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    • br The mode of binding of ligands to GPR

    2022-01-18


    The mode of binding of ligands to GPR35 As noted above, although kynurenic Capsazepine 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 Weinstein [27] (in which the most conserved residue in transmembrane domain X is designated X.50, whereas the amino acid X.49 is one residue closer to the N terminus and X.51 is one closer to the C terminus). This residue was predicted to provide an ionic interaction with the carboxylate [26]. Following alteration of this residue to alanine, neither rat nor human GPR35 responded to kynurenic acid [13] (Figure 3). Furthermore, the agonist action of zaprinast at each orthologue was also eliminated by this mutation [13] (Figure 3). Although lacking a formal negative charge, zaprinast does contain an acid bioisostere (a group with similar physical or chemical properties that provides functional characteristics broadly similar to a chemical compound). Furthermore, alteration of the tyrosine residue to alanine at position 3.32, which is predicted to be on the same face of transmembrane domain III but one turn of the helix further towards the extracellular face of the receptor, also eliminated responses to both kynurenic acid and zaprinast [13]. Although very preliminary, these studies have begun to identify key residues of the binding pocket of GPR35, and this will be investigated further by mutagenesis and analysis of the effects of a wider range of ligands at such mutants.
    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 stimulation of [35S]GTPγS binding by kynurenic acid in membranes of CHO cells expressing human GPR35, by prior treatment of the cells with pertussis toxin [8], was consistent with this. Furthermore, following heterologous introduction of the human isoforms of GPR35 into rat sympathetic neurons, the ability of both kynurenic acid and zaprinast to inhibit N-type calcium channels was blocked by prior treatment with pertussis toxin [5]. The ability of endogenously expressed GPR35 to inhibit forskolin-stimulated cAMP levels in rat dorsal root ganglion was also ablated by pertussis toxin pretreatment [11]. Despite these observations, Jenkins et al.[13] reported that, following expression in HEK293 cells, human GPR35 generated only very modest increases in binding of [35S]GTPγS in response to kynurenic acid; therefore, they explored possible interactions with other G proteins. Although they were unable to record elevation of [Ca2+]i in cells cotransfected with Gαq and either human or rat GPR35, the presence of a Gαq-Gα13 chimera generated robust [Ca2+]i responses to zaprinast via both orthologues, whereas equivalent experiments with a Gαq-Gα12 chimera did not [13]. Use of an antibody able to identify only the GTP-bound, active state of Gα13 provided further support for interaction with this G protein [13]. Subsequent development of an immunocapture assay using an epitope-tagged form of Gα13 confirmed ligand stimulation of [35S]GTPγS binding to this G protein [23] (Figure 4). The ability of GPR35 expressed in HEK293 cells to promote binding of GTP to Rho A [14] is also consistent with a role for Gα13 because activation of Gα13 is generally upstream of this effect [30]. By contrast, pertussis toxin-mediated inhibition of interleukin 4 release from alpha-galactosylceramide-activated human invariant natural killer T cells via GPR35 [31], and of ERK activation in U2OS cells expressing GPR35 [12], both support a role for Gi-family G proteins (as does the capacity of both kynurenic acid and zaprinast to reduce forskolin-elevated cAMP levels in cultured mouse glial cells [32]). It appears, therefore, that GPR35 can couple to both Gα13 and pertussis toxin-sensitive Gi-family G proteins. It will be instructive to determine whether there is ligand bias (Box 2) between these pathways or predominance of one over another in different cells and tissues, because such effects might generate distinct signals from GPR35 in different cell types. Although interactions between GPR35 and β-arrestin-2 have been employed to develop assays to identify novel GPR35 ligands, and presumably occur in cells that express GPR35 endogenously, their possible role in generating G protein-independent signals also remains to be investigated.