Exploring endosomal signalling of G protein coupled receptors (GPCRs) using luciferase complementation and luminosensing optogenetics (360G-Wellcome-211432_Z_18_Z)

Recently G protein coupled receptors (GPCRs) have been shown to signal from intracellular compartments such as endosomes, as well as the cell surface. There is increasing evidence that some synthetic drugs can activate such intracellular located GPCRs directly, but the functional consequences of this activation have been difficult to isolate. This project will study endosomal signalling from engineered optogenetic receptors (OptoXRs), activated by light and coupled to the Gq pathway linked to calcium mobilisation. I will deliver the light stimulus to OptoXR using bioluminescence from a luciferase enzyme, in a local manner to receptors either at the cell surface or within endosomes. This will be achieved using a complementation system, in which luciferase fragments are tagged to the OptoXR and cell surface or early endosomal markers respectively. Active luciferase will therefore only be reconstituted to deliver the light when the OptoXR is present in the same location as the expressed marker. I will then be able to compare the effect of adding the substrate on OptoXR calcium responses in which case, to determine whether calcium signals are produced from receptors in different locations. This will be backed up by confocal and luminescence microscopy to identify reeptor and luciferase locations.