Characterization of Gaq-coupled receptor signalling via the novel effector PKC?

Resumen

The cell is a dynamic entity with highly intertwined biochemical networks responding to internal and external perturbations in an orchestrated manner. The elements in those networks are usually proteins that build complex circuits through protein-protein interactions. G proteins are essential cellular components for the signalling events following G protein-coupled receptor (GPCR) activation. This function is achieved through specific and evolutionary conserved interactions between activated G proteins and a number of cellular effectors. We have recently reported that ERK5 activation by Gq-coupled GPCR requires participation of a novel G¿q effector, PKC¿. Here we present a biochemical and cellular characterization of the G¿q/PKC¿ signalling axis. We demonstrate that the G¿q/PKC¿ cascade is responsible for ERK5 activation in response to Angiotensin II in the heart. Also, it was show that this pathway displays G¿q-biased properties which has potentially interesting therapeutic implications. Next, we performed a cellular and biochemical characterization of the G¿q/PKC¿ complex. We determined the occurrence and the specificity of the G¿q/PKC¿ complex in living cells. Also, we identified lysine 19, located in the PB1 domain of PKC¿, and two glutamic acids at positions 234 and 245, located in a novel region of G¿q termed pseudo-PB1 domain, to be essential for the formation of the G¿q/PKC¿ complex. Introduction of the double mutation (E234/ E245-AA) in G¿q completely abrogated ERK5 activation, demonstrating requirement of an efficient G¿q/PKC¿ complex for the activation of the pathway. Additionally, we found that GRK2, but not RGS2/4, prevents the association of PKC¿ to G¿q due to sequestering of the G protein. This effect, together with receptor desensitisation, impaires the downstream activation of ERK5 by Gq-coupled GPCR. Next, we demostrated that PKC¿ is not phosphorylated in response to Gq-coupled GPCR stimulation, but instead we provide the first evidence for a transient dimerisation of PKC¿. This protein was found to physically link activated G¿q and ERK5. Finally, we studied the role of the G¿q/PKC¿ complex in G¿qdependent cellular effects. The formation this protein complex was shown to be required for muscarinic M3 receptor-induced membrance blebbing and cell growth arrest. Additionally, the G¿q/PKC¿ complex was found to be necessary for apoptosis promotion. Overall, this study provides important biochemical and cellular insight into Gq-coupled receptor signalling via the novel effector PKC¿.