Figure The coupling of Stachel activated GPR56 and BFPP associated mutations of the receptor with Gα11.

The effect of BFPP mutations on β-Arrestin recruitment

It was previously reported that NTF truncated human GPR56 from amino acids 1–342 gave enhanced interactions with β-arrestin compared to the full-length receptor [36]. In this work, GPR56 or BFPPmutants of the receptor were co-expressed with β-arrestin (Rluc8-arrestin3-Sp1) and GRK-based biosensors [30] to assess the effect of each mutation on the β-arrestin recruitment, upon receptor activation with the Stachel peptide. For this, HEK293 cells were treated with 1mM P7 Stachel peptide and β-arrestin recruitment was measured as a BRET signal. The arrestin biosensor assay utilized in this study gave increased plasma membrane recruitment for the NTF truncated GPR56 however stimulation of wild-type receptor orBFPP mutants with the Stachel peptide did not lead to any measurable recruitment.