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First published on December 2, 2004; DOI: 10.1124/mol.104.007559

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Petrine Wellendorph
Kasper B Hansen
Anders Balsgaard
Jan Egebjerg
Hans Brauner-Osborne
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Received for publication September 24, 2004.
Revised November 29, 2004.
Accepted for publication December 2, 2004.

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

Petrine Wellendorph 1, Kasper B Hansen 2, Anders Balsgaard 1, Jeremy R Greenwood 1, Jan Egebjerg 3, Hans Brauner-Osborne 1*

1 The Danish University of Pharmaceutical Sciences 2 The Danish University of Pharmaceutical Sciences and H. Lundbeck A/S 3 H. Lundbeck A/S

* Address correspondence to: E-mail: hbo{at}molpharm.net

Abstract

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 orthologue 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.


Key words: Gq/11 family, Receptor synthesis/trafficking, Mutagenesis/Chimeric approaches


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