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’.