Figure The coupling of Stachel-activated GPR56 and disease associated mutations of the receptor with Gα13.

The effect of BFPP mutations on Gα11coupling

GPR56 was previously shown to couple with Gαq/11 as a GPR56-CD81-Gαq/11 complex [34]. In human glioma cells, stimulation of GPR56 with its agonistic monoclonal antibodies resulted in signaling through Gαq dependent Rho pathway [35]. GPR56 or BFPP mutants of the receptor were co-expressed with Gα11 and GRK-based Gβδ biosensors to assess the effect of each mutation on the G protein coupling when the receptor is stimulated with its Stachel peptide. For this, HEK293 cells were treated with the 1mM P7 Stachel peptide and Gα11coupling and heterotrimeric G protein activation was measured as nanoBRET signal. GPR56C91S gave statistically indistinguishable nanoBRET compared with wild-type receptor. A decrease in nanoBRET indicative of G protein coupling defect was measured in all otherBFPP mutants, however GPR56R565W did not show any Gα11 coupling.