Figure Western blot showing the expression of GPR56 and the BFPP mutants in transiently transfected HEK293 cells. The first row corresponds to the expression of GPR56 and the mutant receptors detected by anti-GPR56 antibody targeting the NTF. The lower row corresponds to β-actin expression detected by anti-β-actin antibody.

The effect of BFPP mutations on Gα12coupling

GPR56 or GPR56R38W, GPR56Y88C, GPR56C91S, GPR56R565W, GPR56L640RBFPP mutants were co-expressed with Gα12 and GRK-based Gβδ biosensors to assess the effect of each mutation on the G protein coupling, upon receptor activation with the Stachelpeptide. For this, HEK293 cells were treated with 1mM P7 Stachelpeptide and Gα12 coupling and heterotrimeric G protein activation was measured as nanoBRET signal. The most severe coupling defect between the receptor and Gα12 was observed for GPR56L640R (p<0.01 compared with GPR56). Gα12coupling defects were also measured for GPR56R565W (p<0.01 compared with GPR56) and GPR56C91S (p<0.01 compared with GPR56). GPR56R38W and GPR56Y88C BFPP mutants showed Gα12 coupling upon Stachel peptide stimulation with no significant change compared with the wild-type receptor.