Mechanisms of GPCR signal regulation in fission yeast
Cells communicate with each other and respond to environmental cues by sending and receiving signals. Many external signals (ligands) are detected through G protein-coupled receptors (GPCRs), a major class of transmembrane proteins. GPCRs transduce these external signals into appropriate intracellular responses, enabling the cell to adapt to its environment. Malfunctions in these signalling pathways can lead to a range of human diseases and hence GPCRs have become attractive candidates for pharmacological design.\ud \ud The activation of a single receptor has the ability to induce numerous intracellular responses. Coupling this with the great number of different GPCR-types expressed in human cells means that understanding the basic principles of signal transduction and termination in humans is complicated. This study utilises the more simplistic eukaryotic yeast Schizosaccharomyces pombe (S. pombe) to overcome this complexity, as it contains only two GPCR types and hence the cross-talk between pathways is greatly reduced, whilst the structure and signalling functions of GPCRs are often evolutionarily conserved between yeast and humans.\ud \ud Mathematical modelling was used to aid the understanding of GPCR signalling in S. pombe and to inform experimental design. Specifically, an ordinary differential equation model first developed by Croft et al. (2013) was extended to include all known downstream signal transduction, regulation and termination events. This model is the first of its kind to describe a whole GPCR signalling pathway within S. pombe. Although it accurately predicts the cellular response to GPCR signalling it could only reproduce the biological plateau in temporal response with the addition of a 'yet unknown mechanism' GPCR degradation term. This motivated the investigation of how GPCRs in S. pombe are internalised from the plasma membrane in response to ligand stimulation.\ud \ud The primary mechanism for signal termination is via internalisation of the GPCR. This study identified three potential casein kinases (Cki1, Cki2 and Cki3) that promote internalisation of the S. pombe GPCR Mam2. Microscopy analyses in combination with quantitative transcriptional, cell growth and cell cycle position assays uncovered a novel role for these kinases: that Cki2 regulates cell size during vegetative growth, Cki1 and Cki3 regulate the GPCR-response pathway and that Cki3 is essential for completing cytokinesis in S. pombe that have already undergone formation of a conjugation tube in response to ligand. Confocal microscopy of fluorescent labelled Mam2 indicated a role for Cki2 in the internalisation and hence termination of the GPCR-response pathway. These findings add to the growing body of evidence that casein kinases are implicated in GPCR desensitisation.