Abstract
G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.
Highlights
OGR1-family G protein-coupled receptors (GPCRs), including ovarian cancer G proteincoupled receptor 1 (OGR1 or GPR68), G protein-coupled receptor 4 (GPR4), T-cell death-associated gene 8 (TDAG8 or GPR65), and G2A, have initially been reported as receptors for lysolipids, such as sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) [1,2,3]; lipid actions have not always been confirmed [4, 5]
Compound 1 almost completely inhibited action induced by any proton concentration employed (Fig 5F). These results suggest that action modes of psychosine and the GPR4 modulator are different with respect to their susceptibility to proton-sensing histidine residues in GPR4
We have shown that some imidazopyridine compounds and negatively modulate GPR4-mediated actions in response to acidic pH without appreciable effects on OGR1- and TDAG8-mediated actions
Summary
OGR1-family G protein-coupled receptors (GPCRs), including ovarian cancer G proteincoupled receptor 1 (OGR1 or GPR68), G protein-coupled receptor 4 (GPR4), T-cell death-associated gene 8 (TDAG8 or GPR65), and G2A, have initially been reported as receptors for lysolipids, such as sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) [1,2,3]; lipid actions have not always been confirmed [4, 5]. Proton sensitivity was reported for TDAG8 [6]. Protonation of histidine residues on the extracellular domains of receptors has been suggested to cause conformational changes in the receptors, thereby facilitating the coupling with G proteins [4, 6, 7]. As for G2A, proton sensitivity was detected, the receptor is constitutively active even at a neutral or alkaline pH [8]. It is controversial whether G2A senses changes in the extracellular pH in native cells that endogenously express G2A [9,10,11]
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