Investigating the role of the Mam2 C-terminal tail in the pheromone-signalling pathway of Schizosaccharomyces pombe

G protein-coupled receptors (GPCRs) allow cells to respond to extracellular stimuli and are involved in virtually all major physiological processes in eukaryotes. Consequently, aberrant GPCR signalling can lead to disease, making them attractive candidates for research and the development of pharmacological interventions. However, GPCR signalling responses in higher eukaryotes are complex, with the presence of multiple signalling pathways hindering the isolation and study of specific signalling components. The model organism, Schizosaccharomyces pombe, provides a simplified system with which to investigate GPCR signalling in the pheromoneresponse pathway. Pheromone stimulation in Sz. pombe causes the production of proteins necessary for the mating process, including the carboxypeptidase Sxa2. This study utilises Sz. pombe reporter strains in which sxa2 has been replaced with the bacterial reporter lacZ, thus enabling quantification of the level of signalling through the pheromoneresponse pathway. Using this system, the pheromone-responsive Mam2 receptor was investigated, with specific focus on the large intracellular C-terminal tail. Truncating Mam2 to remove the C-terminal tail altered signalling by increasing the level of ligand-independent signalling and decreasing the level of maximal signalling. Similar effects were observed when a regulator of G protein signalling, Rgs1, was removed from strains containing the full-length Mam2 receptor. This suggested a relationship between these two signalling components, which was confirmed through yeast 2-hybrid analysis. Using this approach, an 8-residue section of the Mam2 C-terminal tail was found to be necessary for a direct interaction with Rgs1. Further characterisation of the Mam2 tail revealed an additional role for this region in receptor sensitivity to pheromone stimulation. Mutational analysis implicated three serine residues in receptor sensitivity, suggesting that the C-terminal tail of Mam2 may contribute to receptor internalisation from the plasma membrane therefore enabling desensitisation to prolonged pheromone stimulation. Similar techniques applied to the study of Rgs1 revealed that two conserved DEP domains in the N-terminus of Rgs1 are important for the interaction with the Mam2 C-terminal tail. Fluorescent tagging of Rgs1 and subsequent microscopic analysis indicated that Rgs1 localises to endomembranous structures surrounding the nucleus, contrary to predictions made by mathematical models developed by this group, which suggest that Rgs1 requires plasma membrane-localisation in order to function. A better understanding of the action of Mam2 and Rgs1 in the Sz. pombe pheromone response can inform mathematical models and future studies involving the more complex mammalian signalling cascades.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Investigating the role of the Mam2 C-terminal tail in the pheromone-signalling pathway of Schizosaccharomyces pombe

G protein-coupled receptors (GPCRs) allow cells to respond to extracellular stimuli and are involved in virtually all major physiological processes in eukaryotes. Consequently, aberrant GPCR signalling can lead to disease, making them attractive candidates for research and the development of pharmacological interventions. However, GPCR signalling responses in higher eukaryotes are complex, with the presence of multiple signalling pathways hindering the isolation and study of specific signalling components. The model organism, Schizosaccharomyces pombe, provides a simplified system with which to investigate GPCR signalling in the pheromoneresponse pathway. Pheromone stimulation in Sz. pombe causes the production of proteins necessary for the mating process, including the carboxypeptidase Sxa2. This study utilises Sz. pombe reporter strains in which sxa2 has been replaced with the bacterial reporter lacZ, thus enabling quantification of the level of signalling through the pheromoneresponse pathway. Using this system, the pheromone-responsive Mam2 receptor was investigated, with specific focus on the large intracellular C-terminal tail. Truncating Mam2 to remove the C-terminal tail altered signalling by increasing the level of ligand-independent signalling and decreasing the level of maximal signalling. Similar effects were observed when a regulator of G protein signalling, Rgs1, was removed from strains containing the full-length Mam2 receptor. This suggested a relationship between these two signalling components, which was confirmed through yeast 2-hybrid analysis. Using this approach, an 8-residue section of the Mam2 C-terminal tail was found to be necessary for a direct interaction with Rgs1. Further characterisation of the Mam2 tail revealed an additional role for this region in receptor sensitivity to pheromone stimulation. Mutational analysis implicated three serine residues in receptor sensitivity, suggesting that the C-terminal tail of Mam2 may contribute to receptor internalisation from the plasma membrane therefore enabling desensitisation to prolonged pheromone stimulation. Similar techniques applied to the study of Rgs1 revealed that two conserved DEP domains in the N-terminus of Rgs1 are important for the interaction with the Mam2 C-terminal tail. Fluorescent tagging of Rgs1 and subsequent microscopic analysis indicated that Rgs1 localises to endomembranous structures surrounding the nucleus, contrary to predictions made by mathematical models developed by this group, which suggest that Rgs1 requires plasma membrane-localisation in order to function. A better understanding of the action of Mam2 and Rgs1 in the Sz. pombe pheromone response can inform mathematical models and future studies involving the more complex mammalian signalling cascades

A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways