Modelling the role of desensitisation mechanisms within fission yeast

Kathryn Richardson1,2, David Rand1,2, Graham Ladds1. 1The University of Warwick, Warwick Medical School, The University of Warwick, Coventry, CV4 7AL, UK, 2The University of Warwick, Systems Biology DTC, The University of Warwick, Coventry, CV4 7AL, UK

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors that allow cells to respond to extracellular stimuli, and are involved in virtually all-major physiological processes in eukaryotes. Consequently, malfunctions in GPCR signalling can lead to a range of diseases, making them attractive candidates for research and the development of pharmacological treatments. However, GPCR signalling responses in higher eukaryotes are complex and as such a more tractable system is preferred, such as yeast. The pheromone-response pathway in the fission yeast Schizosaccharomyces pombe provides a simplified system for studying signalling through GPCRs because it can be manipulated and controlled easily in comparison to more complex eukaryotes. This research utilises a combination of in vivo experimentation with computational modelling, to aid the mechanistic understanding and the development of an ordinary differential equation model of the molecules that regulate signalling networks mediated by heterotrimeric G proteins.

Specifically, our research focuses on understanding the mechanisms of terminating the pheromone-response pathway in Sz. pombe. There are various mechanisms by which the cell can become desensitised to a signal, ranging from the removal of the external signal to the deactivation of intracellular signalling components or transcription factors. A potential mechanism of desensitisation is the internalisation of the Sz. pombe receptor, Mam2, in response to pheromone. In the homologous yeast system Saccharomyces cerevisiae, two yeast casein kinase (Yck1 and Yck2) proteins have been associated with the internalisation of the receptor, Ste2. The homologous proteins in the Sz. pombe system (Cki1, Cki2 and Cki3) are characterised to distinguish their role in receptor internalisation of Mam2 and their role in pheromone signalling. Deletion of Cki3 indicates a significant (p<0.05) increase in sensitivity to pheromone (pEC50 from 7.56±0.11 (n = 6) to 8.2±0.17 (n = 8)) following treatment for 16 h. Removal of the tail of Mam2 prevents internalisation as previously determined by microscopy. This causes a significant (p<0.001) increase in sensitivity to pheromone (pEC50 from 6.88±0.01 (n = 5) to 8.01±0.27 (n = 7)) consistent with the ΔCki3 mutant. This implies the tail may play an essential role in receptor internalisation and degradation (ubiquitination) and that Cki3 may interact with the receptor tail prolonging the receptor at the cell membrane.