Supplementary Materials

Supplementary Materials for:

Decoding Signaling and Function of the Orphan G Protein–Coupled Receptor GPR17 with a Small-Molecule Agonist

Stephanie Hennen, Haibo Wang, Lucas Peters, Nicole Merten, Katharina Simon, Andreas Spinrath, Stefanie Blättermann, Rhalid Akkari, Ramona Schrage, Ralf Schröder, Daniel Schulz, Celine Vermeiren, Katrin Zimmermann, Stefan Kehraus, Christel Drewke, Alexander Pfeifer, Gabriele M. König, Klaus Mohr, Michel Gillard, Christa E. Müller, Q. Richard Lu, Jesus Gomeza,* Evi Kostenis*

*Corresponding author. E-mail: (E.K.); (J.G.)

This PDF file includes:

  • Fig. S1. Sequence alignment of hGPR17 with phylogenetically related P2Y, CysLT, and small carboxylic acid receptors.
  • Fig. S2. GPR17 is properly trafficked to the cell surface.
  • Fig. S3. Native HEK293 cells are viable and respond to stimuli of endogenously expressed receptors but not to MDL29,951.
  • Fig. S4. Uracil nucleotides and CysLTs do not activate the long isoform of hGPR17 but promote robust activation of their established receptor targets.
  • Fig. S5. Pharmacological silencing of Gαq proteins attenuates GPR17-mediated cAMP formation.
  • Fig. S6 HEK-hGPR17 cells are unresponsive to ASINEX ligands 1 to 5 in holistic label-free DMR and Ca2+ mobilization assays.
  • Fig. S7. Pharmacological silencing of Gαq proteins does not abolish the capacity of MDL29,951 for dual modulation of adenylyl cyclase.
  • Fig. S8. GPR17-Rluc fusion protein is targeted to the cell surface and signals via G protein–mediated pathways.
  • Fig. S9. CysLT1 antagonists effectively inhibit intracellular Ca2+ mobilization by the CysLT1 receptor.
  • Fig. S10. Mouse and rat orthologs of GPR17 are targeted to the plasma membrane in HEK293 cells engineered to stably express these receptors.
  • Fig. S11. MDL29,951 productively interacts with rodent GPR17 orthologs.
  • Fig. S12. MDL29,951, but not ASINEX ligands 1 to 5, display functional activity on rat GPR17.
  • Fig. S13. Pranlukast and montelukast do not activate rat and mouse GPR17.
  • Fig. S14. Pranlukast and montelukast do not dampen Ca2+ signaling of muscarinic M3 receptors.
  • Fig. S15. MDL29,951 does not stimulate loading of [35S]GTPγS to membranes expressing biogenic amine histamine H3 and dopamine D2 receptors.
  • Fig. S16. MDL29,951 does not trigger cell activation across 16 distinct ATP-responsive immortalized and primary cell lines in a receptor panning approach.
  • Fig. S17. HEK293 cells express functional P2Y receptors as evidenced by their responsiveness to purinergic agonists.
  • Fig. S18. Forced expression of a cocktail containing eight purinergic receptors enhances functional DMR responses to purinergic agonists in HEK293 cells.
  • Fig. S19. Immunophenotypes of oligodendroglial cells during differentiation.
  • Fig. S20. Pranlukast abolishes GPR17-mediated Ca2+ mobilization but does not blunt Ca2+ signaling induced by muscarinic M3 receptors in primary rat oligodendrocytes.
  • Fig. S21. Gαi inhibitor PTX and Gαq inhibitor FR900359 do not affect viability of primary rat oligodendrocytes.
  • Fig. S22. Introduction of a triple HA tag at the N termini of hGPR17 and hGPR17-Rluc does not alter signaling capacity of the receptor constructs.
  • Fig. S23. In-frame fusion of GFP2 to the C terminus of GPR17 does not affect receptor functionality.
  • Fig. S24. 1H-NMR spectrum of FR900359 in CDCl3.
  • Fig. S25. 13C-NMR spectrum of FR900359 in CDCl3.
  • Table S1. Chemical structures of test compounds selected for biomolecular screening at GPR17.
  • Table S2. Potencies (pEC50 values) of MDL29,951 at recombinant hGPR17 cell lines.
  • Table S3. Functional activity of GPR17 agonist MDL29,951 on purinergic and CysLT1 receptors.

[Download PDF]

Technical Details

Format: Adobe Acrobat PDF

Size: 2.79 MB

Citation: S. Hennen, H. Wang, L. Peters, N. Merten, K. Simon, A. Spinrath, S. Blättermann, R. Akkari, R. Schrage, R. Schröder, D. Schulz, C. Vermeiren, K. Zimmermann, S. Kehraus, C. Drewke, A. Pfeifer, G. M. König, K. Mohr, M. Gillard, C. E. Müller, Q. R. Lu, J. Gomeza, E. Kostenis, Decoding Signaling and Function of the Orphan G Protein–Coupled Receptor GPR17 with a Small-Molecule Agonist. Sci. Signal. 6, ra93 (2013).

© 2013 American Association for the Advancement of Science