G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

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.

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.

Therapeutic Opportunities and Challenges in Targeting the Orphan G Protein-Coupled Receptor GPR35.

GPR35 is a class A, rhodopsin-like G protein-coupled receptor (GPCR) first identified more than 20 years ago. In the intervening period, identification of strong expression in the lower intestine and colon, in a variety of immune cells including monocytes and a variety of dendritic cells, and in dorsal root ganglia has suggested potential therapeutic opportunities in targeting this receptor in a range of conditions. GPR35 is, however, unusual in a variety of ways that challenge routes to translation. These include the following: (i) Although a substantial range and diversity of endogenous ligands have been suggested as agonist partners for this receptor, it officially remains defined as an "orphan" GPCR. (ii) Humans express two distinct protein isoform sequences, while rodents express only a single form. (iii) The pharmacologies of the human and rodent orthologues of GPR35 are very distinct, with variation between rat and mouse GPR35 being as marked as that between either of these species and the human forms. Herein we provide perspectives on each of the topics above as well as suggesting ways to overcome the challenges currently hindering potential translation. These include a better understanding of the extent and molecular basis for species selective GPR35 pharmacology and the production of novel mouse models in which both "on-target" and "off-target" effects of presumptive GPR35 ligands can be better defined, as well as a clear understanding of the human isoform expression profile and its significance at both tissue and individual cell levels.

Structure-Activity Relationship Studies of Tetrahydroquinolone Free Fatty Acid Receptor 3 Modulators.

Free fatty acid receptor 3 (FFA3, previously GPR41) is activated by short-chain fatty acids, mediates health effects of the gut microbiota, and is a therapeutic target for metabolic and inflammatory diseases. The shortage of well-characterized tool compounds has however impeded progress. Herein, we report structure-activity relationship of an allosteric modulator series and characterization of physicochemical and pharmacokinetic properties of selected compounds, including previous and new tools. Two representatives, 57 (TUG-1907) and 63 (TUG-2015), showed improved solubility and preserved potency. Of these, 57, with EC50 = 145 nM and a solubility of 33 µM, showed high clearance in vivo but is a preferred tool in vitro. In contrast, 63, with EC50 = 162 nM and a solubility of 9 µM, showed lower clearance and seems better suited for in vivo studies. Using 57, we demonstrate for the first time that FFA3 activation leads to calcium mobilization in murine dorsal root ganglia.

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.

PETAL LOSS and ROXY1 Interact to Limit Growth Within and between Sepals But to Promote Petal Initiation in Arabidopsis thaliana.

The activity of genes controlling organ development may be associated with the redox state of subregions within the meristem. Glutaredoxins react to the level of oxidative potential and can reduce cysteine dithiols, in some cases to activate specific transcription factors. In Arabidopsis, loss of function of the glutaredoxin ROXY1 or the trihelix transcription factor PETAL LOSS (PTL) each results in reduced numbers of petals. Here, genetic studies have revealed that loss of petals in ptl mutant plants depends on ROXY1 function. The two genes also act together to restrain stamen-identifying C function from entering the outer whorls. On the other hand, they suppress growth between sepals and in sepal margins, with ROXY1 action partially redundant to that of PTL. Genetic interactions with aux1 mutations indicate that auxin activity is reduced in the petal whorl of roxy1 mutants as in ptl mutants. However, it is apparently increased in the sepal whorl of triple mutants associated with the ectopic outgrowth of sepal margins, and of finger-like extensions of inter-sepal zones that in 20% of cases are topped with bunches of ectopic sepals. These interactions may be indirect, although PTL and ROXY1 proteins can interact directly when co-expressed in a transient assay. Changes of conserved cysteines within PTL to similar amino acids that cannot be oxidized did not block its function. It may be in some cases that under reducing conditions ROXY1 binds PTL and activates it by reducing specific conserved cysteines, thus resulting in growth suppression.

A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures.

The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60(190), N3.43(240), Q7.49(394), and H6.52(363) as key residues involved in peptide-mediated biased agonism, with R2.60(190), N3.43(240), and Q7.49(394) predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53(364)A, N3.43(240)Q, Q7.49(394)N, and N3.43(240)Q/Q7.49(394)N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53(364) and R2.60(190) was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49(394), but not R2.60(190)/E6.53(364) was critical for calcium mobilization for all three peptides. Mutation of N3.43(240) and Q7.49(394) had differential effects on individual peptides, providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.

PETAL LOSS, a trihelix transcription factor that represses growth in Arabidopsis thaliana, binds the energy-sensing SnRK1 kinase AKIN10.

Organogenesis in plants involves differential growth. Rapidly growing primordia are distinguished from the meristem and each other by slower growing boundaries. PETAL LOSS (PTL) is a trihelix transcription factor of Arabidopsis that represses growth in boundaries between newly arising sepals. To identify partners involved in this growth limitation, a young inflorescence cDNA library was screened by yeast two-hybrid technology with PTL as bait. The most frequent prey identified was AKIN10, the catalytic α-subunit of the Snf1-related kinase1 (SnRK1). Interaction was mapped to the C-terminal (non-kinase) half of AKIN10 and the N-terminal portion of PTL. Binding of PTL was specific to AKIN10 as there was little binding to the related AKIN11. The interaction was confirmed by co-immunoprecipitation in vitro. Fluorescently tagged products of 35S:YFP-AKIN10 and 35S:CFP-PTL also interacted when transiently expressed together in leaf cells of Nicotiana benthamiana. In this case, most of the cytoplasmic AKIN10 was preferentially moved to the nucleus where PTL accumulated, possibly because a nuclear export sequence in AKIN10 was now masked. During these experiments, we observed that AKIN10 could variably accumulate in the Golgi, shown by its co-localization with a tagged Golgi marker and through its dispersal by brefeldin A. Tests of phosphorylation of PTL by AKIN10 gave negative results. The functional significance of the PTL-AKIN10 interaction remains open, although a testable hypothesis is that AKIN10 senses lower energy levels in inter-sepal zones and, in association with PTL, promotes reduced cell division.

Functional domains of the PETAL LOSS protein, a trihelix transcription factor that represses regional growth in Arabidopsis thaliana.

PETAL LOSS (PTL) is a trihelix transcription factor that represses growth, especially between sepal primordia. As one of 30 trihelix proteins in Arabidopsis, it falls in the GT2 clade with duplicated trihelix DNA-binding domains and a long α-helical central domain. PTL orthologs occur in all angiosperm genomes examined except grasses, and sequence comparisons reveal that there are two further short conserved domains at each end. GT2 itself carries two nuclear localization sequences, but PTL has an additional nuclear localization sequence (NLS). We show that PTL can act as a transcriptional activator in yeast and in planta, with the latter tested by two different functional assays. Specific deletions revealed that the activation region is C-terminal. Site-directed mutagenesis of the DNA-binding domains has shown that a conserved tryptophan and two downstream acidic amino acids in the second trihelix, predicted to promote folding, are each required for PTL function. Also, three basic residues in the third helix, near the DNA interaction sites, support its function. PTL was found to dimerize in yeast. This was confirmed and extended by jointly expressing differentially tagged forms of PTL in a transient expression system in Nicotiana benthamiana leaves. Cytoplasmic PTL (with mutant NLS sequences) was carried into the nucleus upon binding with nuclear-localized PTL, providing each partner carried intact central domains. As this 90-amino acid domain is conserved in most trihelix family members, it seems likely that they all function in dimeric form.

The trihelix family of transcription factors--light, stress and development.

GT factors are the founding members of the trihelix transcription factor family. They bind GT elements in light regulated genes, and their nature was uncovered in a burst of activity in the 1990s. Study of the trihelix family then slowed. However, interest is now re-awakening. Genomic studies have revealed 30 members of this family in Arabidopsis and 31 in rice, falling into five clades. Newly discovered functions involve responses to salt and pathogen stresses, the development of perianth organs, trichomes, stomata and the seed abscission layer, and the regulation of late embryogenesis. Thus the time is ripe for a review of the genomic and functional information now emerging for this neglected family.