GPR80/99, proposed to be the P2Y(15) receptor activated by adenosine and AMP, is not a P2Y receptor

Abstract

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y(15) receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca(2+) mobilization (Inbe et al. J Biol Chem 2004; 279: 19790-9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A(2A) and A(2B) adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188-93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, alpha-ketoglutarate. Consistent with this report, alpha-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts alpha-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by alpha-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.

Figure 1

Lack of adenosine- and AMP-promoted inositol phosphate accumulation in COS-7 cells transiently expressing GPR80. COS-7 cells were transfected with pcDNA3 or pcDNA3-HA-GPR80 and analyzed for cell surface expression and receptor activity. A) Quantitation of cell surface expression of HA-tagged GPR80 by RIA. B) Cells were transfected with the indicated plasmids and the capacity of adenosine (Ado) and AMP to increase inositol phosphate accumulation over 10 min was measured. C) GPR80-expressing COS-7 cells were incubated for 30 min with adenosine deaminase (ADA), suramin, or PPADS at the indicated concentrations, followed by addition of LiCl and incubation for a further 30 min. Data shown are the mean from triplicate assays from three separate experiments.

Figure 2

Lack of adenosine- and AMP-promoted second messenger signaling in CHO cells stably expressing GPR80. GPR80 was stably expressed in CHO cells by retroviral infection and selection of G418-resistant cells. A) Quantitation of cell surface expression of HA-tagged receptors by RIA. P2Y₁₁ refers to CHO cells stably expressing the HA-P2Y₁₁ receptor. B) Adenosine (Ado), AMP and carbachol were added to wild-type and GPR80-expressing CHO cells, and their capacity to promote [³H]inositol phosphate accumulation was assessed. C) ATP (100 µM) was added to wild-type and P2Y₁₁ receptor-expressing CHO cells for 10 min, followed by measurement of [³H]inositol phosphates accumulation. D) The capacity of adenosine (Ado), AMP and forskolin to increase [³H]cyclic AMP accumulation in wild-type and GPR-80 expressing CHO cells was measured. Data shown are the mean from triplicate assays from three separate experiments.

Figure 3

Lack of adenosine- and AMP-promoted second messenger signaling in GPR80-expressing HEK293 cells. HEK293 cells were transiently transfected with empty vector or pcDNA3-HA-GPR80, and analyzed for cell surface expression and receptor activity. A) Quantitation of cell surface expression of HA-tagged GPR80 by RIA. B) Quantitation of [³H]inositol phosphate accumulation in empty vector- and GPR80-transfected HEK293 cells in response to the indicated agents. C) Quantitation of [³H]cyclic AMP accumulation in empty vector- and GPR80-transfected cells in response to the indicated agents. Data shown are the mean from triplicate assays from three separate experiments.

Figure 4

GPR80 is activated by α-ketoglutarate, a citric acid cycle intermediate. CHO and HEK293 cells expressing GPR80 were challenged with α-ketoglutarate and [³H]inositol phosphate accumulation was measured. Data shown are the mean from triplicate assays from three separate experiments.

Figure 5

Pre-incubation of COS-7 cells expressing GPR80 with glutamate dehydrogenase decreases basal accumulation of inositol phosphates. Transfected COS-7 cells labeled overnight with myo-[³H]inositol were pre-incubated for 60 min with 50 µl PBS, reaction buffer (RB; NADH 135 µM, NH₄Cl 200 µM, EDTA 85 µM, and ADP 100 µM, final concentration), or reaction buffer plus GluDH. Accumulation assays in the absence of added agonist were initiated by adding LiCl (10 mM final) and allowed to proceed for 30 min. Data shown are the mean from triplicate assays from three separate experiments. ^*P < 0.05 (reaction buffer +GluDH versus PBS and reaction buffer +GluDH versus reaction buffer alone, unpaired t-test).