Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells

Abstract

G2A, T cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) form a group of structurally related G protein-coupled receptors (GPCRs) originally proposed to bind proinflammatory lipids. More recent studies have challenged the identification of lipid agonists for these GPCRs and have suggested that they function primarily as proton sensors. We compared the ability of these four receptors to modulate pH-dependent responses by using transiently transfected cell lines. In accordance with previously published reports, OGR1 was found to evoke strong pH-dependent responses as measured by inositol phosphate accumulation. We also confirmed the pH-dependent cAMP production by GPR4 and TDAG8. However, we found the activity of the human G2A receptor and its mouse homolog to be significantly less sensitive to pH fluctuations as measured by inositol phosphate and cAMP accumulation. Sequence homology analysis indicated that, with one exception, the histidine residues that were previously shown to be important for pH sensing by OGR1, GPR4, and TDAG8 were not conserved in the G2A receptor. We further addressed the pH-sensing properties of G2A and TDAG8 in a cellular context where these receptors are coexpressed. In thymocytes and splenocytes explanted from receptor-deficient mice, TDAG8 was found to be critical for pH-dependent cAMP production. In contrast, G2A was found to be dispensable for this process. We conclude that members of this GPCR group exhibit differential sensitivity to extracellular protons, and that expression of TDAG8 by immune cells may regulate responses in acidic microenvironments.

Fig. 1.

Localization of GPCR-EGFP fusion proteins ectopically overexpressed in 293T cells. 293T cells expressing muG2A-, TDAG8-, OGR1-, and GPR4-EGFP fusion proteins were evaluated by confocal fluorescent microscopy as described in Materials and Methods.

Fig. 2.

IP and cAMP production by GPCR-expressing cells exposed to acidic pH. IP production by transfected 293T (A) or retroviral-transduced RAT-1 cells (B) was measured after exposure to DMEM-HEM solutions with different pH, as described in Materials and Methods. (C) cAMP levels in transfected 293T cells exposed to acidic pH. Note the use of a logarithmic scale to display the range of cAMP production. Each experimental sample was done in duplicate or triplicate. These data were repeated in a similar pattern in two (IP production) or three (cAMP production) independent experiments.

Fig. 3.

Several OGR1-defined histidine residues required for pH-sensing are absent in muG2A. Sequences of human OGR1, GPR4, TDAG8, and G2A (huG2A) receptors, as well as the mouse G2A (muG2A) orthologue, were aligned by using clustalw. The putative transmembrane domains (I–VII) are highlighted in gray. IC, intracellular loops; EC, extracellular loops. The histidine residues that have been shown to be important for proton sensing in OGR1 (H17, H20, H84, H169, and H269) and TDAG8 (H10 and H14) are highlighted in black (17, 18). Two of these residues, H20 and H169, are replaced by positively charged amino acids, arginine (R) and lysine (K), respectively, in G2A (highlighted in green). Residues in GPR4 that are assumed to be critical for proton-sensing based on alignment with OGR1 are highlighted in red. H174 in huG2A was found to be required for proton-sensing (highlighted in black) (11). The corresponding residue in muG2A is replaced by an asparagine (N, highlighted in green). H245 of OGR1 (highlighted in yellow) is critical for protein folding and is highly conserved in all five receptors.

Fig. 4.

TDAG8 and G2A are expressed in WT immune cells and are absent in receptor-deficient mice. DNA-free RNA was prepared from thymocytes and splenocytes explanted from WT, TDAG8-deficient (TDAG8 KO), and G2A-deficient (G2A KO) mice. The expression of G2A, TDAG8, OGR1, and GPR4 was evaluated by RT-PCR. β-actin served as a positive control. The sizes of the PCR products were as follows: β-actin, ≈466 bp; G2A, ≈542 bp; TDAG8, ≈492 bp; OGR1, 442 bp; and GPR4, ≈498 bp.

Fig. 5.

TDAG8 is required for pH-induced cAMP production in thymocytes. Thymocytes from WT and TDAG8-deficient (TDAG8 KO) 4- to 6-week-old female mice were incubated either in the absence (A) or presence (B) of 1 μM DEX for 4 h. Thymocytes were then exposed to DMEM-HEM solutions (pH 7.4, 6.9, and 6.4) (A and B) or 1 μM PGE₂ at pH 7.4 (C) for 30 min and lysed for cAMP quantification. Data presented are representative of six independent experiments. Two to three mice per group were examined in each experiment. The total numbers of mice examined were 14 WTs and 14 TDAG8 KOs.

Fig. 6.

G2A is dispensable for pH-induced cAMP production in thymocytes. Thymocytes from WT and G2A-deficient (G2A KO) 4- to 6-week-old female mice were incubated either in the absence (A) or presence (B) of 1 μM DEX for 4 h. Thymocytes were then exposed to DMEM-HEM solutions (pH 7.4, 6.9, and 6.4) (A and B) or 1 μM PGE₂ at pH 7.4 (C) for 30 min and lysed for cAMP quantification. Data presented are representative of two independent experiments. Three mice per group were examined in each experiment.

Fig. 7.

TDAG8 is required for pH-induced cAMP production in splenocytes. Splenocytes from WT (Left), G2A-deficient (G2A KO) (Center), and TDAG8-deficient (TDAG8 KO) (Right) 6- to 8-week-old female mice were exposed to DMEM-HEM solutions (pH 7.4, 6.9, and 6.4) (A) or 1 μM PGE₂ at pH 7.4 (B) and lysed for cAMP quantification. Data shown are representative of two to four independent experiments, each with three mice per group. A total of 12 WT, 12 TDAG8 KO, and 6 G2A KO mice were analyzed.