GPR35 is a novel lysophosphatidic acid receptor

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Abstract

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca2+ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.

Introduction

GPR35 is a Class A (rhodopsin-like) G protein-coupled receptor identified in 1998 [1]. The human GPR35 gene encodes a protein of 309 amino acids. GPR35 is expressed in various mammalian tissues, such as the gastrointestinal tissues, lymphoid tissues and the central and peripheral nervous tissues [1], [2], [3], [4]. Several investigators have reported GPR35 to be involved in the development of gastric cancer [4], the regulation of neuronal excitability and synaptic release [5], nociception [3], the pathogenesis of brachydactyly-mental retardation syndrome [6] and the regulation of blood pressure [7], although details are yet to be clarified.

The endogenous ligand for GPR35 has long remained to be identified. Recently, Wang et al. [2] demonstrated that kynurenic acid, a tryptophan metabolite, is a possible endogenous ligand for GPR35. Kynurenic acid elicited several cellular responses in HEK293 cells and CHO cells expressing GPR35 [2]. Nevertheless, it remains unclear whether GPR35 is a specific receptor for kynurenic acid. Of note, relatively high concentrations of kynurenic acid (above 10 μM) are necessary for eliciting apparent cellular responses [2], [3], [5], [8]. It seems curious that a G protein-coupled receptor would require such high concentrations of endogenous ligand for its activation. Thus, there may be other naturally occurring ligands for GPR35.

In the present study, we conducted a search for other possible endogenous ligands for GPR35. We focused on lysophospholipids because of the homology (30%) between GPR35 and GPR55 whose endogenous ligand is lysophosphatidylinositol (LPI) [9], especially 2-arachidonoyl LPI [10]. Notably, the GPR35 gene and GPR55 gene are located in close proximity on human chromosome 2 (q37.3 and q37.1, respectively). These observations suggest that the two receptors are evolutionally related and there may exist structural similarity between the GPR35 ligand and the GPR55 ligand.

We examined the activity of 2-arachidonoyl LPI, the natural ligand for GPR55, using HEK293 cells expressing GPR35. However, 2-arachidonoyl LPI did not trigger an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in the GPR35-expressing cells or vector-transfected control cells, suggesting that it is not the endogenous ligand for GPR35. Finally, we found that the magnitude of the cellular response induced by 2-arachidonoyl-lysophosphatidic acid (LPA) in the GPR35-expressing cells was markedly greater than that in the vector-transfected control cells. Similar results were obtained with 2-linoleoyl LPA and 2-oleoyl LPA. Several lines of evidence strongly suggest that GPR35 is a functional receptor for LPA, especially 2-acyl LPA.

Section snippets

Chemicals

LPA was prepared from phosphatidic acid (PA), available commercially or obtained from the corresponding phosphatidylcholine (PC) following treatment with Streptomyces chromofuscus phospholipase D, using Rhizopus delemar lipase or Naja naja atra phospholipase A2. 2-Arachidonoyl LPI was prepared as described [10]. 2-Arachidonoyl lysophosphatidylcholine (LPC) was prepared from 1-palmitoyl-2-arachidonoyl PC, and 2-oleoyl lysophosphatidylethanolamine (LPE) and 2-oleoyl lysophosphatidylserine

Effects of zaprinast, kynurenic acid, several cannabinoid receptor ligands and LPA on [Ca2+]i in the GPR35-expressing HEK293 cells

Several investigators have demonstrated that zaprinast, a phosphodiesterase 5A inhibitor, acts as an agonist toward GPR35 [8]. We first examined the effect of zaprinast on [Ca2+]i in HEK293 cells stably expressing GPR35. As demonstrated in Fig. 1A, zaprinast did not elicit any response in the vector-transfected cells. On the other hand, submicromolar or micromolar concentrations of zaprinast (above 300 nM) induced modest increases in [Ca2+]i in the GPR35-expressing cells (Fig. 1B).

We then

Discussion

To date, a number of specific receptors for LPA have been identified, including LPA1, LPA2, LPA3, LPA4, LPA5 and P2Y5 [12]. There is mounting evidence that LPA plays a variety of essential roles in various mammalian tissues and cells by acting on these receptors [12]. Nevertheless, the physiological and pathophysiological significance as well as the mechanism of action of LPA are yet to be fully elucidated and the possibility remains that additional unidentified LPA receptors exist in mammalian

References (23)

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