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And Khivorin-Derivatives Are Small Molecule Partial Agonists for Adhesion G Protein Coupled Receptors GPR56 / ADGRG1 and GPR114 / ADGRG5

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And Khivorin-Derivatives Are Small Molecule Partial Agonists for Adhesion G Protein Coupled Receptors GPR56 / ADGRG1 and GPR114 / ADGRG5 Supplementary Data Gedunin- and Khivorin-Derivatives are Small Molecule Partial Agonists for Adhesion G protein Coupled Receptors GPR56 / ADGRG1 and GPR114 / ADGRG5. Hannah M. Stoveken, Scott D. Larsen, Alan V. Smrcka, and Gregory G. Tall Molecular Pharmacology Supplemental Figure 1 Quinacrine Nonspecifically Activates SRE-Luciferase. (A) Quinacrine activated GPR56 A386M-7TM-expressing HEK293 cells ~2-fold over non- receptor cells in the high throughput screen (values were taken from Figs. 1C-D). (B) Increasing concentrations of quinacrine were tested in a directed SRE-luciferase assay with the truncated tethered agonist receptor, GPR56 M389-7TM. Results are expressed as the firefly luciferase (FLuc) values normalized to the Renilla luciferase internal control (RLuc). Error bars are the mean ± S.D. of three technical replicates. Supplemental Figure 2 Synaptamide does not Enhance G protein Activation Kinetics by Intact GPR110. Urea-dissociated GPR110 robustly activated, (A) Gq, and did not activate, (B) Gs in biochemical G protein reconstitution assays. Mock-extracted GPR110 membrane preparations (i.e. intact NTF:GPCR domain GPR110) showed moderate Gq activation and no Gs activation, whether treated with ethanol vehicle (EtOH), or with 1 µM synaptamide attained from two independent commercial sources. The apparent increased activity of holo-receptor GPR110 (Mock extraction) over dissociated GPR110 (Urea extraction) for Gs activation is actually background activity contributed by endogenous insect cell membrane GTPases present in the mock extracted 1 membrane preparation. Urea removes a substantial portion of this background GTPgS binding activity. Supplemental Figure 3 GPR114 Synthetic Agonist Peptides Cross-Activate GPR56. (A) Amino acid sequence alignment of the tethered-peptide-agonist regions of GPR56 and GPR114 in comparison with the GPR56 / GPR114 P7 peptide and the GPR114 P18, P19, and P20 activating peptides (Wilde et al., 2016). (B) GPR114 P18-20 peptides were tested at increasing concentrations with the truncated tethered-peptide-agonist receptor, GPR114 A230M-7TM in a cAMP Response Element-luciferase (CRE-Luc) gene reporter assay (Chepurny and Holz, 2007). HEK293T cells were transfected with receptor or empty vector (350 ng), CRE-Luc (100 ng) and Renilla luciferase (1 ng) in a 24-well plate format. Twenty-four hours post transfection, cells were treated with peptide or DMSO and incubated for an additional five hours. CRE-Luc signal was normalized to RLuc and expressed as fold increase over cells transfected with CRE-Luc alone. Error bars are the mean ± S.D. of three technical replicates. (C) GPR114 activating peptides (20 µM) were incubated for five hours with HEK293 cells expressing GPR56 A386M-7TM receptor in a directed SRE-luciferase reporter assay. Error bars are the mean ± S.D. of three technical replicates. (D) GPR114 P18 peptide enhanced the kinetics of GPR56 A386M-7TM- mediated G protein 13 activation in the receptor reconstitution assay. Error bars are the mean ± S.D. of three technical replicates. 2 Chepurny OG and Holz GG (2007) A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts. Journal of biomolecular screening 12(5): 740-746. Wilde C, Fischer L, Lede V, Kirchberger J, Rothemund S, Schoneberg T and Liebscher I (2016) The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 30(2): 666-673. 3 Supplemental Figure 1 A Quinacrine-Mediated Luciferase ) Activity in HTS 1.5x106 1.0x106 alues (RLUs V 0.5x105 Raw FLuc 0 Counter-screen Screen B GPR56 M389 7TM SRE +/- Quinacrine SRE M389 7TM 15 c 10 FLuc/ RLu 5 0 1 5 20 0 1 2 5 10 20 [Quinacrine] (µM) Supplemental Figure 2 Gq 1.0 q G l S/ mo 0.5 P mol GT Urea GPR110 0.0 0 10 20 30 Mock GPR110 + EtOH Time (min) Mock GPR110 + Sigma Synaptamide Mock GPR110 + 1.0 Gs s Cayman Chemical Synaptamide G l S/ mo 0.5 P mol GT 0.0 0 10 20 30 Time (min) Supplemental Figure 3 Tethered A Agonist GPR56: MTYFAVLMVSSVEVDAVHKHYLSLLS-TM1 GPR114: MTYFAVLMQLSPALVPAELLAPLT-TM1 P7: TYFAVLM GPR114 P18: TYFAVLMQLSPALVPAEL GPR114 P19: TYFAVLMQLSPALVPAELL GPR114 P20: TYFAVLMQLSPALVPAELLA B GPR114 A230M 7TM +GPR114 P18-P20 30 P18 P19 P20 20 10 Fold Increase over CRE-Luc 0 0 1 5 1020 0 1 5 1020 0 1 5 1020 [GPR114 Peptides] (µM) C GPR56 A386M 7TM +GPR114 Activating Peptides GPR56 40 No Receptor A386M 7TM E 30 20 10 Fold Increase Over SR 0 P7P18P19P20 P7P18P19P20 DMSO DMSO 20µM Peptide D GPR56 A386M 7TM : G protein 13 1.0 A386M 7TM + 20µM P18 13 A386M 7TM + DMSO α 0.8 0.6 S / mol G γ 0.4 0.2 mol GTP 0.0 0 10 20 30 Time (min).
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