Conformational biosensors reveal GPCR signalling from endosomes

These results suggest that β2-AR activation initiates a precisely choreographed series of events: Nb80–GFP is first recruited from the cytoplasm to the plasma membrane, β2-ARs internalize devoid of Nb80–GFP, followed by a second phase of Nb80–GFP recruitment to the internalized β2-ARs

Roshanak Irannejad; Jin C. Tomshine; Jon R. Tomshine; Michael Chevalier; Jacob P. Mahoney; Jan Steyaert; Søren G. F. Rasmussen; Roger K. Sunahara; Hana El-Samad; Bo Huang; Mark von Zastrow

2013

Scholarcy highlights

  • MP response within several minutes after agonist application
  • We generated a biosensor of activated β2-AR based on a conformationspecific single-domain camelid antibody used in recent structural studies. We reasoned that this nanobody, which selectively binds the agonist-occupied β2-AR and is able to stabilize an activated receptor conformation when present in vitro at high concentration, might act as a sensor of receptor activation when expressed at relatively low concentration in intact cells. This proved to be the case; in cells maintained in the absence of agonist, Nb80 fused to enhanced GFP localized to the cytoplasm and not with β2-ARs present in the plasma membrane(Fig. 1c, 0 min, top row; Pearson’s coefficient =
  • These results suggest that β2-AR activation initiates a precisely choreographed series of events: Nb80–GFP is first recruited from the cytoplasm to the plasma membrane, β2-ARs internalize devoid of Nb80–GFP, followed by a second phase of Nb80–GFP recruitment to the internalized β2-ARs
  • Nb80–GFP recruitment to β2-AR-containing endosomes cannot represent an artefact of persistent nanobody binding from the plasma membrane; instead, this observation reveals that β2-ARs present in early endosomes are in an activated conformation
  • The results provide direct evidence supporting the hypothesis that β2AR endocytosis contributes to a second phase of the acute cellular cAMP response, which represents a significant component of the overall biochemical signal developed within several minutes after the initial agonist application
  • More generally, that nanobody-based biosensors represent a versatile strategy for probing other types of dynamic conformational change with high spatiotemporal resolution in living cells
  • For estimating changes in Nb80–GFP surface fluorescence over time in total internal reflection fluorescence images, individual cells were selected manually and fluorescence values measured over the entire stack

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