A Simple and Efficient CRISPR Technique for Protein Tagging.

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.

UTS2B Defines a Novel Enteroendocrine Cell Population and Regulates GLP-1 Secretion Through SSTR5 in Male Mice.

The gut-pancreas axis plays a key role in the regulation of glucose homeostasis and may be therapeutically exploited to treat not only type 2 diabetes but also hypoglycemia and hyperinsulinemia. We identify a novel enteroendocrine cell type expressing the peptide hormone urotensin 2B (UTS2B). UTS2B inhibits glucagon-like peptide-1 (GLP-1) secretion in mouse intestinal crypts and organoids, not by signaling through its cognate receptor UTS2R but through the activation of the somatostatin receptor (SSTR) 5. Circulating UTS2B concentrations in mice are physiologically regulated during starvation, further linking this peptide hormone to metabolism. Furthermore, administration of UTS2B to starved mice demonstrates that it is capable of regulating blood glucose and plasma concentrations of GLP-1 and insulin in vivo. Altogether, our results identify a novel cellular source of UTS2B in the gut, which acts in a paracrine manner to regulate GLP-1 secretion through SSTR5. These findings uncover a fine-tuning mechanism mediated by a ligand-receptor pair in the regulation of gut hormone secretion, which can potentially be exploited to correct metabolic unbalance caused by overactivation of the gut-pancreas axis.

Adhesion G protein-coupled receptors: opportunities for drug discovery.

Adhesion G protein-coupled receptors (aGPCRs) - one of the five main families in the GPCR superfamily - have several atypical characteristics, including large, multi-domain N termini and a highly conserved region that can be autoproteolytically cleaved. Although GPCRs overall have well-established pharmacological tractability, currently no therapies that target any of the 33 members of the aGPCR family are either approved or in clinical trials. However, human genetics and preclinical research have strengthened the links between aGPCRs and disease in recent years. This, together with a greater understanding of their functional complexity, has led to growing interest in aGPCRs as drug targets. A framework for prioritizing aGPCR targets and supporting approaches to develop aGPCR modulators could therefore be valuable in harnessing the untapped therapeutic potential of this family. With this in mind, here we discuss the unique opportunities and challenges for drug discovery in modulating aGPCR functions, including target identification, target validation, assay development and safety considerations, using ADGRG1 as an illustrative example.

DPP9 enzyme activity controls survival of mouse migratory tongue muscle progenitors and its absence leads to neonatal lethality due to suckling defect.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9ki/ki mice). We show that DPP9ki/ki mice die within 12-18h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors.

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia.

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6.

Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options.

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

Transcriptional regulation and functional characterization of the oxysterol/EBI2 system in primary human macrophages.

Oxysterols such as 7 alpha, 25-dihydroxycholesterol (7α,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein-coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses. Activation of EBI2 by specific oxysterols leads to chemotaxis of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adaptive immune response is established by a stromal cells subset here we investigate the involvement of the oxysterol/EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes needed for ligand production such as cholesterol 25-hydroxylase (CH25H), sterol 27-hydroxylase (CYP27A1), and oxysterol 7α-hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharides (LPS) triggers a strong up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Stimulation of EBI2 expressed on macrophages leads to calcium mobilization and to directed cell migration. Supernatants of LPS-stimulated macrophages are able to stimulate EBI2 signaling indicating that an induction of CH25H, CYP27A1, and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is a study characterizing the oxysterol/EBI2 pathway in primary monocyte-derived macrophages. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity.

Second generation S1P pathway modulators: research strategies and clinical developments.

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Identification of the C3a receptor (C3AR1) as the target of the VGF-derived peptide TLQP-21 in rodent cells.

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

An oral sphingosine 1-phosphate receptor 1 (S1P(1)) antagonist prodrug with efficacy in vivo: discovery, synthesis, and evaluation.

A prodrug approach to optimize the oral exposure of a series of sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists for chronic efficacy studies led to the discovery of (S)-2-{[3'-(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-carbonyl]methylamino}-4-dimethylaminobutyric acid methyl ester 14. Methyl ester prodrug 14 is hydrolyzed in vivo to the corresponding carboxylic acid 15, a potent and selective S1P(1) antagonist. Oral administration of the prodrug 14 induces sustained peripheral blood lymphocyte reduction in rats. In a rat cardiac transplantation model coadministration of a nonefficacious dose of prodrug 14 with a nonefficacious dose of sotrastaurin (19), a protein kinase C inhibitor, or everolimus (20), an mTOR inhibitor, effectively prolonged the survival time of rat cardiac allografts. This demonstrates that clinically useful immunomodulation mediated by the S1P(1) receptor can be achieved with an S1P(1) antagonist generated in vivo after oral administration of its prodrug.

A potent and selective S1P(1) antagonist with efficacy in experimental autoimmune encephalomyelitis.

Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.

Gαi2 signaling promotes skeletal muscle hypertrophy, myoblast differentiation, and muscle regeneration.

Skeletal muscle atrophy results in loss of strength and an increased risk of mortality. We found that lysophosphatidic acid, which activates a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor, stimulated skeletal muscle hypertrophy through activation of Gα(i2). Expression of a constitutively active mutant of Gα(i2) stimulated myotube growth and differentiation, effects that required the transcription factor NFAT (nuclear factor of activated T cells) and protein kinase C. In addition, expression of the constitutively active Gα(i2) mutant inhibited atrophy caused by the cachectic cytokine TNFα (tumor necrosis factor-α) by blocking an increase in the abundance of the mRNA encoding the E3 ubiquitin ligase MuRF1 (muscle ring finger 1). Gα(i2) activation also enhanced muscle regeneration and caused a switch to oxidative fibers. Our study thus identifies a pathway that promotes skeletal muscle hypertrophy and differentiation and demonstrates that Gα(i2)-induced signaling can act as a counterbalance to MuRF1-mediated atrophy, indicating that receptors that act through Gα(i2) might represent potential targets for preventing skeletal muscle wasting.

Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors.

Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.