title: 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 no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs 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 GPCR, 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.
authors.affiliation: Department of Psychiatry, University of California, San Francisco, California 94158, USA.
authors.forename: Roshanak
authors.initials: R
authors.lastname: Irannejad
authors.forename: Jin C
authors.initials: JC
authors.lastname: Tomshine
authors.forename: Jon R
authors.initials: JR
authors.lastname: Tomshine
authors.forename: Michael
authors.initials: M
authors.lastname: Chevalier
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Mahoney
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: Søren G F
authors.initials: SG
authors.lastname: Rasmussen
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: Hana
authors.initials: H
authors.lastname: El-Samad
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
authors.forename: Mark
authors.initials: M
authors.lastname: von Zastrow
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D022163:Clathrin-Coated Vesicles; D000242:Cyclic AMP / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008954:Models, Biological; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D018343:Receptors, Adrenergic, beta-2; D061905:Single-Domain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol