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Structure and functional interaction of the extracellular domain of human GABAB receptor GBR2

Abstract

Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABAB receptor is a G protein–coupled receptor central to mammalian brain function. Malfunction of GABAB receptor has been implicated in several neurological disorders. GABAB receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimeric interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABAB receptor that is unique to the GABAergic system.

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Figure 1: Interaction between GBR1bVFT and GBR2VFT.
Figure 2: Crystal structure of human GBR2VFT.
Figure 3: Comparison of interdomain hinge angles.
Figure 4: Analysis of N-glycosylated GBR2 mutants.
Figure 5: Heterodimerization interface of GBR2VFT.
Figure 6: Effect of the GBR2 mutation Y118A on receptor function.
Figure 7: Effect of the GBR1b mutations Y113A and Y117A on receptor function.
Figure 8: Effect of the GBR1b mutation G151V on ligand binding.

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Acknowledgements

We thank W.A. Hendrickson for advice and for generously providing lab space at the initial stage of this project, R. Kass for support and advice on electrophysiology experiments, L. Jan (University of California, San Francisco) for the cDNA clones encoding GIRK channels, V. Frasca for isothermal titration calorimetry and DSC measurements, C. Brouillette and I. Protassevitch for DSC measurements, L. Shapiro and J. Hunt for advice, M. Gawinowicz for mass spectrometry and protein sequencing, K. Rajashankar at the Advanced Proton Source and J. Schwanof at the National Synchrotron Light Source for help with data collection, L. Geneve for analyzing the glycosylation mutants, the ARPEGE platform facility at Institute of Functional Genomics (Montpellier, France) for inositol phosphate and Tag-lite (Cisbio) assays, and W.A. Hendrickson and M. Evelyn for critical reading of the manuscript. This work was supported by American Heart Association grant SDG0835183N, US National Institutes of Health grant R01GM088454 (both to Q.R.F.) and Agence Nationale de la Recherche (France) grant ANR-09-BLANC-0272 (to J.-P.P.). Q.R.F. is an Irma Hirschl Career Scientist, Pew Scholar and McKnight Scholar.

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Q.R.F. conceived the study, designed the experiments and wrote the paper; Q.R.F., Y.G., D.X., L.M., D.L.M., Y.C., S.B. and M.B. performed experiments and analyzed data; J.K. and J.-P.P. designed and carried out studies of glycosylation mutants; M.Q. and J.A.J. supervised SPA experiments.

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Correspondence to Qing R Fan.

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Geng, Y., Xiong, D., Mosyak, L. et al. Structure and functional interaction of the extracellular domain of human GABAB receptor GBR2. Nat Neurosci 15, 970–978 (2012). https://doi.org/10.1038/nn.3133

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