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A monoclonal antibody for G protein–coupled receptor crystallography

Nature Methods volume 4, pages 927–929 (2007)Cite this article

Abstract

G protein–coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human β2 adrenergic (β2AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

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Figure 1: Binding characteristics of β2AR-specific antibodies.
Figure 2: Fab 5 binds IL3 of the β2AR but does not effect structural changes associated with G-protein activation.

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Acknowledgements

This study was supported by a US National Institutes of Health Vascular Biology Training Grant 5 T32 HL007708 (to P.W.D.), the Lundbeck Foundation (to S.G.F.R.), National Institute of General Medical Sciences grant GM56169 (to W.I.W.), National Institute of Neurological Disorders and Stroke grant NS28471 (to B.K.K.), the Mather Charitable Foundation (to B.K.K.) and a generous gift from 7TM Pharma (to B.K.K.). B.K.K. and W.I.W. are grateful for the advice of R. MacKinnon in the preparation and use of antibody fragments for crystallography.

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Author notes

  1. Peter W Day and Søren G F Rasmussen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, Palo Alto, 94305, California, USA

    Peter W Day, Søren G F Rasmussen, Charles Parnot, Juan José Fung, Tong Sun Kobilka, Xiao-Jie Yao, Hee-Jung Choi, William I Weis & Brian K Kobilka

  2. Medarex, Inc., 521 Cottonwood Drive, Milpitas, 95035, California, USA

    Asna Masood & Daniel K Rohrer

  3. Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, Palo Alto, 94305, California, USA

    Hee-Jung Choi & William I Weis

Authors
  1. Peter W Day
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Contributions

P.W.D. characterized antibody binding to the β2AR on HEK-293 cells and spotted on nitrocellulose. S.G.F.R. purified antibody 5, prepared Fab 5, determined the receptor binding surface of Fab 5 by tryptic digestion, determined ligand binding affinities for the β2AR in the presence of Fab 5 and crystallized the β2AR-TMR-Fab 5 complex. C.P. and J.J.F. characterized antibody-induced fluorescence changes of β2AR-TMR. A.M. and D.K.R. generated the antibodies and performed initial antibody screening. T.S.K. prepared monoclonal antibody 5. X.-J.Y. performed bimane fluorescence experiments. H.-J.C. and W.I.W. performed isothermal titration calorimetry experiments on β2AR and Fab 5. P.W.D. and B.K.K. prepared the manuscript.

Corresponding author

Correspondence to Brian K Kobilka.

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A.M. and D.K.R. are employees of Medarex and own Medarex stock.

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Supplementary Figures 1–3, Supplementary Table 1, Supplementary Methods (PDF 1066 kb)

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Day, P., Rasmussen, S., Parnot, C. et al. A monoclonal antibody for G protein–coupled receptor crystallography. Nat Methods 4, 927–929 (2007). https://doi.org/10.1038/nmeth1112

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