Non-canonical β-adrenergic activation of ERK at endosomes.

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival 1 . However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood 2-4 . Here we investigated how spatially organized β 2 -adrenergic receptor (β 2 AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors 5 , we show that β 2 AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gα s and requires ligand-stimulated β 2 AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gα s , RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β 2 AR and Gα s signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.