TY - JOUR
T1 - Deorphanization of GPRC6A
T2 - A promiscuous L-α-amino acid receptor with preference for basic amino acids
AU - Wellendorph, Petrine
AU - Hansen, Kasper B.
AU - Balsgaard, Anders
AU - Greenwood, Jeremy R.
AU - Egebjerg, Jan
AU - Bräuner-Osborne, Hans
PY - 2005/3
Y1 - 2005/3
N2 - One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRCGA. To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with α-amino acids and was employed to rationally select potential ligands. Measurement of Ca 2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/ 5.24 and identification of L-α-amino acids as agonists. The most active agonists were basic L-α-amino acids, L-Arg, L-Lys, and L-ornithine, suggesting that these may function as endogenous signaling molecules. Measurement of intracellular calcium in tsA cells expressing h6A/5.24 allowed determination of EC50 values, which confirmed the agonist preferences observed in oocytes. Cloning, cell surface expression and deorphanization of the mouse ortholog further reinforces the assignment of the agonist preferences of hGPRC6A. This study demonstrates the utility of a chimeric receptor approach in combination with molecular modeling, for elucidating agonist interaction with GPRC6A, a novel family C G-protein-coupled receptor.
AB - One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRCGA. To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with α-amino acids and was employed to rationally select potential ligands. Measurement of Ca 2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/ 5.24 and identification of L-α-amino acids as agonists. The most active agonists were basic L-α-amino acids, L-Arg, L-Lys, and L-ornithine, suggesting that these may function as endogenous signaling molecules. Measurement of intracellular calcium in tsA cells expressing h6A/5.24 allowed determination of EC50 values, which confirmed the agonist preferences observed in oocytes. Cloning, cell surface expression and deorphanization of the mouse ortholog further reinforces the assignment of the agonist preferences of hGPRC6A. This study demonstrates the utility of a chimeric receptor approach in combination with molecular modeling, for elucidating agonist interaction with GPRC6A, a novel family C G-protein-coupled receptor.
UR - http://www.scopus.com/inward/record.url?scp=14944341203&partnerID=8YFLogxK
U2 - 10.1124/mol.104.007559
DO - 10.1124/mol.104.007559
M3 - Article
C2 - 15576628
AN - SCOPUS:14944341203
SN - 0026-895X
VL - 67
SP - 589
EP - 597
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 3
ER -