Intracellular targeting of a light-activated GPCR elicits spatially selective signalling

G protein-coupled receptors (GPCRs) initiate unique cell responses dependent on their sub-cellular location. Major advances in our understanding of sub-cellular GPCR signalling have been driven by experimental approaches that manipulate receptor trafficking, block signalling at specific intracellular sites, or compare receptor signalling in response to ligands with differing membrane permeability. Here, we describe an optogenetic platform that allows a single stimulus to activate the same GPCR at different intracellular membranes, enabling direct comparisons of spatial signalling in intact cells. To achieve this, we have directed a light-activated GPCR chimera to endosomal, mitochondrial, Golgi and nuclear membranes using C-terminal targeting sequences. We demonstrate activation of cAMP and ERK signalling in response to light at all sites; however, each targeted receptor generates a unique transcriptional fingerprint dependent on its sub-cellular location. Our engineered optogenetic platform reveals that GPCRs targeted to distinct intracellular locations are self-sufficient signalling platforms and highlights GPCR positioning as a critical driver of signalling. TeaserControlling GPCR signals by light and location reveals how intracellular positioning shapes cellular responses.