The fate of P2Y-related orphan receptors: GPR80/99 and GPR91 are receptors of dicarboxylic acids

Abstract

Several orphan G protein-coupled receptors are structurally close to the family of P2Y nucleotide receptors: GPR80/99 and GPR91 are close to P2Y(1/2/4/6/11) receptors, whereas GPR87, H963 and GPR34 are close to P2Y(12/13/14). Over the years, several laboratories have attempted without success to identify the ligands of those receptors. In early 2004, two papers have been published: One claiming that GPR80/99 is an AMP receptor, called P2Y(15), and the other one showing that GPR80/99 is a receptor for alpha-ketoglutarate, while GPR91 is a succinate receptor. The accompanying paper by Qi et al. entirely supports that GPR80/99 is an alpha-ketoglutarate receptor and not an AMP receptor. The closeness of dicarboxylic acid and P2Y nucleotide receptors might be linked to the negative charges of both types of ligands and the involvement of conserved Arg residues in their neutralization.

Figures

Figure 1

Phylogenetic tree illustrating the relatedness of the two groups of P2Y receptors, dicarboxylic acid receptors, receptors for short-chain monocarboxylic acids and remaining orphan P2Y-like receptors. The following receptors are included in the tree: P2Y receptors, group I: P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11; P2Y receptors, group II: P2Y12, P2Y13 and P2Y14; Dicarboxylic acid receptors: succinate (GPR91) and α-ketoglutarate (GPR80/99); Monocarboxylic acid (propionate) receptors: GPR41 and GPR43; Orphan P2Y-like receptors: GPR87, H963 and GPR34. Alignment was performed using ClustalX algorithm and the dendrogram was constructed using the TreeView algorithm.

Figure 2

Role of conserved positively charged residues in the activation of P2Y and dicarboxylic acid receptors by their respective ligands. Conserved residues among P2Y and/or dicarboxylic acids receptors are shown in a higher size font. The residues that have been mutated in the studies of Erb et al. [17], Jiang et al. [14] and He et al. [10] are underlined. Those residues that are crucial in the activation of those receptors are in bold.

Figure 3

Comparison of the responses to α-ketoglutarate (A) and AMP (B) in an aequorin-based functional assay using CHO-K1 cells coexpressing human GPR80/99, apo-aequorin and Gα16. The plasmid was transfected into CHO-K1 cells expressing Gα16 and a mitochondria-targeted form of apoaequorin. Clonal cell lines were established, and functional responses were analyzed using a bioassay based on the luminescence of aequorin, in the presence of 5 µM coelenterazine H, as a result of intracellular calcium release, as described previously [15, 16]. Briefly, cells (25,000 cells in 50 µl) were added to 50-µl samples in 96-well plates, and the luminescence was recorded for 30 s (Berthold luminometer). Maximal response was evaluated following cell lysis by Triton X-100. The ATP response is due to endogenous P2Y2 receptors.