The rat GPRC6A: cloning and characterization

TY - JOUR

T1 - The rat GPRC6A

T2 - cloning and characterization

AU - Wellendorph, Petrine

AU - Burhenne, Nicole

AU - Christiansen, Bolette

AU - Walter, Bettina

AU - Schmale, Hartwig

AU - Bräuner-Osborne, Hans

PY - 2007/7/15

Y1 - 2007/7/15

N2 - GPRC6A is a novel member of family C of G protein-coupled receptors with so far elusive biological function. GPRC6A has been described in human and mouse as a promiscuous l-alpha-amino acid receptor. We now report the cloning, expression analysis and, functional characterization of the rat orthologue of GPRC6A. Full-length cloning of rat GPRC6A (rGPRC6A) was accomplished using amplification of cDNA from taste tissue, and the identity of rGPRC6A confirmed at both the genomic and the protein level by similarity studies. Using selective primers, reverse transcriptase polymerase chain reaction showed that the mRNA is widely but weakly distributed, except for a high expression in the soft palate, the so-called geschmacksstreifen. On the protein level, rGPRC6A was shown to be glycosylated and most likely oligomeric, and using immunochemistry we observed that rGPRC6A is expressed at the plasma membrane of mammalian cell lines. Utilizing co-expression of rGPRC6A and the promiscuous Galpha(q)(G66D) protein in an engineered cell-based inositol phosphate turnover assay, we were able to study the ligand profile of the receptor. We found that l-ornithine is the most potent and efficacious l-amino acid agonist with an EC(50) value of 264 microM, followed by several other aliphatic, neutral, and basic amino acids. Furthermore, the divalent cation Mg(2+) was found to be a positive modulator of the l-ornithine response. The presented quantitative pharmacological data underlines the evolutionary conservation of GPRC6A to the rat and signifies the physiological importance and emerging pharmacological potential of GPRC6A.

AB - GPRC6A is a novel member of family C of G protein-coupled receptors with so far elusive biological function. GPRC6A has been described in human and mouse as a promiscuous l-alpha-amino acid receptor. We now report the cloning, expression analysis and, functional characterization of the rat orthologue of GPRC6A. Full-length cloning of rat GPRC6A (rGPRC6A) was accomplished using amplification of cDNA from taste tissue, and the identity of rGPRC6A confirmed at both the genomic and the protein level by similarity studies. Using selective primers, reverse transcriptase polymerase chain reaction showed that the mRNA is widely but weakly distributed, except for a high expression in the soft palate, the so-called geschmacksstreifen. On the protein level, rGPRC6A was shown to be glycosylated and most likely oligomeric, and using immunochemistry we observed that rGPRC6A is expressed at the plasma membrane of mammalian cell lines. Utilizing co-expression of rGPRC6A and the promiscuous Galpha(q)(G66D) protein in an engineered cell-based inositol phosphate turnover assay, we were able to study the ligand profile of the receptor. We found that l-ornithine is the most potent and efficacious l-amino acid agonist with an EC(50) value of 264 microM, followed by several other aliphatic, neutral, and basic amino acids. Furthermore, the divalent cation Mg(2+) was found to be a positive modulator of the l-ornithine response. The presented quantitative pharmacological data underlines the evolutionary conservation of GPRC6A to the rat and signifies the physiological importance and emerging pharmacological potential of GPRC6A.

KW - Animals

KW - Cations

KW - Cell Line

KW - Cell Membrane

KW - Cloning, Molecular

KW - Databases, Protein

KW - Glycosylation

KW - Humans

KW - Magnesium

KW - Ornithine

KW - Palate, Soft

KW - RNA, Messenger

KW - Rats

KW - Receptors, G-Protein-Coupled

KW - Tissue Distribution

U2 - 10.1016/j.gene.2007.03.008

DO - 10.1016/j.gene.2007.03.008

M3 - Journal article

C2 - 17478059

SN - 0378-1119

VL - 396

SP - 257

EP - 267

JO - Gene

JF - Gene

IS - 2

ER -