Conformational biosensors reveal GPCR signalling from endosomes

Abstract

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or non-subcellular resolution1. It has been subsequently proposed that signalling by internalized GPCR is restricted to G-protein-independent mechanisms such as scaffolding by arrestins2,3, or GPCR activation elicits a discrete form of persistent G protein activation4–9, or that internalized GPCR can indeed contribute to the acute G protein-mediated response10. Evidence supporting these various latter hypotheses is indirect or subject to alternate interpretation, and it remains unknown if endosome-localized GPCR are even present in an active form. Here we describe the application of conformation-specific single domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR11, and its cognate G protein, Gs (ref. 12) in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane; and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States

“…While still activating intracellular cAMP, Gαs DREADDs act as a 'generic' Gαs GPCR, it is currently unclear whether this tool exhibits identical intracellular signaling cascades comparable to the endogenous Gαs receptors expressed within the BLA. We now know, for instance, that all pools of Gαs are not identical and that receptors signal to G-proteins in a very selective microdomain-dependent manner that is limited by receptor subtype (Irannejad et al, 2013;Puthenveedu et al, 2010). Studies thoroughly comparing the pharmacodynamic properties of Gαs DREADDs to canonical GPCRs, such as β 2 AR, are needed to provide further validation.…”

Spatio‐Temporal Parameters of Endosomal Signaling in Cancer: Implications for New Treatment Options

“…The current model proposes that activated cell surface GPCRs redistribute into early endosomes, from where signaling can be extended. Direct evidence that GPCRs and cognate G protein activation indeed occurs in endosomes came from recent experiments employing nanobody‐based biosensors in living mammalian cells [Irannejad et al, 2013]. In this report von Zastrow and colleagues used conformational active‐state‐specific single‐domain antibodies and detected two temporally and spatially separated waves of β2AR signaling and its associated Gs protein.…”

“…Both signaling waves led to the accumulation of cAMP by the enzyme adenylyl cyclase, reaching a maximum of two signaling waves within approximately 10 min. Interestingly, the second discrete phase of β2AR signaling is separated from the first wave by an endocytosis event and appears to begin shortly after the delivery of receptors to early endosomes [Irannejad et al, 2013]. …”

The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block

“…Third, the spatial pattern of heterotrimeric G protein activation by GIV also poses a stark contrast to canonical heterotrimeric G protein signaling. Canonical G protein activation by ligand-activated GPCRs is initiated exclusively at the PM and may continue on PM-contiguous endocytic compartments [32][33][34] By contrast, non-canonical heterotrimeric G protein activation by GIV-GEM can be triggered at multiple intracellular compartments, including centrosomes, focal adhesions, cell-cell junctions, early endosomes, exocytic vesicles, autophagosomes, and more recently, on Golgi membranes (summarized in 30 ) (Fig. 1).…”

“…Such activation is triggered primarily at the plasma membrane (PM) via a finite process that is rapid and completes within a few hundred milliseconds, and may continue on PM-contiguous endocytic compartments. [32][33][34] Right: Non-canonical G protein signaling that is triggered by GEMs are characterized by 4 key differences (from top to bottom) -(A) GEMs can coordinately modulate heterotrimeric G protein signaling downstream of diverse classes of receptors (signal input). (B) GEMs can activate monomeric G i a (as a GEF) or inhibit stimulatory G s a (as a GDI) using the same short motif, thereby coordinately reducing cellular cAMP.…”