Deorphanization of GPRC6A: a promiscuous L-alpha-amino acid receptor with preference for basic amino acids

TY - JOUR

T1 - Deorphanization of GPRC6A: a promiscuous L-alpha-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 hGPRC6A. 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 alpha-amino acids and was employed to rationally select potential ligands. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/5.24 and identification of L-alpha-amino acids as agonists. The most active agonists were basic L-alpha-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 hGPRC6A. 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 alpha-amino acids and was employed to rationally select potential ligands. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/5.24 and identification of L-alpha-amino acids as agonists. The most active agonists were basic L-alpha-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.

KW - Amino Acid Sequence

KW - Amino Acids

KW - Amino Acids, Diamino

KW - Animals

KW - Cell Line

KW - Cloning, Molecular

KW - Goldfish

KW - Humans

KW - Mammals

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Conformation

KW - Receptors, G-Protein-Coupled

KW - Recombinant Proteins

KW - Signal Transduction

KW - Substrate Specificity

U2 - 10.1124/mol.104.007559

DO - 10.1124/mol.104.007559

M3 - Journal article

C2 - 15576628

SN - 0026-895X

VL - 67

SP - 589

EP - 597

JO - Molecular Pharmacology

JF - Molecular Pharmacology

IS - 3

ER -