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Measurement of the millisecond activation switch of G protein–coupled receptors in living cells

Abstract

Hormones and neurotransmitters transduce signals through G protein–coupled receptors (GPCR). Despite their common signaling pathways, however, the responses they elicit have different temporal patterns. To reveal the molecular basis for these differences we have developed a generally applicable fluorescence-based technique for real-time monitoring of the activation switch of GPCRs in living cells. We used such direct measurements to investigate the activation of the α2A-adrenergic receptor (α2AAR; neurotransmitter) and the parathyroid hormone receptor (PTHR; hormone) and observed much faster kinetics than expected: 40 ms for the α2AAR and 1 s for the PTHR. The different switch times are in agreement with the different receptors' biological functions. Agonists and antagonists could rapidly switch the receptors on or off, whereas a partial agonist caused only a partial signal. This approach allows the comparison of agonist and partial agonist intrinsic activities at the receptor level and provides evidence for millisecond activation times of GPCRs.

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Figure 1: FRET efficiency and time-resolved changes in the FRET signal of PTHR-cam.
Figure 2: Pharmacological properties of the GPCR-cam constructs.
Figure 3: Agonist-induced decrease in FRET signal corresponds to receptor activation.
Figure 4: Action of the partial agonist clonidine on α2AAR-cam.
Figure 5: Comparison between the dynamics of receptor activation and desensitization of PTHR-cam.
Figure 6: Dynamics of agonist-mediated receptor conformational change.

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Acknowledgements

This paper is dedicated to R.J. Lefkowitz on the occasion of his 60th birthday. We thank Manfred Bernhard for support with the radioligand binding studies and Christian Dees for the G protein preparation. We are grateful to Martin Heck for help with computer simulations and Ernst J.M. Helmreich, Lutz Hein, Ursula Quitterer and Henry R. Bourne for critical comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (grants to M.J.L.) and fellowships from the Alexander von Humboldt foundation and Ministerio de Ciencia y Tecnología, Spain (to M.C.).

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Correspondence to Martin J Lohse.

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The University of Würzburg has applied for a patent covering the use of the technology described in this paper for drug development purposes.

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Vilardaga, JP., Bünemann, M., Krasel, C. et al. Measurement of the millisecond activation switch of G protein–coupled receptors in living cells. Nat Biotechnol 21, 807–812 (2003). https://doi.org/10.1038/nbt838

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