RESUMO
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.
Assuntos
Autoimunidade/imunologia , Imunidade Inata/imunologia , Receptores Acoplados a Proteínas G/imunologia , Baço/imunologia , Animais , HumanosRESUMO
Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.
Assuntos
Quimiocina CCL20/genética , Dermatite/terapia , Mutagênese Sítio-Dirigida/métodos , Psoríase/terapia , Receptores CCR6/metabolismo , Animais , Terapia Biológica/métodos , Células COS , Quimiocina CCL20/imunologia , Quimiocina CCL20/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Dermatite/imunologia , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/metabolismo , Humanos , Interleucina-23/imunologia , Camundongos , Psoríase/imunologia , Receptores CCR6/imunologia , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Citomegalovirus/isolamento & purificação , Receptores de Quimiocinas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Virais/genética , Latência Viral , Antígenos CD34/imunologia , Morte Celular , Células Cultivadas , Quimiocinas/metabolismo , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Reservatórios de Doenças , Endocitose , Genes Virais , Transplante de Células-Tronco Hematopoéticas , Humanos , Receptores de Lipopolissacarídeos/imunologia , Monócitos/imunologia , Monócitos/virologia , Receptores de Quimiocinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Células-Tronco/imunologia , Células-Tronco/virologia , Carga Viral , Proteínas Virais/metabolismo , Ativação ViralRESUMO
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 125I-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.
Assuntos
Polipeptídeo Inibidor Gástrico/fisiologia , Glucagon/metabolismo , Insulina/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Somatostatina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Polipeptídeo Inibidor Gástrico/química , Polipeptídeo Inibidor Gástrico/farmacologia , Humanos , Secreção de Insulina , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Ratos , Ratos Wistar , Homologia de Sequência de AminoácidosRESUMO
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, 42(6):665-673, 2016.
Assuntos
Intolerância à Glucose/tratamento farmacológico , Glicerídeos/administração & dosagem , Receptores Acoplados a Proteínas G/metabolismo , Administração Oral , Animais , Linhagem Celular , Colecistocinina/metabolismo , Feminino , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Intolerância à Glucose/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMO
BACKGROUND AND PURPOSE: Specific, high potency 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 pancreata both from wild type and GIP receptor-deficient rodents, insulin-releasing, glucagon-releasing and somatostatin-releasing properties in response to species-specific GIP and (Pro3)GIP analogues were evaluated. KEY RESULTS: Human (Pro3)GIP is a full agonist at human GIP receptors with similar efficacy (Emax ) for 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 in the presence of 7 mM glucose, both rodent (Pro3)GIP analogues induced modest insulin, glucagon and somatostatin secretion, corresponding to the partial agonist activities observed in cAMP production. CONCLUSIONS AND IMPLICATIONS: When evaluating new compounds, 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 was not an antagonist at human GIP receptors, so there is still a need for a potent antagonist in order to elucidate the physiology of human GIP.
Assuntos
Agonismo Parcial de Drogas , Polipeptídeo Inibidor Gástrico/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Animais , Ligação Competitiva/efeitos dos fármacos , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Polipeptídeo Inibidor Gástrico/análogos & derivados , Glucagon/metabolismo , Humanos , Insulina/metabolismo , Radioisótopos do Iodo/metabolismo , Masculino , Camundongos , Pâncreas/metabolismo , Ensaio Radioligante , Ratos , Somatostatina/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND AND PURPOSE: Glucose-dependent insulinotropic polypeptide (GIP) affects lipid, bone and glucose homeostasis. High-affinity ligands for the GIP receptor are needed to elucidate the physiological functions and pharmacological potential of GIP in vivo. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here, we have characterized eight N-terminal truncations of human GIP(1-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with human GIP receptors and assessed for cAMP accumulation upon ligand stimulation or competition binding with (125) I-labelled GIP(1-42), GIP(1-30)NH2 , GIP(2-30)NH2 or GIP(3-30)NH2 . KEY RESULTS: GIP(1-30)NH2 displaced (125) I-GIP(1-42) as effectively as GIP(1-42) (Ki 0.75 nM), whereas the eight truncations displayed lower affinities (Ki 2.3-347 nM) with highest affinities for GIP(3-30)NH2 and GIP(5-30)NH2 (5-30)NH2 . Only GIP(1-30)NH2 (Emax 100% of GIP(1-42)) and GIP(2-30)NH2 (Emax 20%) were agonists. GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and Schild plot analyses identified GIP(3-30)NH2 and GIP(5-30)NH2 as competitive antagonists (Ki 15 nM). GIP(3-30) NH2 was a 26-fold more potent antagonist than GIP(3-42). Binding studies with agonist ((125) I-GIP(1-30)NH2 ), partial agonist ((125) I-GIP(2-30)NH2 ) and competitive antagonist ((125) I-GIP(3-30)NH2 ) revealed distinct receptor conformations for these three ligand classes. CONCLUSIONS AND IMPLICATIONS: The N-terminus is crucial for GIP agonist activity. Removal of the C-terminus of the endogenous GIP(3-42) creates another naturally occurring, more potent, antagonist GIP(3-30)NH2 , which like GIP(5-30)NH2 , was a high-affinity competitive antagonist. These peptides may be suitable tools for basic GIP research and future pharmacological interventions.
Assuntos
Polipeptídeo Inibidor Gástrico/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Animais , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismoRESUMO
Treatment with liraglutide leads to weight loss. We investigated whether blood-to-cerebrospinal fluid (CSF) transfer of liraglutide occurs, and if so, whether it associates with clinical weight loss following liraglutide treatment in humans. We performed lumbar puncture and blood sampling in eight patients with type 2 diabetes (mean (range)): age 63 (54-79) years; actual body weight: 90 (75-118) kg treated with 1.8 mg liraglutide for 14 (5-22) months and with a treatment-induced weight loss of 8.4 (7-11) kg. We measured liraglutide in plasma and CSF with a radioimmunoassay specific for the N-terminus of the GLP-1 moiety of liraglutide. Mean plasma liraglutide was 31 (range: 21-63) nmol l(-1). The mean CSF-liraglutide concentration was 6.5 (range: 0.9-13.9) pmol l(-1). Ratio of CSF: plasma-liraglutide concentrations was 0.02 (range: 0.07-0.002)% and plasma liraglutide did not correlate with CSF-liraglutide levels (P=0.67). Body weight loss tended to correlate with plasma-liraglutide levels (P=0.06), but not with CSF-liraglutide levels (P=0.69). In conclusion, we measured very low concentrations of liraglutide in CSF, and the levels of CSF liraglutide did not correlate with the actual clinical weight loss in these patients. The amount of liraglutide in plasma tended to correlate with the clinical weight loss.
Assuntos
Fármacos Antiobesidade/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Liraglutida/farmacologia , Idoso , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/sangue , Feminino , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
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.
Assuntos
Peptídeos Cíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Animais , Ligação Competitiva , Células COS , Quimiocina CXCL12/farmacologia , Chlorocebus aethiops , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutagênese , Ligação Proteica , Receptores CXCR4/genéticaRESUMO
BACKGROUND AND PURPOSE: A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. EXPERIMENTAL APPROACH: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and ß-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation. KEY RESULTS: [L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and ß-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist. CONCLUSIONS AND IMPLICATIONS: The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the ß2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.
Assuntos
Ativação do Canal Iônico , Isoleucina/fisiologia , Receptores CCR5/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Receptores CCR5/química , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. KEY RESULTS: All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies. CONCLUSION AND IMPLICATIONS: Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.
Assuntos
Receptores CCR8/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , Quimiocina CCL1/metabolismo , Quimiocinas CC/metabolismo , Chlorocebus aethiops , Ligantes , Naftalenos/metabolismo , Receptores CCR8/agonistas , Receptores CCR8/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
BACKGROUND AND PURPOSE: The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1. EXPERIMENTAL APPROACH: Single point mutations were introduced in CCR1 and CCR8, and their effect on small molecule ligand-induced receptor activation was examined through inositol trisphosphate (IP(3) ) accumulation. The molecular interaction profile of the agonist was verified by molecular modeling. KEY RESULTS: The chemokine receptor conserved glutamic acid in TM-VII served as a common anchor for the positively charged amine in the piperidine ring. However, whereas the phenyl-tetrazol group interacted with TyrIV:24 (Tyr(172) ) and TyrIII:09 (Tyr(114) ) in the major binding pocket (delimited by TM-III to VII) of CCR8, it also interacted with TrpII:20 (Trp(90) ) and LysII:24 (Lys(94) ) in the minor counterpart (delimited TM-I to III, plus TM-VII) in CCR1. A straightening of TM-II by Ala-substitution of ProII:18 confirmed its unique role in CCR1. The extracellular loop 2 (ECL-2) contributed directly to the small molecule binding site in CCR1, whereas it contributed to efficacy, but not potency in CCR8. CONCLUSION AND IMPLICATIONS: Despite high ligand potency and efficacy and receptor similarity, this dual-active and bitopic compound binds oppositely in CCR1 and CCR8 with different roles of ECL-2, thereby expanding and diversifying the influence of extracellular receptor regions in drug action.
Assuntos
Inositol 1,4,5-Trifosfato/metabolismo , Piperidinas/farmacologia , Receptores CCR1/metabolismo , Receptores CCR8/metabolismo , Tetrazóis/farmacologia , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Modelos Moleculares , Piperidinas/química , Piperidinas/metabolismo , Mutação Puntual , Receptores CCR1/agonistas , Receptores CCR1/genética , Receptores CCR8/agonistas , Receptores CCR8/genética , Tetrazóis/química , Tetrazóis/metabolismoRESUMO
Epstein-Barr virus (EBV) open reading frame BILF1 encodes a seven trans-membrane (TM) G protein-coupled receptor that signals with high constitutive activity through Galpha(i) (Beisser et al., 2005; Paulsen et al., 2005). In this paper, the transforming potential of BILF1 is investigated in vitro in a foci formation assay using retrovirally transduced NIH3T3 cells, as well as in vivo by using nude mice. BILF1 revealed a substantial transforming potential that was dependent on constitutive signaling, as a signaling-deficient mutant completely lost its ability to transform cells in vitro, and an intermediately active triple-mutated receptor possessed an intermediate transforming potential. Furthermore, BILF1 expression induced vascular endothelial growth factor secretion in a constitutively active manner. In nude mice, BILF1 promoted tumor formation in 90% of cases, ORF74 (from Kaposi's sarcoma-associated herpes virus) in 100% of cases, whereas the signaling-deficient receptor resulted in tumor establishment in 40% of cases. These data suggest that BILF1, when expressed during EBV infection, could indeed be involved in the pathogenesis of EBV-associated diseases and malignancies. Furthermore, the correlation between receptor activity and the ability to mediate cell transformation in vitro and tumor formation in vivo supports the idea that inverse agonists for BILF1 could inhibit cell transformation and be relevant therapeutic candidates.
Assuntos
Transformação Celular Viral/genética , Regulação Neoplásica da Expressão Gênica , Receptores Acoplados a Proteínas G/fisiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Transfecção , Transplante Heterólogo , Proteínas Virais/genéticaRESUMO
BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [(35)S]-GTPgammaS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC(4) and LTD(4). Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively. KEY RESULTS: The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC(50). Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Galpha(i). CONCLUSIONS AND IMPLICATIONS: We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.
Assuntos
Encéfalo/metabolismo , Expressão Gênica , Receptores Acoplados a Proteínas G/genética , Ligação Competitiva , Linhagem Celular , Humanos , Rim/metabolismo , Leucotrienos/metabolismo , Ligantes , Miocárdio/metabolismo , Ligação Proteica , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Receptores Acoplados a Proteínas G/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Inibidores da Fusão de HIV/uso terapêutico , Receptores CXCR4/antagonistas & inibidores , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Antagonistas dos Receptores CCR5 , Quimiocinas/metabolismo , Inibidores da Fusão de HIV/química , Infecções por HIV/tratamento farmacológico , Humanos , Dados de Sequência Molecular , Monobactamas/química , Monobactamas/uso terapêutico , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMO
A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting, and possibly for cell entry. In addition, many virally-encoded chemokine 7TM receptors have been suggested to be causally involved in pathogenic phenotypes like Kaposi sarcoma, atherosclerosis, HIV-infection and tumour development. The role of these receptors during the viral life cycle and in viral pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we highlight the emerging impact of these receptor on virus-mediated diseases.
Assuntos
Receptores CCR7/biossíntese , Receptores CCR7/genética , Viroses/genética , Vírus/genética , Animais , Humanos , Receptores CCR7/química , Receptores CCR7/fisiologia , Rodopsina/química , Relação Estrutura-Atividade , Viroses/fisiopatologiaRESUMO
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.
Assuntos
Quimiocinas/fisiologia , Receptores de Quimiocinas/fisiologia , Sarcoma de Kaposi/etiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Dados de Sequência Molecular , Neovascularização Patológica/etiologia , Sarcoma de Kaposi/prevenção & controle , Transdução de SinaisRESUMO
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.
Assuntos
Quimiocinas/imunologia , Herpesviridae/imunologia , Poxviridae/imunologia , Receptores de Quimiocinas/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Quimiocinas/genética , Herpesviridae/genética , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Poxviridae/genética , Estrutura Secundária de Proteína , Receptores de Quimiocinas/química , Receptores de Quimiocinas/classificação , Receptores de Quimiocinas/genética , Proteínas Virais/genéticaRESUMO
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.