Conformational biosensors reveal GPCR signalling from endosomes

Area of Science:

• Molecular Pharmacology
• Cell Biology
• Biochemistry

Background:

• Traditionally, G-protein-coupled receptor (GPCR) signaling was believed to be confined to the plasma membrane.
• Previous evidence for signaling from internalized receptors was indirect or open to interpretation.
• The active state of endosome-localized GPCRs remained unconfirmed.

Purpose of the Study:

• To directly investigate the subcellular localization and activity of GPCRs and their cognate G proteins in living cells.
• To determine if internalized GPCRs contribute to canonical G-protein-mediated signaling.
• To introduce a novel method for probing dynamic conformational changes of receptors in vivo.

Main Methods:

• Utilized conformation-specific single-domain antibodies (nanobodies) to visualize receptor and G protein activation.
• Studied the prototypical β2-adrenoceptor and its G protein, Gs, in living mammalian cells.
• Applied adrenergic agonist isoprenaline to stimulate signaling pathways.

Main Results:

• Demonstrated simultaneous activation of the β2-adrenoceptor and Gs protein at the plasma membrane and early endosome membranes.
• Showed that internalized receptors contribute to the cellular cyclic AMP response within minutes of agonist application.
• Confirmed the presence and activity of GPCRs on endosomes.

Conclusions:

• Canonical GPCR signaling occurs from both the plasma membrane and endosomes.
• Internalized GPCRs actively participate in G-protein-mediated cellular responses.
• Nanobody technology offers a versatile approach to study dynamic receptor conformational changes in living systems.