In the present study we

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 486 2 (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.
Materials and methods
Results
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 tissues. The results of the present investigation indicated that GPR35 is a novel functional receptor for LPA, especially for 2-acyl LPA. We obtained evidence that 2-acyl LPA markedly enhanced the Ca2+ response, the activation of RhoA and the phosphorylation of ERK in GPR35-expressing cells (Fig. 1, Fig. 2, Fig. 3). 2-Acyl LPA also induced the internalization of the receptor molecule (Fig. 4). Similar results were obtained with another clone of HEK293 cells stably expressing GPR35 (data not shown). To the best of our knowledge, this is the first report showing that GPR35 acts as a 2-acyl LPA receptor. We also found that GPR35 may function as a receptor or binding site for Δ9-THC (Fig. 1F), despite that the effect of Δ9-THC was less prominent toward the GPR35-expressing HEK293 cells than NG108-15 cells expressing the CB1 receptor [13], and that other cannabinoid receptor ligands, such as CP55940 and WIN55212-2, were almost inactive. Wang et al. [2] reported that GPR35 acts as a receptor for kynurenic acid, a tryptophan metabolite. Kynurenic acid elicited the mobilization of Ca2+ and production of inositol phosphate in GPR35-expressing CHO cells or HEK293 cells and inhibition of the lipopolysaccharide-induced release of tumor-necrosis factor-α in peripheral blood mononuclear cells and monocytes [2]. It also stimulated the binding of [35S]GTPγS to the membranes of GPR35-expressing CHO cells [2]. However, the concentration of kynurenic acid required for the cellular response was relatively high; the response was detectable above 10μM, and the EC50 was 39μM for the Ca2+ response and 36μM for the binding of [35S]GTPγS [2]. Several investigators also reported that kynurenic acid induced the inhibition of N-type Ca2+ channels in rat sympathetic neurons [5] and adenylyl cyclase in rat dorsal root ganglion neurons [3] at relatively high concentrations; above 50μM in the former case and above 100μM in the latter. We also found that a high concentration of kynurenic acid (100μM) induced a weak response in GPR35-expressing HEK293 cells (Fig. 1D), while the effect of 2-acyl LPA was detectable from the nanomolar range (Fig. 1H). The reason why high concentrations of kynurenic acid are required for various biological activities is not known. One possible explanation may be that both 2-acyl LPA and kynurenic acid act as GPR35 ligands, yet interact with the receptor in different ways, such as with different affinities.