Fig 2: 10C7 potentiates activation of Gα12/13 with a preference for Gα13 and GAIN cleavage is not necessary for 10C7 activation of GPR56-T383A. a, b Time course of 10C7-mediated p115-RhoGEF (a) or PDZ-RhoGEF (b) recruitment to the PM. HEK293 ΔGα12/13 cells were co-transfected with 125 ng of GPR56-WT, p115-RhoGEF-RlucII (a) or PDZ-RhoGEF-RlucII (b) and rGFP-CAAX alone or with Gα12 or Gα13 respectively, and stimulated with 20nM 10C7 or vehicle (arrow). c, d Time course of 3αDOG-mediated activation of Gα12/13 reflected by p115-RhoGEF (c) or PDZ-RhoGEF (d) recruitment to PM. HEK293 ΔGα12/13 cells co-transfected with 125 ng of WT and p115-RhoGEF-RlucII (c) or PDZ-RhoGEF-RlucII (d) and rGFP-CAAX, alone or with Gα12 or Gα13 subunits and stimulated with 10 µM of 3αDOG or vehicle (DMSO 0,1%) (arrow). e, f Time course of 10C7-mediated p115-RhoGEF (e) or PDZ-RhoGEF (f) recruitment to the PM. HEK293 ΔGα12/13 cells were co-transfected with 125 ng GPR56-T383A, p115 or PDZ-RhoGEF-RlucII and rGFP-CAAX alone or with Gα12 or Gα13 subunits and stimulated with 20 nM 10C7 or vehicle (arrow). Data are represented as the means ± SD of triplicate in a representative experiment that was repeated three times with similar results. Note that some control curves and error bars overlap with each other

Fig 3: BRET G protein coupling profile of GPR56-WT and Mutants. a Schematic representation of GPR56-WT, autocleavage deficient mutant GPR56-T383A and GPR56-Δ1-385 mutant in which 3 amino acids were deleted from the N terminus of the Tethered Agonist (TA). GPS is indicated by a red arrow and Tethered Agonist sequence is TYFAVLM in which T (red) is the GPS P1’ residue. Abbreviations are, ECD, Extracellular domain; NTF, N-terminal fragment; CTF, C-terminal fragment, GAIN, GPCR Autoproteolysis Inducing domain; TA, Tethered Agonist, GPS, GPCR Proteolysis Site; ICD, Intracellular Domain. b Schematic diagram of the G protein BRET biosensor (GABY) which measures the activation of G protein resulting in distancing of RlucII from GFP10 and a decrease of BRET signal, created with BioRender.com. c BRET-based G protein activation profile in the presence of 125ng GPR56-WT cDNA represented by the BRET ratio normalized with that measured in the absence of GPR56-WT using empty vector (Mock). d Representative GPR56-WT and mutants BRET2 ratio curves with the Gα12 BRET sensor in relation to increasing receptor DNA concentrations (0-250ng). Data are represented as the mean values of at least three independent experiments of three replicates each (mean ± SD)

Fig 4: 3αDOG activates GPR56-Δ1-385 but not GPR56-T383A. a, c Time course of 3αDOG-mediated activation of Gα12/13 proteins reflected by p115-RhoGEF or b, d PDZ-RhoGEF recruitment to the PM. HEK293T ΔGα12/13 cells were co-transfected with either 400 ng of Δ1-385 (a, b) or 125 ng of T383A (c, d), and p115-RhoGEF-RlucII (a, c) or PDZ-RhoGEF-RlucII (b, d) and rGFP-CAAX, alone or in presence of Gα12 or Gα13 subunits and stimulated with 10 µM of 3αDOG or vehicle (DMSO 0,1%) (arrow). Data are represented as the means ± SD of triplicate in a representative experiment that was repeated three times with similar results Note that some control curves and error bars overlap with each other

Fig 5: 10C7 and 3αDOG trigger the recruitment of β-arrestin2 to the PM and GPR56-WT is internalized in a β-arrestin-independent but clathrin-dependent manner. a Schematic representation of the β-arrestin-based biosensors to monitor β-Arr1/2 recruitment to the PM. Upon agonist stimulation, the β-Arr1/2-RlucII-tagged is recruited by the receptor to the PM inducing an increase BRET with the membrane-anchored rGFP (rGFP-CAAX), created with BioRender.com. b, c Time course of 10C7 and 3αDOG-mediated β-arrestin1 (b) or β-arrestin2 (c) recruitment to PM by GPR56-WT. HEK293 cells were co-transfected with 125 ng GPR56-WT and β-arrestin1 or β-arrestin2 -RlucII/rGFP-CAAX sensors before stimulation with 20 nM of 10C7, 10 µM of 3αDOG or Vehicle (arrow). Data are represented as the means ± SD of triplicate in a representative experiment that was repeated three times with similar results. d Myc-GPR56 stable HEK293 cells were treated with non-targeting siRNA (CTL-siRNA) or β-arrestin1 and 2 specific siRNA alone (β-arr1/2-siRNA) or together with clathrin siRNA (Clathrin-siRNA), and also transfected with Flag-AT1R. Cell surface GPR56 and AT1R were labeled with anti-Myc polyclonal antibody and anti-Flag monoclonal antibody, respectively. To allow internalization, GPR56 and AT1R were stimulated with 10C7 (15 µg/mL)) and Ang II (1µM), respectively, for 30 min at 37 °C. Cells were fixed, processed for immunofluorescence and analyzed by confocal microscopy. Scale bars:10 μm