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Multiple ligand-specific conformations of the β2-adrenergic receptor

Nature Chemical Biology volume 7, pages 692–700 (2011)Cite this article

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Abstract

Seven-transmembrane receptors (7TMRs), also called G protein–coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β2-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

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Figure 1: Labeling of cysteines and lysines in the β2AR to monitor conformational changes.
Figure 2: Schematic two-dimensional topology of human β2AR showing location of sites of study.
Figure 3: Schematic illustration of time-dependent, residue-specific labeling experiment designed to monitor conformational changes in the β2AR.
Figure 4: Reactivities of residues in β2AR featuring conformational rearrangements of classic receptor activation.
Figure 5: Reactivities of residues in β2AR featuring ligand-specific conformational rearrangements.
Figure 6: Proposed models illustrating the conformational rearrangements observed in different structural elements of the β2AR.

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Acknowledgements

R.J.L. is an investigator with the Howard Hughes Medical Institute. This work was supported in part by grants from the US National Institutes of Health (HL16037 and HL70631) to R.J.L. T.G.O. is supported by a grant from the US National Institute of General Medical Sciences (5RO1GM081666). We gratefully acknowledge T. Haystead (Duke University) and D. Loiselle for valuable assistance with the mass spectrometry experiments, and Theravance, Inc. (South San Francisco, California, USA) for the supply of THRX-144877; we are also grateful to R.T. Strachan, J. Kovacs, R. Dror (D.E. Shaw Research, New York, New York, USA), C.H. Borchers (University of Victoria, Victoria, British Columbia, Canada), I.A. Kaltashov (University of Massachusetts), B. Donald (Duke University) and members of his laboratory for stimulating ideas and helpful discussions; we also thank X. Jiang and C.M. Lam for excellent technical assistance and D. Addison and Q. Lennon for secretarial assistance.

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  1. Alem W Kahsai and Kunhong Xiao: These authors contributed equally to this work.

  2. khxiao@receptor-biol.duke.edu

Authors and Affiliations

  1. Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA

    Alem W Kahsai, Kunhong Xiao, Sudarshan Rajagopal, Seungkirl Ahn, Arun K Shukla, Jinpeng Sun & Robert J Lefkowitz

  2. Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA

    Alem W Kahsai, Arun K Shukla & Robert J Lefkowitz

  3. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA

    Terrence G Oas & Robert J Lefkowitz

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Contributions

A.W.K., K.X., T.G.O. and R.J.L. designed the experiments; A.W.K., K.X., S.A. and A.K.S. conducted experiments; A.W.K., K.X., S.R., S.A., A.K.S., J.S., T.G.O. and R.J.L. analyzed data; A.W.K., K.X. and R.J.L. wrote the paper; all authors read, edited and discussed the paper.

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Correspondence to Kunhong Xiao or Robert J Lefkowitz.

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Kahsai, A., Xiao, K., Rajagopal, S. et al. Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat Chem Biol 7, 692–700 (2011). https://doi.org/10.1038/nchembio.634

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