1 Stem Cell Institute, Ankara University, Çankaya 06520 Ankara, Türkiye 2 Department of Biological Sciences, Middle East Technical University, Çankaya 06800 Ankara, Türkiye 3 Department of Chemistry, Middle East Technical University, Çankaya 06800 Ankara, Türkiye * Two authors contributed to the work equally. Correspondence Orkun Cevheroğlu, Stem Cell Institute, Ankara University, Çankaya 06520 Ankara, Türkiye Email: cevheroglu@ankara.edu.tr Funding Information This work was supported by Tübitak 118Z590 and Tübitak 118Z694 grants. Abstract GPR56/ADGRG1 is an adhesion GPCR and mutations on this receptor cause cortical malformation due to the over-migration of neural progenitor cells on the brain surface. At the pial surface, GPR56 interacts with collagen III, induces Rho dependent activation through Gα12/13 and inhibits the neuronal migration. In human glioma cells, GPR56 inhibits cell migration through Gαq/11 dependent Rho pathway. GPR56-tetraspanin complex is known to couple with Gαq/11. GPR56 is an aGPCR that couples with various G proteins and signals through different downstream pathways. In this study, BFPP mutants disrupting GPR56 function but remain to be expressed on plasma membrane were used to study receptor signaling through Gα12, Gα13 and Gα11 with BRET biosensors. GPR56 showed coupling with all three G proteins and activated heterotrimeric G protein signaling upon stimulation with Stachel peptide. However, BFPP mutants showed different signaling defects for each G protein indicative of distinct activation and signaling properties of GPR56 for Gα12, Gα13 or Gα11. β-arrestin recruitment was also investigated following the activation of GPR56 with Stachel peptide using BRET biosensors. N-terminally truncated GPR56 showed enhanced β-arrestin recruitment, however neither wild-type receptor nor BFPP mutants gave any measurable recruitment upon Stachel stimulation, pointing different activation mechanisms for β-arrestin involvement. KEYWORDS GPR56, ADGRG1, Bilateral frontoparietal polymicrogyria (BFPP), GPCR, Adhesion GPCR, G protein, β-arrestin, biosensor