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Cdk5 induces constitutive activation of 5-HT6 receptors to promote neurite growth

Abstract

The serotonin6 receptor (5-HT6R) is a promising target for treating cognitive deficits of schizophrenia often linked to alterations of neuronal development. This receptor controls neurodevelopmental processes, but the signaling mechanisms involved remain poorly understood. Using a proteomic strategy, we show that 5-HT6Rs constitutively interact with cyclin-dependent kinase 5 (Cdk5). Expression of 5-HT6Rs in NG108-15 cells induced neurite growth and expression of voltage-gated Ca2+ channels, two hallmarks of neuronal differentiation. 5-HT6R–elicited neurite growth was agonist independent and prevented by the 5-HT6R antagonist SB258585, which behaved as an inverse agonist. Moreover, it required receptor phosphorylation at Ser350 by Cdk5 and Cdc42 activity. Supporting a role of native 5-HT6Rs in neuronal differentiation, neurite growth of primary neurons was reduced by SB258585, by silencing 5-HT6R expression or by mutating Ser350 into alanine. These results reveal a functional interplay between Cdk5 and a G protein–coupled receptor to control neuronal differentiation.

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Figure 1: Proteomic characterization of the 5-HT6R–associated complex.
Figure 2: 5-HT6R expression induces neurite growth of NG108-15 cells through a Cdk5-dependent mechanism.
Figure 3: 5-HT6R expression induces NG108-15 cell differentiation.
Figure 4: Neurite growth elicited by 5-HT6R expression requires receptor phosphorylation at Ser350.
Figure 5: Neurite growth elicited by 5-HT6R expression requires Cdc42 activity.
Figure 6: 5-HT6Rs promote neurite growth of cultured hippocampal and striatal neurons Primary cultured striatal and hippocampal neurons were infected with viral constructs encoding either HA-tagged 5-HT6-IRES-GFP or GFP alone.

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Acknowledgements

This work was supported by grants from the Fondation pour la Recherche Médicale (Contract Equipe FRM2009), Fondation FondaMental (fondation de coopération scientifique), ANR (contract no. ANR-11-BSV4-0008), CNRS, INSERM, University Montpellier 1 and la Région Languedoc-Roussillon. F.D. was a recipient of a fellowship from Fonds National de la Recherche (Luxembourg). MS analyses were performed using the facilities of the Functional Proteomic Platform of Montpellier Languedoc-Roussillon, and confocal microscopy was performed using the facilities of Montpellier RIO Imaging Platform. The BRET and 96-well FRET experiments were performed using the ARPEGE Pharmacology Screening Interactome platform facility (Institut de Génomique Fonctionnelle de Montpellier).

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Contributions

C.M.l.C., M.J.M., J.B., P.M. and S.C.-D. designed experiments; M.S. performed MS experiments, F.D. and F.R. studied neurite growth in neuronal cultures and explants; F.D., S.M.-L. and S.C.-D. performed BRET and FRET experiments; S.C.-D. and P.M. performed calcium imaging experiments; F.D., P.D. and S.C.-D. performed biochemistry experiments; M.J.M., P.M. and S.C.-D. wrote the manuscript.

Corresponding authors

Correspondence to Philippe Marin or Séverine Chaumont-Dubel.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–13 and Supplementary Tables 1 and 2. (PDF 9807 kb)

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Duhr, F., Déléris, P., Raynaud, F. et al. Cdk5 induces constitutive activation of 5-HT6 receptors to promote neurite growth. Nat Chem Biol 10, 590–597 (2014). https://doi.org/10.1038/nchembio.1547

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