Molecular Determinants of Signaling and Posttranslational Modifications of GPR64

Abstract

Some of the adhesion G protein‐coupled receptors (aGPCRs) show constitutive activity in the absence of their large N‐terminal fragment (NTF). We previously demonstrated that GPR64/ADGRG2, an orphan aGPCR, regulates the secretion of parathyroid hormone by interacting with the calcium‐sensing receptor and elevating the cAMP levels in parathyroid adenoma cells. In HEK293 cells, we described the role of beta‐arrestins, dynamin, and GPCR kinases (GRKs) in the signaling of NTF‐truncated GPR64. Whether structural domains and motifs other than the NTF also regulate signaling, trafficking, and posttranslational modifications of GPR64 remains unknown. In the current study, we mutated a number of extracellular motifs that are conserved among zebrafish, mouse, and human GPR64. We measured cAMP production (time‐resolved FRET), surface expression (ELISA and biotinylation assays), and ubiquitination (co‐immunoprecipitation) of WT and mutant GPR64 in HEK293 cells. We discovered that several residues in the extracellular domains alter the surface expression, ubiquitination, and degradation of GPR64 at basal and tethered agonist‐stimulated conditions. The molecular mechanisms by which these residues regulate trafficking, signaling, and ubiquitination of GPR64 are currently under investigation.Support or Funding InformationThis work was supported by NIH grant 1R01GM13061701A1 (to N.B.). Student support was provided by an NIH‐NIGMS Initiative for Maximizing Student Development Grant (R25‐GM55036) (to A.A.G.).