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Spatial regulation of GPR64/ADGRG2 signaling by ?-arrestins and GPCR kinases.

ABSTRACT: Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 signaling and endocytosis, we generated mutants that lack either NTF alone (?NTF) or NTF and tethered agonist (P622). We discover that unlike full-length GPR64, ?NTF and P622 mutants interact with ?-arrestin1 and ?-arrestins2 and are constitutively internalized in steady states. However, only ?NTF shows exaggerated basal activation of the G?s -cAMP-CRE signaling cascade. Neither ?NTF nor P622 shows constitutive activation of the G?13 -SRE pathway, but both mutants respond to exogenously added agonistic peptide via CRE and SRE. GPCR kinases and dynamin mediate the constitutive internalization of ?NTF and P622 to early endosomes, where ?NTF constantly induces CRE. These data suggest that NTF not only shields the tethered agonist to prevent G protein signaling but also confers a conformation that inhibits the interaction with ?-arrestins and the consequent endocytosis and sustained signaling from endosomes.

SUBMITTER: Azimzadeh P

PROVIDER: S-EPMC7236788 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Spatial regulation of GPR64/ADGRG2 signaling by β-arrestins and GPCR kinases.

Azimzadeh Pedram P Talamantez-Lyburn Sarah C SC Chang Katarina T KT Inoue Asuka A Balenga Nariman N

Annals of the New York Academy of Sciences 20190909 1

Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 sig ...[more]

PMID: 31502283

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Project description:Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β-catenin), the molecular basis of abnormal calcium sensing by calcium-sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein-coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N-terminally truncated human GPR64 is constitutively active and a 15-amino acid-long peptide C-terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full-length GPR64 physically interacts with CaSR and attenuates the CaSR-mediated intracellular Ca2+ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.

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Spatial regulation of GPR64/ADGRG2 signaling by ?-arrestins and GPCR kinases.

Publications

Spatial regulation of GPR64/ADGRG2 signaling by β-arrestins and GPCR kinases.

Similar Datasets

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