Downstream signaling of disease-associated mutations on GPR56/ADGRG1

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.

ADGRG1, an adhesion G protein‐coupled receptor, forms oligomers

“…2A, Fig. S1) [73]. Autoproteolysis‐deficient ADGRG1H381S constructs were resolved on SDS‐PAGE as higher bands corresponding to their molecular weights using anti‐GPR56 antibody (Fig.…”

Re‐routing GPR56 signalling using Gα12/13 G protein chimeras

“…The stronger activation of GPR56-CTF with P19 is in line with previous work by Stoveken and colleagues that showed a roughly 1.5-fold increase of P19 stimulation over P7 stimulation for the related GPR56 A386M CTF construct. 35 While GPR56-FL was lacking the ability to be activated on membranes using P7 in GTPγS binding assays, other studies have reported signalling activation of GPR56-FL by the P7 peptide in BRET-based assays in HEK293 cells, 66 and activation of Ca 2+ signalling in MIN6 mouse pancreatic beta cells. 67 Additionally, murine GPR56-FL could be activated by the GPR56/GPR114 peptide ligand P19, 68 and administration of P7 or P19 resulted in GPR56-mediated antidepressant-like effects in mice and upregulated related signalling pathways.…”

Adhesion G protein‐coupled receptor's structure, function and role in biology—Status from the 10th adhesion GPCR workshop in Copenhagen, 2022

“…Thorough signalling profiling requires assays that cover the full panel of G protein signalling pathways, which previously has been challenging for receptors coupled to the G12/13 pathway due to a lack of efficient and pathway‐specific downstream assays. In this issue, Faas et al (Rosenkilde group) 23 describe a new strategy for detecting ADGRG1 G12/13 signalling, which nicely complement the BRET strategies laid forward by Cevheroğlu et al 22 and Ojeda‐Muñiz et al 21 Using chimeric G proteins, Faas and co‐workers reroute GPR56 G12/13 signalling activity to a convenient Gq readout and show that synthetic Stachel peptide ligands in combination with these chimeras enhance the signalling in conventional gene transcription factor and second messenger assays. As such, the authors offer an additional readout for signalling profiling of adhesion GPCRs in the G12/13 pathway.…”

“…ADGRG1 (GPR56) is associated with the monogenetic disease bilateral frontoparietal polymicrogyria (BFPP) as well as cancer and represent a potential promising drug target. Cevheroğlu et al (Son Group) 22 utilize synthetic Stachel peptide activation and BRET biosensors to investigate the effect of BFPP disease associated mutations on ADGRG1 signalling. The authors investigate the peptide‐dependent activation of G proteins and β‐arrestin membrane recruitment.…”