MATERIAL AND METHODS

The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [31].

Chemical reagents and other materials

General laboratory chemicals were purchased from Sigma–Aldrich (Darmstadt, Germany) or Merck (Darmstadt, Germany). Cell culture media components, Lipofectamine™ LTX Reagent with PLUS™, GeneJET Plasmid Miniprep Kit, GeneJET PCR Purification Kit were purchased from Thermo Fisher Scientific (MA, USA) or Invitrogen (MA, USA). Q5 Hot Start High-Fidelity DNA Polymerase was purchased from New England Biolabs (MA, USA). Primers used in this study were purchased from IDT Europe (Munich, Germany). Protease inhibitor cocktail tablets were from Roche Diagnostics, (Mannheim, Germany). Antibodies (anti-GPR56, anti-β-actin and secondary horseradish peroxidase conjugated antibody) were from Santa Cruz Biotechnology (TX, USA). Immobilon-P membrane was from Millipore (Millipore Corporation, Bedford, MA). ECL reagent was from Pierce (MA, USA). White 96-well Lumitrac microplates were purchased from Greiner Bio-One (Kremsmünster, Austria), Nano-Glo®Luciferase Assay System which includes Furimazine was from Promega (Promega, USA), Coelenterazine was from Invitrogen (MA, USA). Berthold Mithras2 LB 943 multimode plate reader was used in bioluminescence/fluorescence based experiments.

Synthesis of P7 Stachelpeptide

The Stachel peptide for GPR56 P7, “TYFAVLM ” was synthesized using solid phase peptide synthesis based on Fmoc strategy using HBTU and DIEA as the coupling agents. 20% piperidine-DMF solution was used as deprotection solution for Fmoc group and Rink amide resin was used as solid support. To cleave the side chain protecting groups and the peptide from resin, TFA-TIPS-H2O (95 : 2.5 : 2.5) system was used. Analytical HPLC was performed on a Dionex instrument, with ThermoScientific ODS Hypersil, 5µ C18 column. 0.1% TFA-water as solvent A and 0.08% TFA-acetonitrile as solvent B were used. The gradient was 0-90% B 20 min and peptide was used without the necessity of any further purification. The purity of synthesized peptide was >90%.

DNA Constructs

The plasmid carrying the full-length human cDNA of GPR56/ADGRG1 was a generous gift from Assoc. Prof. Dr. Demet Araç (University of Chicago, USA). GRK-based G protein BRET biosensors (Venus 156-239-Gβ1, Venus 1-155-Gɣ2 and masGRK3ct-NanoLuc) were kind gifts from Prof. Dr. Kirill Martemyanov (University of Florida, USA). Finally, β-arrestin BRET biosensors (Rluc8-Arrestin-3-Sp1 and mem-linker-citrine-SH3) were generously donated by Prof. Dr. Nevin A. Lambert (Augusta University, USA).

Site directed mutagenesis

Five missense mutations from BFPP patients, which were shown not to fully block the surface expression of GPR56 [32] were introduced using site-directed mutagenesis with the double primer method. For this, both forward and reverse primers were designed to carry the targeted base change and to prevent the primer-primer dimers, eight non-overlapping bases were introduced at the 3’ end of each primer. All primers used in the mutagenesis are shown in Table 1 (see supplementary materials for more). All constructs were verified by sequencing.
Table List of primers used to construct BFPP mutants of GPR56. For each mutation first lane corresponds forward primer, second lane gives reverse primer. All primers were given in 5’ > 3’.