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Modular composition and dynamics of native GABAB receptors identified by high-resolution proteomics

Abstract

GABAB receptors, the most abundant inhibitory G protein–coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-β A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.

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Figure 1: ME-AP proteomics identify the protein constituents of native GABAB receptors.
Figure 2: Deletions of core subunits unravel protein interactions and diversity in GABAB receptor complexes.
Figure 3: GABAB receptors forming during postnatal development identify time-dependent dynamics in complex assemblies.
Figure 4: Assembly of HCN2 channels into GABAB(1a,2) receptor complexes and their functional coupling in DA neurons.
Figure 5: AJAP1 and A4 are distinctly assembled into heterologously reconstituted GABAB receptor complexes and can be effectively co-purified via extracellularly administered sushi domains (SDI/II).
Figure 6: AP series identify two distinct sets of sushi-domain interactors.
Figure 7: Nano-architecture and modular assembly of native GABAB receptors derived from proteomic and biochemical analyses, as well as from properties reported in literature.

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Acknowledgements

We thank J.P. Adelman, G. Zolles, J. Bischofberger, C. Lüscher and H.R. Brenner for discussions and/or critical reading of the manuscript, N. Nevian for reconstruction of neurons, and A. Haupt and D. Böning for support in bioinformatics and software development. This work was supported by grants of the Swiss Science Foundation (31003A-152970) the National Center for Competences in Research (NCCR) 'Synapsy, Synaptic Basis of Mental Health Disease' to B.B. and by grants of the Deutsche Forschungsgemeinschaft to B.F. (SFB 746/TP16; Fa 332/9-1,2).

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J.S., U.S., E.P.-G., B.B. and B.F. conceived the project. J.S., E.P.-G., A.S., A.K., W.B., A.G.-K., T.F., A.R., M.C.D., A.H., J.K., M.G., U.S. and B.F. performed experiments and analyzed data. J.S., B.B. and B.F. wrote the manuscript with the support of all of the authors.

Corresponding authors

Correspondence to Bernhard Bettler or Bernd Fakler.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Table 2 (PDF 17991 kb)

Supplementary Methods Checklist (PDF 351 kb)

Supplementary Table 1: PV ratios (rPVs) obtained for the identified GABAB receptor constituents in APs.

Summary of PV ratios (rPVs) obtained for the identified GABAB receptor constituents in APs with the indicated anti-B1, anti-B2 and anti-KCTD ABs upon solubilization with CL-91 and CL-47; source material was rat brain (rb), or mouse brain from wildtype (mb) or target knock-out animals (KO). rPV threshold (values in red) indicates the minimal PV ratio for considering a given protein specifically co-purified in the respective AP. Empty fields indicate that no PV ratio could be calculated. (XLSX 44 kb)

Supplementary Table 3: Summary of Mascot search results of the datasets underlying comprehensive MS analysis of the ME-APs in Fig. 1.

Columns (from left to right) indicate: entry name of the protein in the UniProtKB/Swiss-Prot database, its accession number, recommended protein name, theoretical molecular weight and length, total number of identified non-redundant peptides and the number of identified non-redundant protein-specific peptides. (XLSX 166 kb)

Supplementary Table 4: Data underlying the heatmap shown in Figure 2.

(XLSX 13 kb)

Supplementary Table 5: Data underlying the graphs shown in Figures 6a and b

(XLSX 41 kb)

Supplementary Table 6: Summary of MS results and specificity evaluation determined in ME-APs for the GABAB interactors reported in literature.

Accession numbers refer to the UniProtKB/Swiss-Prot database; peptides denote the number of MS-retrieved peptides identifying the respective protein in anti-B1/B2 APs. Abbreviations used for annotation of the (non)-specificity and (non)-consistency of co-purification are indicated at the bottom. (XLSX 41 kb)

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Schwenk, J., Pérez-Garci, E., Schneider, A. et al. Modular composition and dynamics of native GABAB receptors identified by high-resolution proteomics. Nat Neurosci 19, 233–242 (2016). https://doi.org/10.1038/nn.4198

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