have used yeast two-hybrid studies to show that Rhes binds selectively to Gβ1, Gβ2 and Gβ3 subunits. Binding to the Gβ subunits involves the cationic regions of AGS1 and Rhes, and we used Rhes-AGS1 chimeras to show that their different cationic regions determine the Gβ-specificity of the interactions. Possible implications of this interaction for the activity of Rhes are discussed

Differential effects of RGS proteins on Gαq and Gα11 activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human Gαq and Gα11 spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of Gα-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both Gαq and Gα11 but, in contrast, RGS5 and RGS16 were much less effective against Gα11 than Gαq. Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active Gαq QL/Gα11 QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-Gα specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of Gαq/Gα11 enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both Gα subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential Gα activity, further highlighting that RGS proteins can discriminate between two very closely related Gα subunits

Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells.

We have used three established human glioblastoma (GBM) cell lines-U87MG, A172, and T98G-as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come

Hyperglycaemia does not affect antigen-specific activation and cytolytic killing by CD8+ T cells in vivo

Metabolism is of central importance for T cell survival and differentiation. It is well known that T cells cannot function in the absence of glucose, but it is less clear how they respond to excessive levels of glucose. In the present study, we investigated how increasing levels of glucose affect T-cell-mediated immune responses. We examined the effects of increased levels of glucose on CD8+ T-cell behaviour in vitro by assessing activation and cytokine production, as well as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and intracellular signalling. In addition, we assessed in vivo proliferation, cytokine production and cytolytic activity of cells in chemically induced diabetic C57BL/6 mice. Elevated levels of glucose in in vitro cultures had modest effects on proliferation and cytokine production, while in vivo hyperglycaemia had no effect on CD8+ T-cell proliferation, interferon γ (IFNγ) production or cytolytic killing

Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition.

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy

Dual positive and negative regulation of GPCR signaling by GTP hydrolysis

G protein-coupled receptors (GPCRs) regulate a variety of intracellular pathways through their ability to promote the binding of GTP to heterotrimeric G proteins. Regulator of G protein signaling (RGS) proteins increase the intrinsic GTPase activity of G-subunits and are widely regarded as negative regulators of G protein signaling. Using yeast we demonstrate that GTP hydrolysis is not only required for desensitization, but is essential for achieving a high maximal (saturated level) response. Thus RGS-mediated GTP hydrolysis acts as both a negative (low stimulation) and positive (high stimulation) regulator of signaling. To account for this we generated a new kinetic model of the G protein cycle where GGTP enters an inactive GTP-bound state following effector activation. Furthermore, in vivo and in silico experimentation demonstrates that maximum signaling output first increases and then decreases with RGS concentration. This unimodal, non-monotone dependence on RGS concentration is novel. Analysis of the kinetic model has revealed a dynamic network motif that shows precisely how inclusion of the inactive GTP-bound state for the G produces this unimodal relationship

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

The effects of RAMPs upon cell signalling.

G protein-coupled receptors (GPCRs) play a vital role in signal transduction. It is now clear that numerous other molecules within the cell and at the cell surface interact with GPCRs to modulate their signalling properties. Receptor activity modifying proteins (RAMPs) are a group of single transmembrane domain proteins which have been predominantly demonstrated to interact with Family B GPCRs, but interactions with Family A and C receptors have recently begun to emerge. These interactions can influence cell surface expression, ligand binding preferences and G protein-coupling, thus modulating GPCR signal transduction. There is still a great deal of research to be conducted into the effects of RAMPs on GPCR signalling; their effects upon Family B GPCRs are still not fully documented, in addition to their potential interactions with Family A and C GPCRs. New interactions could have a significant impact on the development of therapeutics

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase