A Conserved Extracellular Motif Regulates Signaling and Trafficking of ADGRG2

Abstract

ADGRG2(GPR64) is an orphan adhesion G protein-coupled receptor (aGPCR), which is expressed in the epididymis and parathyroid glands. We have previously shown that ADGRG2 regulates the secretion of parathyroid hormone by parathyroid adenoma cells via interaction with and altering calcium-sensing receptor signaling. Generally, dissociation of a cleaved N-terminal Fragment (NTF) from the C-terminal fragment is a prerequisite for the activation of aGPCRs. Our previous data confirmed this mechanism in human ADGRG2, as well as the role of b-arrestins, dynamin, and GPCR kinases in the signaling of NTF-truncated ADGRG2. However, the structural motifs other than the NTF that regulate signaling and trafficking of ADGRG2 remain unknown. Herein, we mutated residues in the extracellular loop 2 (ECL2) that are conserved among zebrafish, mouse, and human ADGRG2, as well as the whole family of aGPCRs. Using HEK293 cells as a cell model, we elucidated how these mutations alter cAMP production and CRE induction (Biosensor and Luciferase Reporter Assays), cell resistance (label-free impedance assay), receptor surface expression (ELISA, Immunofluorescence imaging), and receptor degradation (Western blotting). We discovered that mutations in tryptophan and isoleucine of ECL2 significantly decrease surface and total expression of ADGRG2. Treatment with a translation inhibitor, cycloheximide, revealed that these mutants are degraded at a much higher rate compared to the WT receptor. Having adjusted the dose of plasmids to reach similar surface expression, we discovered that ECL2-mutated receptors do not respond to the activating synthetic peptide, P-15, in any of the signaling readouts. Using a fluorophore-conjugated P-15, we found that ECL2 plays a critical role in the binding of the activating peptide to ADGRG2. These data suggest a major role for ECL2 and its conserved residues in signaling, trafficking, and stability of ADGRG2.