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Orthologue selectivity and ligand bias: translating the pharmacology of GPR35

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GPR35 is a poorly characterized G protein-coupled receptor (GPCR) that has been suggested as a potential therapeutic target for the treatment of diabetes, hypertension and asthma. Two endogenously produced ligands have been suggested as activators of GPR35, although the relevance of these remains unclear. Recently, a series of surrogate agonist ligands and the first antagonists of GPR35 have been identified. However, marked differences in the potency of agonists at species orthologues of GPR35 have been noted, and this presents substantial challenges in translating the pharmacology at the cloned human receptor to ex vivo and in vivo studies of the physiological function of this receptor in animal models. Currently identified agonists will probably not display high selectivity for GPR35. By contrast, comparisons of the potency of ligands at species orthologues of GPR35 have provided insight into the nature of the ligand binding pocket and could result in the identification of more potent and selective ligands.

Introduction

GPR35 is a poorly characterized 7-transmembrane domain G protein-coupled receptor (GPCR) first identified more than 10 years ago. It was derived from an open reading frame corresponding to 309 amino acids located in humans on chromosome 2, region q37.3 [1]. In these initial studies, expression was examined in a range of tissues but was detected only in the intestine of the rat; it was also reported to be lacking in a number of regions of human brain [1]. Subsequently, this same sequence (and a further sequence encoding a second form of GPR35 that appears to be a differentially spliced isoform containing an N-terminal extension of 31 amino acids) (Figure 1) was identified from a cDNA library produced from human gastric cancer cells [2]. Again, expression was also detected in normal intestinal mucosal cells [2], and because these cDNAs were able to transform NIH-3T3 cells, it was suggested that GPR35 might be oncogenic and play a role in the generation of gastric cancers [2]. The significance of the N-terminally extended form of GPR35 remains to be defined, but messenger RNA encoding this variant has been reported to be present at higher levels than the shorter form [2].

In humans, the GPR35 gene displays significant polymorphic variability, with a number of non-synonymous variants within the open reading frame resulting in alterations in amino acid sequence 3, 4 (Figure 1). However, to date, apart from a very cursory examination of the Ser294Arg variant [5], which has been associated with the propensity to develop coronary artery calcification [6] (Table 1), effects of these variations on signal transduction and pharmacology have yet to be reported. A further single nucleotide polymorphism, located in the 5′’ untranslated region of the GPR35 gene, has been linked to early-onset inflammatory bowel disease in a genome-wide association study [7] but no further information on this is currently available.

Section snippets

Potential endogenous agonists of GPR35

The first endogenously produced chemical that was shown to be able to activate GPR35 was the tryptophan metabolite kynurenic acid [8]. When human GPR35 was expressed along with a mixture of promiscuous and chimeric G proteins 9, 10 (Box 1) in CHO cells, addition of kynurenic acid elevated [Ca2+]i in a concentration-dependent fashion [8]. Importantly, other intermediates of tryptophan metabolism, including the non-carboxylate kynurenine, were inactive [8]. This demonstrated the probable

Surrogate ligands for GPR35

Although identification of endogenously produced chemicals with agonist action at GPR35 is of considerable importance, the ligands described above are far from ideal to probe the roles of GPR35. Surrogate ligands are therefore required. Until recently, the key GPR35 agonist has been zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one) (Table 1). Zaprinast was first identified as a GPR35 agonist by Tanaguchi et al. [22]. Like kynurenic acid, zaprinast was considerably more potent at rat than human

The mode of binding of ligands to GPR35

As noted above, although kynurenic acid is an agonist at GPR35, this is true for neither kynurenine [8] nor kynurenic acid ethyl ester [13]. This implicates a key role for the carboxylate group in binding and/or activation of GPR35. Importantly, in studies of the l-lactate receptor GPR81 [26], a number of receptors related to GPR35 (and which have acidic ligands) were noted to have a conserved arginine in transmembrane domain III. This is at position 3.36 in the nomenclature of Ballesteros and

G protein-coupling profile of GPR35

Although some of the earliest studies of GPR35 detected ligand activation via transfection of a mixture of chimeric and promiscuous G proteins 8, 22, they noted selective interaction of GPR35 with chimeric G proteins containing the receptor recognition regions of Gαo and Gαi [8]. By contrast the promiscuous G protein Gα16 (Box 1) did not appear to couple to GPR35 [8]. Standard [35S]GTPγS binding studies are most suited to detect activation of Gi-family G proteins 28, 29. Prevention of

Expression profile of GPR35

As noted above, initial studies indicated expression of GPR35 in rat intestine [1] and stomach [2]. Subsequent studies have confirmed significant expression levels in the small intestine, colon and stomach, and this might be relevant in the association between a GPR35 polymorphic variant and early-onset inflammatory bowel disease [7]. GPR35 is also expressed in a range of other rat tissues including lung, uterus, dorsal root ganglion and spinal cord 22, 32. Wang et al. [8] were the first to

Physiological roles of GPR35 and potential therapeutic opportunities

The expression of GPR35 in pancreatic β-cells, coupled with the ability of thiazolidinedione ligands that have agonist action at GPR35 to enhance glucose-stimulated insulin release in model cell lines and to improve oral glucose tolerance tests [25], has suggested a potential use for agonists of GPR35 in the treatment of diabetes and related metabolic disorders (Table 2). Expression of GPR35 in the intestine and colon might also be relevant because other GPCRs expressed on β-cells that regulate

Concluding remarks

Several orphan GPCRs have expression profiles that indicate they are worthy of consideration as therapeutic targets. This view can be supported via various transgenic techniques, and it would be interesting to have wide-ranging phenotypic information on GPR35 knockout mice. Based on the number of GPR35 active compounds identified recently in very small-scale screens 12, 23, 33, there is reason to hope that more extensive screens, along with follow-up medical chemistry programmes, will rapidly

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