Investigating Gaq/11-coupled receptor signalling diversity at the level of specific PLC isoenzyme activation in neurones
GPCRs coupled to phospholipase C (PLC) mediate Ins(1,4,5)P3/Ca 2+ release and DAG/PKC activation are capable of generating a multitude of signalling outcomes in vivo. Using a variety of techniques, including single cell imaging, this Thesis will investigate whether Ga q/11PCR specificity is controlled by the activation of particular PLC isoenzymes in a model CHO cell system (CHOm3/alpha1B) and in primary neuronal cells.;The roles of specific PLC isoenzymes were assessed using over-expression or dominant-negative/antisense approaches in the CHOm3/alpha1B cell background. These studies allowed roles for PLCbeta, but not PLCdelta or PLCgamma isoenzymes, to be defined in receptor-mediated responses. Significantly, PLCbeta1 over-expression potentiated Ins(1,4,5)P3/DAG responses to MCh and NA challenge (at sub-maximal agonist concentrations), while PLCbeta3 over-expression attenuated these responses.;The development of rat-specific PLCbeta1 sirRNAs allowed an 80% knockdown of PLCbeta1 protein to be achieved in C6 glioma and cerebellar granule cells. Significantly, M3 mACh receptor-mediated Ca2+ mobilisation was attenuated in C6 glioma cells following siRNA-mediated PLCbeta1 knock-down. In contrast, mACh receptor agonist-stimulated Ins(1,4,5)P3 production was unaffected by PLCbeta1 knock-down in cerebellar granule cells. Further studies revealed that a significant PLCbeta1 knock-down in hippocampal neurones did not attenuate Ins(1,4,5)P3 production mediated by either receptor or concurrent metabotropic/ionotropic stimulation. Interpretation of these data is complicated as PLCbeta3 and PLCbeta4 isoenzymes were shown to be selectively increased by PLCbeta1 siRNA treatment in C6 glioma and cerebellar granule cells, respectively.