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Title: Dissecting Group I metabotropic glutamate receptor signalling at the level of the G-protein
Author: Atkinson, Peter J.
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2005
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This Thesis explores the use of novel strategies aimed at interrogating Gq/11-coupled receptor signalling at the level of the G-protein. The development of an RNAi approach to reduce Gq/11 protein expression enabled an investigation into the role of these proteins in shaping single-cell IP3 and Ca2+ signals mediated by the group I mGlu receptors, mGlu1a and mGlu5a, expressed in CHO cells. In control cells, mGlu1a receptor-activation initiates sustained elevations in Ca2+, which are sensitive to agonist concentration. In contrast, mGlu5a receptor-activation typically elicits Ca2+ oscillations that are largely agonist concentration-independent. Plasmid-encoded RNAi produced a robust knockdown of Gq and G11 mRNA and protein expression that almost abolished mGlu1a and mGlu5a receptor-activated increases in IP3, measured in single-cells using eGFP-PHPLC. For the mGlu1a receptor, RNAi-mediated knockdown of Gq and G11 proteins unmasked agonist-stimulated baseline Ca2+ spikes that persisted even at maximal concentrations of agonist. The mGlu5a receptor-activated Ca2+ oscillations were still observed, but were only initiated at high agonist concentrations. Recombinant over-expression of Gq enhanced IP3 signals following mGlu1a or mGlu5 receptor activation. Although mGlu5a receptor-mediated IP3 and Ca2+ oscillations in control cells were largely insensitive to agonist concentration, increasing Gq expression converted these oscillatory signatures to sustained peak-and --plateau responses in a high proportion of cells. Up- or down- regulation of Gq/11 protein expression also altered the agonist concentration-dependencies of signalling for both mGlula and mGlu5a receptors. These experiments indicate that altering Gq/11 expression levels differentially affects spatiotemporal aspects of IP3 and Ca2+ signalling by group I mGlu receptors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available