Combinatorial expression of GPCR isoforms affects signalling and drug responses.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.

Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Evidence for the Existence of a CXCL17 Receptor Distinct from GPR35.

The chemokine CXCL17 is associated with the innate response in mucosal tissues but is poorly characterized. Similarly, the G protein-coupled receptor GPR35, expressed by monocytes and mast cells, has been implicated in the immune response, although its precise role is ill-defined. A recent manuscript reported that GPR35 was able to signal in response to CXCL17, which we set out to confirm in this study. GPR35 was readily expressed using transfection systems but failed to signal in response to CXCL17 in assays of ß-arrestin recruitment, inositol phosphate production, calcium flux, and receptor endocytosis. Similarly, in chemotaxis assays, GPR35 did not confirm sensitivity to a range of CXCL17 concentrations above that observed in the parental cell line. We subsequently employed a real time chemotaxis assay (TAXIScan) to investigate the migratory responses of human monocytes and the monocytic cell line THP-1 to a gradient of CXCL17. Freshly isolated human monocytes displayed no obvious migration to CXCL17. Resting THP-1 cells showed a trend toward directional migration along a CXCL17 gradient, which was significantly enhanced by overnight incubation with PGE2 However, pretreatment of PGE2-treated THP-1 cells with the well-characterized GPR35 antagonist ML145 did not significantly impair their migratory responses to CXCL17 gradient. CXCL17 was susceptible to cleavage with chymase, although this had little effect its ability to recruit THP-1 cells. We therefore conclude that GPR35 is unlikely to be a bona fide receptor for CXCL17 and that THP-1 cells express an as yet unidentified receptor for CXCL17.

Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.

Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.

G-Protein-Coupled Receptor 35 Mediates Human Saphenous Vein Vascular Smooth Muscle Cell Migration and Endothelial Cell Proliferation.

Vascular smooth muscle cell (VSMC) migration and proliferation is central to neointima formation in vein graft failure following coronary artery bypass. However, there are currently no pharmacological interventions that prevent vein graft failure through intimal occlusion. It is hence a therapeutic target. Here, we investigated the contribution of GPR35 to human VSMC and endothelial cell (EC) migration, using a scratch-wound assay, and also the contribution to proliferation, using MTS and BrdU assays, in in vitro models using recently characterized human GPR35 ortholog-selective small-molecule agonists and antagonists. Real-time PCR studies showed GPR35 to be robustly expressed in human VSMCs and ECs. Stimulation of GPR35, with either the human-selective agonist pamoic acid or the reference agonist zaprinast, promoted VSMC migration in the scratch-wound assay. These effects were blocked by coincubation with either of the human GPR35-specific antagonists, CID-2745687 or ML-145. These GPR35-mediated effects were produced by inducing alterations in the actin cytoskeleton via the Rho A/Rho kinase signaling axis. Additionally, the agonist ligands stimulated a proliferative response in ECs. These studies highlight the potential that small molecules that stimulate or block GPR35 activity can modulate vascular proliferation and migration. These data propose GPR35 as a translational therapeutic target in vascular remodeling.

The antiallergic mast cell stabilizers lodoxamide and bufrolin as the first high and equipotent agonists of human and rat GPR35.

Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.

The pharmacology of TUG-891, a potent and selective agonist of the free fatty acid receptor 4 (FFA4/GPR120), demonstrates both potential opportunity and possible challenges to therapeutic agonism.

TUG-891 [3-(4-((4-fluoro-4'-methyl-[1,1'-biphenyl]-2-yl)methoxy)phenyl)propanoic acid] was recently described as a potent and selective agonist for the long chain free fatty acid (LCFA) receptor 4 (FFA4; previously G protein-coupled receptor 120, or GPR120). Herein, we have used TUG-891 to further define the function of FFA4 and used this compound in proof of principle studies to indicate the therapeutic potential of this receptor. TUG-891 displayed similar signaling properties to the LCFA α-linolenic acid at human FFA4 across various assay end points, including stimulation of Ca²âº mobilization, ß-arrestin-1 and ß-arrestin-2 recruitment, and extracellular signal-regulated kinase phosphorylation. Activation of human FFA4 by TUG-891 also resulted in rapid phosphorylation and internalization of the receptor. While these latter events were associated with desensitization of the FFA4 signaling response, removal of TUG-891 allowed both rapid recycling of FFA4 back to the cell surface and resensitization of the FFA4 Ca²âº signaling response. TUG-891 was also a potent agonist of mouse FFA4, but it showed only limited selectivity over mouse FFA1, complicating its use in vivo in this species. Pharmacologic dissection of responses to TUG-891 in model murine cell systems indicated that activation of FFA4 was able to mimic many potentially beneficial therapeutic properties previously reported for LCFAs, including stimulating glucagon-like peptide-1 secretion from enteroendocrine cells, enhancing glucose uptake in 3T3-L1 adipocytes, and inhibiting release of proinflammatory mediators from RAW264.7 macrophages, which suggests promise for FFA4 as a therapeutic target for type 2 diabetes and obesity. Together, these results demonstrate both potential but also significant challenges that still need to be overcome to therapeutically target FFA4.

High-throughput identification and characterization of novel, species-selective GPR35 agonists.

Drugs targeting the orphan receptor GPR35 have potential therapeutic application in a number of disease areas, including inflammation, metabolic disorders, nociception, and cardiovascular disease. Currently available surrogate GPR35 agonists identified from pharmacologically relevant compound libraries have limited utility due to the likelihood of off-target effects in vitro and in vivo and the variable potency that such ligands exhibit across species. We sought to identify and characterize novel GPR35 agonists to facilitate studies aimed at defining the physiologic role of GPR35. PathHunter ß-arrestin recruitment technology was validated as a human GPR35 screening assay, and a high-throughput screen of 100,000 diverse low molecular weight compounds was conducted. Confirmed GPR35 agonists from five distinct chemotypes were selected for detailed characterization using both ß-arrestin recruitment and G protein-dependent assays and each of the human, mouse, and rat GPR35 orthologs. These studies identified 4-{(Z)-[(2Z)-2-(2-fluorobenzylidene)-4-oxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid (compound 1) as the highest potency full agonist of human GPR35 yet described. As with certain other GPR35 agonists, compound 1 was markedly selective for human GPR35, but displayed elements of signal bias between ß-arrestin-2 and G protein-dependent assays. Compound 1 also displayed competitive behavior when assessed against the human GPR35 antagonist, ML-145 (2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid). Of the other chemotypes studied, compounds 2 and 3 were selective for the human receptor, but compounds 4 and 5 demonstrated similar activity at human, rat, and mouse GPR35 orthologs. Further characterization of these compounds and related analogs is likely to facilitate a better understanding of GPR35 in health and disease.

Antagonists of GPR35 display high species ortholog selectivity and varying modes of action.

Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and ß-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues.

G Protein-Coupled Receptor GPR35 Suppresses Lipid Accumulation in Hepatocytes.

Although prevalent, nonalcoholic fatty liver disease is not currently treated effectively with medicines. Initially, using wild-type and genome-edited clones of the human hepatocyte cell line HepG2, we show that activation of the orphan G protein-coupled receptor GPR35 is both able and sufficient to block liver X-receptor-mediated lipid accumulation. Studies on hepatocytes isolated from both wild-type and GPR35 knock-out mice were consistent with a similar effect of GPR35 agonists in these cells, but because of marked differences in the pharmacology of GPR35 agonists and antagonists at the mouse and human orthologues, as well as elevated basal lipid levels in hepatocytes from the GPR35 knock-out mice, no definitive conclusion could be reached. To overcome this, we generated and characterized a transgenic knock-in mouse line in which the corresponding human GPR35 splice variant replaced the mouse orthologue. In hepatocytes from these humanized GPR35 mice, activation of this receptor was shown conclusively to prevent, and also reverse, lipid accumulation induced by liver X-receptor stimulation. These studies highlight the potential to target GPR35 in the context of fatty liver diseases.

G-protein coupled receptor 35 (GPR35) regulates the colonic epithelial cell response to enterotoxigenic Bacteroides fragilis.

G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.

The emerging pharmacology and function of GPR35 in the nervous system.

G protein-coupled receptor 35 (GPR35) is an orphan G protein-coupled receptor (GPCR) that can be activated by kynurenic acid at high micromolar concentrations. A previously unappreciated mechanism of action of GPR35 has emerged as a Gαi/o-coupled inhibitor of synaptic transmission, a finding that has significant implications for the accepted role of kynurenic acid as a broad-spectrum antagonist of the NMDA, AMPA/kainite and α7 nicotinic receptors. In conjunction with previous findings that link agonism of GPR35 with significant reduction in nociceptive pain, GPR35 has emerged as a potential effector of regulation of mechanical sensitivity and analgesia of the Ret tyrosine kinase, and as a receptor involved in the transmission of anti-inflammatory effects of aspirin- potentially through affecting leucocyte rolling, adhesion and extravasation. Single nucleotide polymorphisms of GPR35 have linked this receptor to coronary artery calcification, inflammatory bowel disease and primary sclerosing cholangitis, while chromosomal aberrations of the 2q37.3 locus and altered copy number of GPR35 have been linked with autism, Albight's hereditary osteodystrophy-like syndrome, and congenital malformations, respectively. Herein, we present an update on both the pharmacology and potential function of GPR35, particularly pertaining to the nervous system. This review forms part of a special edition focussing on the role of lipid-sensing GPCRs in the nervous system. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.

G protein-coupled receptor 35: an emerging target in inflammatory and cardiovascular disease.

G protein-coupled receptor 35 (GPR35) is an orphan receptor, discovered in 1998, that has garnered interest as a potential therapeutic target through its association with a range of diseases. However, a lack of pharmacological tools and the absence of convincingly defined endogenous ligands have hampered the understanding of function necessary to exploit it therapeutically. Although several endogenous molecules can activate GPR35 none has yet been confirmed as the key endogenous ligand due to reasons that include lack of biological specificity, non-physiologically relevant potency and species ortholog selectivity. Recent advances have identified several highly potent synthetic agonists and antagonists, as well as agonists with equivalent potency at rodent and human orthologs, which will be useful as tool compounds. Homology modeling and mutagenesis studies have provided insight into the mode of ligand binding and possible reasons for the species selectivity of some ligands. Advances have also been made in determining the role of the receptor in disease. In the past, genome-wide association studies have associated GPR35 with diseases such as inflammatory bowel disease, type 2 diabetes, and coronary artery disease. More recent functional studies have implicated it in processes as diverse as heart failure and hypoxia, inflammation, pain transduction and synaptic transmission. In this review, we summarize the progress made in understanding the molecular pharmacology, downstream signaling and physiological function of GPR35, and discuss its emerging potential applications as a therapeutic target.

An engineered zinc finger protein reveals a role for the insulin VNTR in the regulation of the insulin and adjacent IGF2 genes.

An engineered zinc finger protein (eZFP) was isolated from a library based on its ability to activate expression of the endogenous insulin gene in HEK-293 cells. Using a panel of insulin promoter constructs, the eZFP was shown to act through the variable number of tandem repeat (VNTR) region located 365 base pairs upstream of the transcription start site. The eZFP also activated expression of the IGF2 gene that lies close to INS on chromosome 11p15. These results demonstrate that the INSVNTR controls expression of the insulin and IGF2 genes and provide a mechanistic explanation for previous studies that demonstrated an association between INSVNTR genotypes and placental levels of IGF2.