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The seven transmembrane G protein-coupled receptor EBV-induced gene 2 (EBI2), also known as GPR183, is expressed in particular in immune cells. Activated by its endogenous ligands, which are a group of oxysterols, it functions as a chemo-attractant receptor, mediating cell migration. In coordination with other receptors, EBI2 plays important roles in controlling the migration of immune cells during the course of a T-dependent Ab response in the spleen. In recent years, it has become clear that EBI2 also has other roles to play in the immune system. Thus, EBI2 seems to be involved in innate immune responses, such as those mediated by TLR signaling, and it has been implicated in regional immune responses, including immune responses in the CNS. In this review, we describe the functions of EBI2 in B cells, T cells, and dendritic cells during the course of a T-dependent Ab response in the spleen. Furthermore, we review the existing evidence supporting a role for EBI2 in local immune responses and in autoimmune diseases, with a special focus on immune responses in the CNS. Finally, we discuss which type of role EBI2 may play in autoimmune diseases, and we give our opinion about the paths of future research in EBI2.

Alternative processing of the precursor protein pro-GIP results in endogenously produced GIP(1-30)NH2, that by DPP-4 cleavage in vivo results in the metabolite GIP(3-30)NH2. We showed previously that GIP(3-30)NH2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species-dependent. Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human (125)I-GIP(1-42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin-releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3-30)NH2 bound with high affinity (Ki of 17nM), in contrast to human GIP(3-30)NH2 (Ki of 250nM). In cAMP studies, rat GIP(3-30)NH2 inhibited GIP(1-42)-induced rat GIPR activation and schild-plot analysis showed competitive antagonism with a pA2 of 13nM and a slope of 0.9±0.09. Alone, rat GIP(3-30)NH2 displayed weak, low-potent partial agonistic properties (EC50>1μM) with an efficacy of 9.4% at 0.32μM compared to GIP(1-42). In perfused rat pancreata, rat GIP(3-30)NH2 efficiently antagonized rat GIP(1-42)-induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3-30)NH2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP-mediated G protein-signaling as well as pancreatic hormone release, while human GIP(3-30)NH2, despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3-30)NH2; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems.

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation.

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation.

The intestinal G protein-coupled receptor GPR119 is a novel metabolic target involving glucagon-like peptide-1 (GLP-1)-derived insulin-regulated glucose homeostasis. Endogenous and diet-derived lipids, including N-acylethanolamines and 2-monoacylglycerols (2-MAG) activate GPR119. The purpose of this work is to evaluate whether 2-oleoyl glycerol (2-OG) improves glucose tolerance through GPR119, using wild type (WT) and GPR 119 knock out (KO) mice. We here show that GPR119 is essential for 2-OG-mediated release of GLP-1 and CCK from GLUTag cells, since a GPR119 specific antagonist completely abolished the hormone release. Similarly, in isolated primary colonic crypt cultures from WT mice, GPR119 was required for 2-OG-stimulated GLP-1 release while there was no response in crypts from KO mice. In vivo, gavage with 2-oleyl glyceryl ether ((2-OG ether), a stable 2-OG analog with a potency of 5.3 µM for GPR119 with respect to cAMP formation as compared to 2.3 µM for 2-OG), significantly (P < 0.05) improved glucose clearance in WT littermates, but not in GPR119 KO mice. Finally, deletion of GPR119 in mice resulted in lower glucagon levels, whereas the levels of insulin and GIP were unchanged. In the present study we show that 2-OG stimulates GLP-1 secretion through GPR119 activation in vitro, and that fat-derived 2-MAGs are potent candidates for mediating fat-induced GLP-1 release through GPR119 in vivo. © 2016 BioFactors, 2016.

BACKGROUND AND PURPOSE: Specific high potent receptor antagonists are valuable tools when evaluating animal and human physiology. Within the glucose-dependent insulinotropic polypeptide (GIP) system, considerable attention has been given to the presumed GIP receptor antagonist (Pro3)GIP and its effect in murine studies. We conducted a pharmacological analysis of this ligand including interspecies differences between the rodent and human GIP system.EXPERIMENTAL APPROACH: Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation and assayed in competition binding using (125) I-human GIP. Using isolated perfused rodent pancreata both from wild type and GIPR-deficient animals, insulin-, glucagon-, and somatostatin-releasing properties in response to species-specific GIP and (Pro3)GIP analogues were evaluated.KEY RESULTS: Human (Pro3)GIP is an efficacious agonist on the human GIPR with similar efficacy (Emax ) in cAMP production as human GIP, while both rat and mouse(Pro3)GIP were partial agonists on their corresponding receptors. Rodent GIPs are more potent and efficacious at their receptors than human GIP. In perfused pancreata at 7 mM glucose, both rodent (Pro3)GIP analogues induced modest insulin, glucagon and somatostatin secretion corresponding to the partial agonism observed in cAMP production. Conclusions and Implications When evaluating compound properties it is important to consider interspecies differences both at the receptor and ligand level. Thus, in rodent models human GIP is a comparatively weak partial agonist. Human (Pro3)GIP is not an effective antagonist, so there is still a need for an effective antagonist for the elucidation of GIP's physiology.

BACKGROUND AND PURPOSE: The cyclopentapeptide FC131 (cyclo(-L-Arg(1) -L-Arg(2) -L-2-Nal(3) -Gly(4) -D-Tyr(5) -)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure-activity relationship (SAR) data; however, none of these have been verified by in vitro experiments.EXPERIMENTAL APPROACH: Heterologous (125) I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data.KEY RESULTS: The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic pocket) respectively. Arg(1) forms charge-charge interactions with Asp(187) in ECL-2, while D-Tyr(5) points to the extracellular side of CXCR4. Furthermore, the backbone of FC131 interacts with the chemokine receptor-conserved Glu(288) via two water molecules. Intriguingly, Tyr(116) and Glu(288) form a H-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity.CONCLUSIONS AND IMPLICATIONS: Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR4 activation by CXCL12 were gained. The combined findings will facilitate future design of novel CXCR4 antagonists.

Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes.

In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms.

Udgivelsesdato: 2010-Jan-21, CXCR4 and CCR5 constitute the two major coreceptors for HIV-1 entry into host cells. In the course of an HIV-infection, a coreceptor switch takes place in approximately half of the patients - from R5 HIV-1 (CCR5 utilizing) strains to X4 HIV-1 (CXCR4 utilizing) strains. Treatment of HIV-infected individuals with CXCR4 antagonists delays the onset of AIDS by preventing the CCR5 to CXCR4 coreceptor switch. In addition to the endogenous CXCR4 and CCR5 ligands, other chemokines, for example the human herpesvirus 8 encoded CC-chemokine, vCCL2, and modifications hereof, have proven efficient HIV-1 cell-entry inhibition through chemokine receptor interaction. However, pharmacokinetic and immunogenic drawbacks of chemokines and peptidic/peptoid compounds have brought the attention towards small-molecule antagonists, such as AMD3100, that displays high specificity and affinity towards CXCR4, but unfortunately no oral bioavailability. The hunt for orally active small-molecule CXCR4 antagonists led to the development of monocyclam-based compounds, and recently to the non-cyclam antagonist AMD070, which is orally active and currently in Phase II clinical trial as anti-HIV treatment. Current review provides an overview of the drug discovery within the field of anti-HIV treatment targeting CXCR4 spanning from natural occurring and modified chemokines, to HIV-mimicking peptides and peptoids ending at the non-peptide antagonists.

During the two past decades tremendous effort has been put into uncovering the activation mechanism of 7TM receptors. The majority of such studies have focused on the major binding pocket, comprised of transmembrane segments (TM) -III through -VII, as most non-peptide and peptide ligands, in addition to biogenic amines and retinal a.m.o. bind to residues in this region. Consequently the major helical movements occur here during activation, as described recently in the Global Toggle Switch Model for Family A (also known as rhodopsin-like) members of the 7TM receptors. As a result, the minor binding pocket, comprised of TM-I, -II and, in part, -III and -VII, has received much less attention. With a few exceptions, such as the highly conserved Asp in position II:10/2.50, the residues in this region have generally been considered insignificant with regard to receptor activation. However, accumulating evidence emphasize that this is not the case. In this review, we focus on TM-II with an emphasis on position II:20/2.60, and present data from structure-activity studies on a range of Family A 7TM receptors including chemokine, ghrelin and melanocortin receptors in addition to the orphan EBI2 suggesting that TM-II has important functions for both ligand-dependent and -independent activation of 7TM receptors.

Udgivelsesdato: 2001-Dec, ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines.

Udgivelsesdato: 2001-Mar-26, A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion, cellular reprogramming, tissue targeting or for cell entry. Through their efficient evolutionary machinery and through in vivo selection performed directly on the human cellular and molecular targets, virus have been able to optimize the encoded receptors for distinct pharmacological profiles to help in various parts of the viral life cyclus. Most of the receptors encoded by human pathogenic virus are still orphan receptors, i.e. the endogenous ligand is unknown. In the few cases where it has been possible to characterize these receptors pharmacologically, they have been found to bind a broad spectrum of either CC chemokines, US28 from human cytomegalovirus, or CXC chemokines, ORF74 from human herpesvirus 8. Nevertheless, US28 has been specifically optimized for recognition of the membrane bound chemokine, fractalkine, conceivably involved in cell-cell transfer of virus; whereas ORF74 among the endogenous CXC chemokines has selected angiogenic chemokines as agonists and angiostatic/modulatory chemokines as inverse agonists. ORF74 possess substantial cell-transforming properties and signals with high constitutive activity through the phospholipase C and MAP kinase pathways. Interestingly, transgenic expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets.

Udgivelsesdato: 2000-Aug-25, Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

A number of CXC chemokines competed with similar, nanomolar affinity against 125I-interleukin-8 (IL-8) binding to ORF-74, a constitutively active seven-transmembrane receptor encoded by human herpesvirus 8. However, in competition against 125I-labeled growth-related oncogene (GRO)-alpha, the ORF-74 receptor was highly selective for GRO peptides, with IL-8 being 10,000-fold less potent. The constitutive stimulating activity of ORF-74 on phosphatidylinositol turnover was not influenced by, for example, IL-8 binding. In contrast, GRO peptides acted as potent agonists in stimulating ORF-74 signaling, whereas IP-10 and stromal cell-derived factor-1alpha surprisingly acted as inverse agonists. These peptides had similar pharmacological properties with regard to enhancing or inhibiting, respectively, the stimulatory effect of ORF-74 on NIH-3T3 cell proliferation. Construction of a high affinity zinc switch through introduction of two His residues at the extracellular end of transmembrane segment V enabled Zn2+ to act as a prototype non-peptide inverse agonist, which eliminated the constitutive signaling. It is concluded that ORF-74, which is believed to be causally involved in the formation of highly vascularized tumors, has been optimized for agonist and inverse agonist modulation by the endogenous angiogenic GRO peptides and angiostatic IP-10 and stromal cell-derived factor-1alpha, respectively. ORF-74 could serve as a target for the development of non-peptide inverse agonist drugs as demonstrated by the effect of Zn2+ on the metal ion site-engineered receptor.

Udgivelsesdato: 1998-Nov-13, In the wild-type tachykinin NK3A receptor histidyl residues are present at two positions in TM-V, V:01 and V:05, at which Zn2+ functions as an antagonist in NK1 and kappa-opioid receptors with engineered metal-ion sites. Surprisingly, in the NK3A receptor Zn2+ instead increased the binding of the agonist 125I-[MePhe7]neurokinin B to 150%. [MePhe7]neurokinin B bound to the NK3A receptor in a two-component mode of which Zn2+ eliminated the subnanomolar binding mode but induced a higher binding capacity of the nanomolar binding mode. Signal transduction was not induced by ZnCl2 but 10 microM ZnCl2 enhanced the effect of neurokinin B. Ala-substitution of HisV:01 eliminated the enhancing effect of Zn2+ on peptide binding. It is concluded that physiological concentrations of Zn2+ have a positive modulatory effect on the binding and function of neurokinin B on the NK3A receptor through a bis-His site in TM-V.