Biomedical subjects

John Davey

Publications and source records attributed to John Davey.

14 recordsLinked to original sources

PubMed2006

Identification of Gnr1p, a negative regulator of G alpha signalling in Schizosaccharomyces pombe, and its complementation by human G beta subunits.

G protein-coupled receptors (GPCRs) are involved in the response of eukaryotic cells to a wide variety of stimuli, traditionally mediating their effects through heterotrimeric G proteins comprised of G alpha, G beta and G gamma subunits. The fission yeast Schizosaccharomyces pombe is an established tool for GPCR research, possessing two G alpha-dependent signalling cascades. A complete G alpha beta gamma complex has been characterised for the glucose-sensing pathway, but only the G alpha subunit, Gpa1p, has been identified in the pheromone-response pathway. Here, we report the use of the yeast two-hybrid system to identify a novel protein, Gnr1p, which interacts with Gpa1p. Gnr1p is predicted to contain seven WD repeats and to adopt a structure similar to typical G beta subunits. Disruption and overexpression studies reveal that Gnr1p negatively regulates the pheromone-response pathway but is not required for signalling. Human G beta subunits complement the loss of Gnr1p, functioning as negative regulators of G alpha signalling in fission yeast.

Amino Acid Sequence↗

PubMed2006

Differential effects of RGS proteins on G alpha(q) and G alpha(11) 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 G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.

GTP-Binding Protein alpha Subunits, Gq-G11↗

PubMed2006

Investigating RGS proteins in yeast.

Regulator of G protein signalling (RGS) proteins are vital in the adaptation of cells to stimulation via G protein-coupled receptors. Yeast have been integral in elucidating the important role that RGS proteins play within cellular processes. In addition to extensive characterisation of the endogenous RGS proteins, these organisms have enabled the identification and analysis of numerous mammalian homologues. The simplicity and plasticity of the yeast pheromone-response pathway has facilitated studies which would have been impossible in mammalian systems and it is certain that yeast will continue to have a great impact on this field of research in the future.

GTP-Binding Protein Regulators↗

PubMed2006

Combined use of two transcriptional reporters improves signalling assays for G protein-coupled receptors in fission yeast.

The biochemical and genetic tractability of yeasts make them ideal hosts for the analysis of signalling from G protein-coupled receptors (GPCRs). Selected modifications to the strains allow the introduction of non-yeast components, while signal-dependent expression of reporter genes provides growth selection or enzyme read-out as assays for signalling. One issue with such systems is reporter expression in the absence of stimulation, usually because of spontaneous activation of intracellular signalling components and/or incomplete repression of the signal-dependent promoter. This limits the difference between reporter activity in the presence and absence of stimulation, often referred to as the signal:background ratio. In an effort to extend the applicability of the yeast system, we generated a Schizosaccharomyces pombe strain containing pheromone-dependent reporters for both growth selection and beta-galactosidase production. Simultaneous use of the two reporters provided several advantages over strains expressing only one reporter, particularly when coupled to the use of a competitive inhibitor of the nutritional reporter. For example, the beta-galactosidase signal:background ratio following stimulation with 10(-6) M P-factor increased from 35 for a strain containing a single lacZ reporter to almost 2500 for the double reporter. The sensitivity of the system was also improved, with higher signal:background ratios allowing detection of lower concentrations of P-factor. Although we have used Sz. pombe and focused on GPCR-based induction of beta-galactosidase, the principles described can be applied to other yeasts, different signalling pathways and alternative reporters.

DNA, Fungal↗

PubMed2005

Specific in vivo binding of activator of G protein signalling 1 to the Gbeta1 subunit.

Activator of G protein signalling 1 (AGS1) is a Ras-like protein that affects signalling through heterotrimeric G proteins. Previous in vitro studies suggest that AGS1 can bind to G(alpha)-GDP subunits and promote nucleotide exchange, leading to activation of intracellular signalling pathways. This model is consistent with in vivo evidence demonstrating that AGS1 activates both G(alpha)- and G(betagamma)-dependent pathways in the absence of ligand. However, it does not easily explain how AGS1 blocks G(betagamma)-dependent, but not G(alpha)-dependent, signalling following receptor activation. We have used yeast two hybrid analysis and co-immunoprecipitation studies in mammalian cells to demonstrate a direct interaction between AGS1 and the G(beta1) subunit of heterotrimeric G proteins. The interaction is specific for G(beta1) and involves the cationic region of AGS1 and the C-terminal region of G(beta1). Possible implications of this novel interaction for the activity of AGS1 are discussed.

Binding Sites↗

PubMed2005

Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2.

To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted alpha-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour. Unlike assays based on beta-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 alpha-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe.

Amino Acid Sequence↗

PubMed2005

Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.

Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates.

DNA, Fungal↗

PubMed2005

Functional analysis of heterologous GPCR signalling pathways in yeast.

G protein-coupled receptors (GPCRs) regulate diverse biological processes in eukaryotes and such conservation allows an almost unrestricted interchange of signalling components between different cell types. Yeasts are attractive hosts in which to study GPCRs--they are amenable to both genetic and biochemical manipulation and their robustness, low cost and our ability to create strains that lack endogenous GPCRs make them ideal starting points for the development of assays suitable for high-throughput screening. Here we introduce readers to the possibilities of using yeast to analyse GPCRs describing the endogenous signalling pathways, the development of assays for heterologous GPCRs and the technology to elucidate GPCR structure and activity, focusing on the budding yeast Saccharomyces cerevisiae and recent developments using the fission yeast Schizosaccharomyces pombe.

Genes, Reporter↗

PubMed2005

A constitutively active GPCR retains its G protein specificity and the ability to form dimers.

G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.

Amino Acid Sequence↗

PubMed2004

An efficient bipartite PCR technique to introduce specific changes in large plasmids.

Amplifying an entire double-stranded plasmid by an inverse polymerase chain reaction (PCR) using a pair of tail-to-tail primers is a particularly efficient approach for introducing changes into DNA sequences. However, the approach generally works best for plasmids less than 5 Kb and it can be difficult to amplify the large multicomponent vectors that are used for protein expression in various eukaryotic cells. We have therefore adopted an alternative approach in which two smaller PCR products are generated and then ligated to produce the complete plasmid. A mutagenic primer is used to introduce the desired change and each reaction includes one of a pair of tail-to-tail primers from within an antibiotic resistance gene contained on the plasmid so that the two PCR products contain complementing parts of the complete gene. Ligating the two products generates various combinations but only the correctly ligated molecules recreate the antibiotic resistance gene and are able to replicate in Escherichia coli. When combined with methods to minimize the carryover of template plasmid, this can be an efficient way of introducing mutations into large plasmids.

Mutagenesis, Site-Directed↗

PubMed2004

G-protein-coupled receptors: new approaches to maximise the impact of GPCRS in drug discovery.

IBC's Drug Discovery Technology Series is a group of conferences highlighting technological advances and applications in niche areas of the drug discovery pipeline. This 2-day meeting focused on G-protein-coupled receptors (GPCRs), probably the most important and certainly the most valuable class of targets for drug discovery. The meeting was chaired by J Beesley (Vice President, European Business Development for LifeSpan Biosciences, Seattle, USA) and included 17 presentations on various aspects of GPCR activity, drug screens and therapeutic analyses. Keynote Addresses covered two of the emerging areas in GPCR regulation; receptor dimerisation (G Milligan, Professor of Molecular Pharmacology and Biochemistry, University of Glasgow, UK) and proteins that interact with GPCRs (J Bockaert, Laboratory of Functional Genomics, CNRS Montpellier, France). A third Keynote Address from W Thomsen (Director of GPCR Drug Screening, Arena Pharmaceuticals, USA) discussed Arena's general approach to drug discovery and illustrated this with reference to the development of an agonist with potential efficacy in Type II diabetes.

Animals↗

PubMed2004

Analysis of human GPCRs in fission yeast.

G protein-coupled receptors (GPCRs) regulate diverse biological processes in all eukaryotes, including yeast, insects, plants and humans. This evolutionary conservation allows an almost unrestricted interchange of signaling components between different cell types. A large number of model systems have been developed for the study of GPCRs, and yeasts provide one of the more attractive hosts since they are amenable to both genetic and biochemical manipulation, while their robustness, low cost and lack of endogenous GPCRs are ideal starting points for the development of assays suitable for high-throughput screening. The purpose of this review is to introduce readers to the possibilities of using the fission yeast Schizosaccharomyces pombe for analysis of GPCRs. We describe the endogenous signaling pathways, the development of assays for heterologous GPCRs, and some of the technology available to elucidate GPCR structure and activity.

Humans↗

PubMed2003

Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins.

G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.

Corticotropin-Releasing Hormone↗

PubMed2002

Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe.

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling. Several different types of mutant were identified. Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2). Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway. Although previously shown to regulate other MAP kinase pathways in Sz. pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.

Cell Cycle↗