My Research
Abstract
Our research is to examine the flow and processing of information from extracellular signals to intracellular responses - to understand how cells receive, amplify, integrate and interpret a variety of stimuli to generate a change in behaviour. Such signal transduction is an integral part of most cellular processes. Quantifying how cells sense and respond to various stimuli and creating a model that will accurately predict these responses are a crucial step to understanding a range of diseases.
Our ultimate objective is a model of a eukaryotic regulatory network, which takes account of currently available experimental observations and facilitates refinement based on new observations. It will allow prediction of the quantitative behaviour in individual cells over time and in response to defined perturbations. Regressive testing, using experiment to guide the model and the model to guide the experiment, will allow refinement of our knowledge of the system.
The particular pathway to which we refer is the G protein coupled-receptor (GPCR) signal transduction pathway which regulates mating in the fission yeast Schizosaccharomyces pombe. In order to achieve our objective, we will combine experimental, analytical and numerical study of the pathway, borrowing a number of techniques from mathematics, computer science and engineering. The modelling techniques we have earmarked have a strong theoretical background and, in some cases, have enjoyed an encouraging degree of success in modelling biological systems similar in nature to this one [1-4]. Fundamental design principles of the modelling system being constructed will be user-friendliness, such that new experimental observations can be input with relative ease; and clarity of representation, such that the hypothetical researcher can obtain an intuitive insight into the functioning of the pathway.
Background and biological motivation
GPCRs are a diverse family of integral membrane proteins that enable cells to respond to extracellular signals. In response to stimulation the receptors act through heterotrimeric G proteins to regulate intracellular effector proteins and bring about changes in cell behaviour. G proteins, which are composed of Ga, Gb and Gg-subunits, play a pivotal role in linking different GPCRs to their intracellular effectors. In unstimulated cells the Ga is bound to GDP and associated with the Gbg dimer but, upon binding agonist, the receptor stimulates the exchange of GDP for GTP and Ga-GTP is released from the Gbg dimer. The dissociated subunits regulate the activity of effector proteins that bring about the changes in cell behaviour.
Understanding GPCRs and their associated signalling pathways is not just an academic exercise. Humans express about 400 non-sensory GPCRs (there are also several hundred olfactory receptors). Most of these are valuable pharmaceutical targets and approximately 30% of marketed drugs act on GPCRs, generating annual sales of US$23 billion. A desire for a more complete understanding of mammalian G protein signalling is required to effectively produce drugs with enhanced specificity. Studies within mammalian cell systems are hampered by the presence of endogenous GPCRs. Indeed it is estimated that a single mammalian cell will express, at any one time, 40-100 different GPCRs upon its cell surface, each of which can interact with one or many different Ga? activating one or many down stream effector molecules. With the potential of a high degree of crosstalk leading to a much greater level of complexity there has been an absolute requirement for simple eukaryotic model organisms. Yeast has proven to be an attractive system in which to analyse GPCRs. Mechanisms of GPCR signalling in yeast are highly homologous to those in mammalian systems and in some cases the proteins involved are functionally interchangeable. Schizosaccharomyces pombe is a haploid yeast existing in one of two mating types and conjugation occurs between cells of opposite mating type by the reciprocal exchange of diffusible pheromones. These peptides are released by cells of one mating type, bind to GPCRs on the surface of a partner of the opposite type, and activate intracellular machinery that includes a G protein, a MAP kinase cascade and a transcription factor (Figure 1). Stimulation by mating pheromone induces expression of the genes required to bring about mating-related changes in cell behaviour, including an arrest of the cell cycle, enhanced cell agglutination, and cell fusion.
While the understanding of GPCR signalling is in an advanced state there remains a vast number of details for which our knowledge is incomplete e.g. the conformational change that occurs upon the GPCR once the ligand has bound. To address such issue researches have utilised mathematical models in ordered to assist their understanding. A detailed model relating to ligand-GPCR-Ga binding has been produced and refined [5,6] and likewise models relating to RGS-Ga function [7]. However, they have not yet been integrated into one complete model for a signalling cascade starting at the ligand-GPCR interface and ending at transcriptional activation. Such models within mammalian systems are difficult to generate and therefore the production of a model within Sz. pombe would represent an initial step to producing a fully integrated model that is applicable to higher eukaryotic organisms.
Through 7 years of research Dr Ladds has amassed an array of data relating to signalling within the Sz. pombe pheromone pathway [e.g. 8 - 14]. He has developed strains which have a number of the components deleted so providing a null background to enable better understanding of each of the individual signalling events. During the course of this work a number of aberrant results have been generated, for which the previous methods of biological enquiry have been unable to provide a satisfactory understanding. Examples of these results are; signalling being reduced in strains lacking rgs1 when exposed to high concentrations of ligand, and pheromone induced signalling even when the GPCR has been deleted. Mathematical models of the pheromone pathway should enable a better understanding of these and other such results. We intend and expect to generate a symbiotic relationship between the mathematical models and the experimental system. This will mean that, as the mathematical models are refined, we will be able to target the experimental work to resolve apparent inconsistencies in our results and provide answers to questions which relate to more complex issues regarding GPCR signalling.
References
[1] Kremling, A., Heermann, R., Centler, F., Jung, K. and Gillesa, E.D. (2004) Analysis of two-component signal transduction by mathematical modeling using the KdpD/KdpE system of Escherichia coli. BioSystems 78: 23-37.
[2] Alur, R., Belta, C., Ivancic, F., Kumar, V., Rubin, H., Schug, J., Sokolsky, O. and Webb, J. (2002) Visual Programming for Modeling and Simulation of Biomolecular Regulatory Networks, 2552: 702-712.
[3] Fisher, J., Piterman, N., Hubbard, E.J.A., Stern, M.J. and Harel, D. (2005) Computational Insight into Caenorhabditis elegans vulval development. PNAS 102: 1951-1956.
[4] Efroni, S., Harel, D. and Cohen, I.R. (2003) Towards Rigorous Comprehension of Biological Complexity: Modeling, Execution, and Visualisation of Thymic T-Cell Maturation. Genome Research 13: 2485-2497.
[5] Kenakin, T. (2004) Principles: Receptor theory in pharmacology. TIPS 25: 186-192.
[6] Kenakin, T. (1997) Agonist-specific receptor conformations. TIPS 18: 416-417.
[7] Yildirim, N., Hao, N., Dohlman, H.G. and Elston, T.C. (2004) Mathematical modeling of RGS and G-protein regulation in yeast. Methods Enzymol. 389: 383-398.
[8] Ladds,G. et al. (1998) Extracellular degradation of agonists as an adaptive mechanism. Semin. Cell Dev. Biol. 9: 111-118
[9] Ladds,G. and Davey,J. (2000) Sxa2 is a serine carboxypeptidase that degrades extracellular P-factor in the fission yeast Schizosaccharomyces pombe. Mol. Microbiol. 36: 377-390
[10] Didmon,M. et al. (2002) Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe. Curr. Genet. 41: 241-253
[11] Ladds,G. et al. (2003) Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins. Mol. Microbiol. 47: 781-792
[12] Ladds,G. and Davey,J. (2004) Analysis of human GPCRs in fission yeast. Curr. Opin. Drug Discov. Devel. 7: 683-691
[13] Ladds,G. et al. (2005) Functional analysis of heterologous GPCR signalling pathways in yeast. Trends Biotechnol. 23: 367-373
[14] Ladds,G. et al. (2005) A constitutively active GPCR retains its G protein specificity and the ability to form dimers. Mol. Microbiol. 55: 482-497
