3,745 results on '"Enzyme-linked receptor"'
Search Results
2. Receptor Adaptation Mechanisms
- Author
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Eugenia V. Gurevich, Bih-Hwa Shieh, and Vsevolod V. Gurevich
- Subjects
G protein-coupled receptor kinase ,Biochemistry ,Chemistry ,Interleukin-21 receptor ,Muscarinic acetylcholine receptor M5 ,Enzyme-linked receptor ,Estrogen-related receptor gamma ,5-HT5A receptor ,sense organs ,Protease-activated receptor 2 ,G protein-coupled receptor ,Cell biology - Abstract
Receptor adaptation mechanisms involve regulation of the number, sensitivity and subcellular localization of receptors that detect environmental cues and convert extracellular signals into a change of intracellular events. Keywords: regulation of receptor desensitization; downregulation and gene expression; pharmacology; G protein-coupled receptors; β-adrenergic receptor; rhodopsin; G protein-coupled receptor kinase; arrestins; nicotinic acetylcholine receptor
- Published
- 2020
3. Structural biology of GABAB receptor
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Qing R. Fan and Aurel Frangaj
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0301 basic medicine ,Pharmacology ,Liver receptor homolog-1 ,Biology ,GABAB receptor ,Cell biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,030104 developmental biology ,Metabotropic receptor ,nervous system ,Enzyme-linked receptor ,5-HT5A receptor ,GABBR2 ,GABBR1 ,G protein-coupled receptor - Abstract
Metabotropic GABAB receptor is a G protein-coupled receptor (GPCR) that mediates slow and prolonged inhibitory neurotransmission in the brain. It functions as a constitutive heterodimer composed of the GABAB1 and GABAB2 subunits. Each subunit contains three domains; the extracellular Venus flytrap module, seven-helix transmembrane region and cytoplasmic tail. In recent years, the three-dimensional structures of GABAB receptor extracellular and intracellular domains have been elucidated. These structures reveal the molecular basis of ligand recognition, receptor heterodimerization and receptor activation. Here we provide a brief review of the GABAB receptor structures, with an emphasis on describing the different ligand-bound states of the receptor. We will also compare these with the known structures of related GPCRs to shed light on the molecular mechanisms of activation and regulation in the GABAB system, as well as GPCR dimers in general. This article is part of the "Special Issue Dedicated to Norman G. Bowery".
- Published
- 2018
4. The cellular and molecular effects of the androgen receptor agonist, Cl-4AS-1, on breast cancer cells
- Author
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Shatha Abu Hammad, Mamoun Ahram, Mariam Y. Al-Hudhud, Randa M. Bawadi, Lubna H. Tahtamouni, Ebtihal Mustafa, Faisal A. Khatib, and Malek Zihlif
- Subjects
0301 basic medicine ,Agonist ,medicine.drug_class ,Gene Expression ,Breast Neoplasms ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Breast cancer ,Cell Line, Tumor ,Gene expression ,medicine ,Enzyme-linked receptor ,Humans ,Cell Proliferation ,Dose-Response Relationship, Drug ,Cell growth ,Chemistry ,Dihydrotestosterone ,General Medicine ,medicine.disease ,Androgen receptor ,030104 developmental biology ,Apoptosis ,030220 oncology & carcinogenesis ,Androgens ,Cancer research ,medicine.drug - Abstract
The androgen receptor (AR) has attracted attention in the treatment of breast cancer. Due to the undesirable side effects of AR agonists, attempts have been undertaken to develop selective AR modulators. One of these compounds is Cl-4AS-1. This study examined this compound more closely at the cellular and molecular levels.Three different breast cancer cell lines were utilized, namely the luminal MCF-7 cells, the molecular apocrine MDA-MB-453 cells, and the triple negative, basal MDA-MB-231 cells.High and significant concordance between dihydrotestosterone (DHT) and Cl-4AS-1 in regulation of gene expression in MDA-MB-453 cells was found. However, some differences were noted including the expression of AR, which was upregulated by DHT, but not Cl-4AS-1. In addition, both DHT and Cl-4AS-1 caused a similar morphological change and reorganization of the actin structure of MDA-MB-453 cells into a mesenchymal phenotype. Treatment of cells with DHT resulted in induction of proliferation of MCF-7 and MDA-MB-453 cells, but no effect was observed on the growth of MDA-MB-231 cells. On the other hand, increasing doses of Cl-4AS-1 resulted in a dose-dependent inhibition on the growth of the three cell lines. This inhibition was a result of induction of apoptosis whereby Cl-4AS-1 caused a block in entry of cells into the S-phase followed by DNA degradation.These results indicate that although Cl-4AS-1 has characteristics of classical AR agonist, it has dissimilar properties that may make it useful in treating breast cancer.
- Published
- 2018
5. From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor
- Author
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Michael J. Stocks, Iain Walters, Steve Thom, Kenneth McKechnie, John Dixon, Nicholas Kindon, Premji Meghani, Iain G. Dougall, Timothy Johnson, and Andrew M. Davis
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0301 basic medicine ,Agonist ,P2Y receptor ,Chemistry ,medicine.drug_class ,Organic Chemistry ,Clinical Biochemistry ,Pharmaceutical Science ,Pharmacology ,Biochemistry ,HYDIA ,03 medical and health sciences ,030104 developmental biology ,Competitive antagonist ,Drug Discovery ,Enzyme-linked receptor ,medicine ,Molecular Medicine ,Selective receptor modulator ,Kidney disorder ,Receptor ,Molecular Biology - Abstract
The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.
- Published
- 2017
6. Parallel Chemistry Approach to Identify Novel Nuclear Receptor Ligands Based on the GW0742 Scaffold
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Adam Yasgar, David J. Maloney, Leggy A. Arnold, Ganesha Rai, Anton Simeonov, Preetpal S. Sidhu, Ajit Jadhav, Kelly A. Teske, Premchendar Nandhikonda, and Belaynesh Feleke
- Subjects
0301 basic medicine ,Agonist ,Transcription, Genetic ,Cell Survival ,medicine.drug_class ,Stereochemistry ,Tetrazoles ,Ligands ,GW0742 ,Calcitriol receptor ,Article ,Compound 32 ,Small Molecule Libraries ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Enzyme-linked receptor ,Humans ,PPAR delta ,Receptor ,Chemistry ,General Chemistry ,General Medicine ,High-Throughput Screening Assays ,Molecular Docking Simulation ,Thiazoles ,HEK293 Cells ,030104 developmental biology ,Nuclear receptor ,Biochemistry ,Drug Design ,030220 oncology & carcinogenesis ,Receptors, Calcitriol ,Bioisostere - Abstract
We describe the parallel synthesis of novel analogs of GW0742, a peroxisome proliferator-activated receptor δ (PPARδ) agonist. For that purpose, modified reaction conditions were applied, such as a solid-phase palladium-catalyzed Suzuki coupling. In addition, tetrazole-based compounds were generated as a bioisostere for carboxylic acid-containing ligand GW0742. The new compounds were investigated for their ability to activate PPARδ mediated transcription and their cross-reactivity with the vitamin D receptor (VDR), another member of the nuclear receptor superfamily. We identified many potent PPARδ agonists that were less toxic than GW0742, where ~65 of the compounds synthesized exhibited partial PPARδ-activity (23-98%) with EC50 values ranging from 0.007 – 18.2 μM. Some ligands, such as compound 32, were more potent inhibitors of VDR-mediated transcription with significantly reduced PPARδ activity than GW0742, however, none of the ligands were completely selective for VDR inhibition over PPARδ activation of transcription.
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- 2017
7. Bivalent ligand that activates mu opioid receptor and antagonizes mGluR5 receptor reduces neuropathic pain in mice
- Author
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Maureen S. Riedl, Eyup Akgün, Mary M. Lunzer, Carolyn A. Fairbanks, George L. Wilcox, Lucy Vulchanova, Cristina D. Peterson, Philip S. Portoghese, and Kelley F. Kitto
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Male ,0301 basic medicine ,Agonist ,medicine.drug_class ,Narcotic Antagonists ,Receptor, Metabotropic Glutamate 5 ,Receptors, Opioid, mu ,Pharmacology ,Ligands ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,mental disorders ,medicine ,Enzyme-linked receptor ,Animals ,Injections, Spinal ,Analgesics ,Chemistry ,Metabotropic glutamate receptor 5 ,030104 developmental biology ,Anesthesiology and Pain Medicine ,nervous system ,Neurology ,Opioid ,Hyperalgesia ,Oxymorphone ,Anesthesia ,Neuropathic pain ,Neuralgia ,Neurology (clinical) ,medicine.symptom ,μ-opioid receptor ,030217 neurology & neurosurgery ,medicine.drug - Abstract
The mu opioid receptor (MOR) and metabotropic glutamate receptor 5 (mGluR5) are well-established pharmacological targets in the management of chronic pain. Both receptors are expressed in the spinal cord. MMG22, a bivalent ligand containing 2 pharmacophores separated by 22 atoms, which simultaneously activates MOR and antagonizes mGluR5, has been shown to produce potent reversal of tactile hypersensitivity in rodent models of lipopolysaccharide (LPS)-and bone cancer-induced chronic pain. This study assessed whether intrathecal MMG22 also is effective in reducing pain of neuropathic origin. Furthermore, we theorized that MMG22 should reduce hyperalgesia in nerve-injured mice in a manner consistent with a synergistic interaction between MOR and mGluR5. Several weeks after spared nerve injury, tactile hypersensitivity was reversed in mice by the intrathecal injection of MMG22 (0.01-10 nmol) but also by its shorter spacer analog, MMG10, with similar potency. The potencies of the bivalent ligands were 10- to 14-fold higher than those of the compounds upon which the bivalent structure was based, the MOR agonist oxymorphone and the mGluR5 antagonist MPEP. Coadministration of oxymorphone and MPEP demonstrated analgesic synergism, an interaction confirmed by isobolographic analysis. This study indicates that in the spared nerve injury-induced model of neuropathic pain, the 2 pharmacophores of the bivalent ligands MMG22 and MMG10 target MOR and mGluR5 as separate receptor monomers. The observed increase in the potency of MMG22 and MMG10, compared with oxymorphone and MPEP, may reflect the synergistic interaction of the 2 pharmacophores of the bivalent ligand acting at their respective separate receptor monomers.
- Published
- 2017
8. Expression of estrogen receptor β in hypothalamic stem cells
- Author
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Li Cui, Zhen He, Merle G. Paule, and Sherry A. Ferguson
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medicine.medical_specialty ,Endocrinology ,Internal medicine ,medicine ,Enzyme-linked receptor ,Estrogen receptor ,Estrogen-related receptor gamma ,Stem cell ,Nestin ,Biology ,Estrogen receptor alpha ,Estrogen receptor beta ,Sexually dimorphic nucleus - Published
- 2017
9. Relative Antagonism of Mutants of the CGRP Receptor Extracellular Loop 2 Domain (ECL2) Using a Truncated Competitive Antagonist (CGRP8–37): Evidence for the Dual Involvement of ECL2 in the Two-Domain Binding Model
- Author
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Sifat J. Uddin, Alex C. Conner, John Simms, Michael J. Woolley, and David R. Poyner
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0301 basic medicine ,Cell signaling ,Receptor Activity-Modifying Protein 1 ,Calcitonin gene-related peptide ,Biology ,Biochemistry ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Competitive antagonist ,Enzyme-linked receptor ,Signal transduction ,Receptor ,030217 neurology & neurosurgery ,G protein-coupled receptor - Abstract
The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor.
- Published
- 2017
10. Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells
- Author
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Tadayuki Oshima, Jiro Watari, Hirokazu Fukui, Hiroto Miwa, and Liping Wu
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Hepatology ,medicine.drug_class ,business.industry ,Gastroenterology ,Pharmacology ,Receptor antagonist ,Adenosine A3 receptor ,03 medical and health sciences ,Interleukin 10 ,030104 developmental biology ,Endocrinology ,Internal medicine ,Interleukin-21 receptor ,medicine ,Enzyme-linked receptor ,Receptor ,business ,Glucagon-like peptide 1 receptor ,Insulin-like growth factor 1 receptor - Abstract
Background and Aim Immune-mediated mucosal inflammation characterized by the release of IL-8 is associated with gastroesophageal reflux disease (GERD). ATP released by human esophageal epithelial cells mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including human esophageal epithelial cells. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Methods Primary human esophageal epithelial cells (HEECs) were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Results ATP-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of ERK but not of PKC blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of ERK, and a P2Y2 receptor antagonist blocked this phosphorylation. Conclusions IL-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory GERD.
- Published
- 2017
11. N-glycosylation of the β2adrenergic receptor regulates receptor function by modulating dimerization
- Author
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Wei Huang, Huaiyu Yang, Mang Zhou, and Xiaona Li
- Subjects
0301 basic medicine ,Receptor expression ,Cell Biology ,Biology ,Alpha-1B adrenergic receptor ,Biochemistry ,Cell biology ,Beta-1 adrenergic receptor ,03 medical and health sciences ,030104 developmental biology ,Enzyme-linked receptor ,5-HT5A receptor ,Alpha-1D adrenergic receptor ,Molecular Biology ,Protease-activated receptor 2 ,Nuclear receptor co-repressor 1 - Abstract
N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β2 adrenergic receptor (β2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Enzymes Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96).
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- 2017
12. Progesterone Receptor Membrane Component-1 is phosphorylated upon treatment with Progestins and binds to Estrogen Receptor α-coregulators PHB1 and PHB2 in breast cancer cells
- Author
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Gereon Poschmann, Kai Stühler, AO Mueck, Tanja Fehm, Hans Neubauer, M Willibald, G Bayer, V Stahlhut, Dieter Niederacher, and H Seeger
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Androgen receptor ,Estrogen-related receptor alpha ,Chemistry ,Progesterone receptor ,Cancer research ,Enzyme-linked receptor ,Estrogen receptor ,Estrogen-related receptor gamma ,Estrogen receptor alpha ,Estrogen receptor beta - Published
- 2017
13. β-arrestins negatively control human adrenomedullin type 1-receptor internalization
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Jiang Danfeng, Sayaka Nagata, Kenji Kuwasako, Kazuo Kitamura, Toshio Sekiguchi, Manabu Murakami, Yuichi Hattori, and Johji Kato
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0301 basic medicine ,media_common.quotation_subject ,B-cell receptor ,Biophysics ,Biology ,Biochemistry ,Receptors, G-Protein-Coupled ,Adrenomedullin ,03 medical and health sciences ,0302 clinical medicine ,Enzyme-linked receptor ,Humans ,5-HT5A receptor ,Receptors, Adrenomedullin ,Internalization ,Molecular Biology ,Protease-activated receptor 2 ,media_common ,Calcitonin Receptor-Like Protein ,Cell Biology ,beta-Arrestin 2 ,Molecular biology ,Cell biology ,HEK293 Cells ,beta-Arrestin 1 ,030104 developmental biology ,RAMP2 ,Interleukin-21 receptor ,Estrogen-related receptor gamma ,030217 neurology & neurosurgery - Abstract
Adrenomedullin (AM) is a potent hypotensive peptide that exerts a powerful variety of protective effects against multiorgan damage through the AM type 1 receptor (AM1 receptor), which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). Two β-arrestin (β-arr) isoforms, β-arr-1 and β-arr-2, play a central role in the agonist-induced internalization of many receptors for receptor resensitization. Notably, β-arr-biased agonists are now being tested in phase II clinical trials, targeting acute pain and acute heart failure. Here, we examined the effects of β-arr-1 and β-arr-2 on human AM1 receptor internalization. We constructed a V5-tagged chimera in which the cytoplasmic C-terminal tail (C-tail) of CLR was replaced with that of the β2-adrenergic receptor (β2-AR), and it was transiently transfected into HEK-293 cells that stably expressed RAMP2. The cell-surface expression and internalization of the wild-type or chimeric receptor were quantified by flow cytometric analysis. The [125I]AM binding and the AM-induced cAMP production of these receptors were also determined. Surprisingly, the coexpression of β-arr-1 or -2 resulted in significant decreases in AM1 receptor internalization without affecting AM binding and signaling prior to receptor internalization. Dominant-negative (DN) β-arr-1 or -2 also significantly decreased AM-induced AM1 receptor internalization. In contrast, the AM-induced internalization of the chimeric AM1 receptor was markedly augmented by the cotransfection of β-arr-1 or -2 and significantly reduced by the coexpression of DN-β-arr-1 or -2. These results were consistent with those seen for β2-AR. Thus, both β-arrs negatively control AM1 receptor internalization, which depends on the C-tail of CLR.
- Published
- 2017
14. Development of Potent and Selective Antagonists for the UTP-Activated P2Y4 Receptor
- Author
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Younis Baqi, Aliaa Abdelrahman, Vigneshwaran Namasivayam, Muhammad Rafehi, Christa E. Müller, Alexander Neumann, Theodor Hanck, and Enas M. Malik
- Subjects
0301 basic medicine ,P2Y receptor ,Chemistry ,Allosteric regulation ,Antagonist ,Pharmacology ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Docking (molecular) ,Drug Discovery ,Enzyme-linked receptor ,Molecular Medicine ,Receptor ,Glucagon-like peptide 1 receptor ,Protease-activated receptor 2 - Abstract
P2Y4 is a Gq protein-coupled receptor activated by uridine-5′-triphosphate (UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective P2Y4 receptor antagonist has been described so far. Therefore, we developed and optimized P2Y4 receptor antagonists based on an anthraquinone scaffold. Potency was assessed by a fluorescence-based assay measuring inhibition of UTP-induced intracellular calcium release in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. The most potent compound of the present series, sodium 1-amino-4-[4-(2,4-dimethylphenylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-16133, 61) exhibited an IC50 value of 233 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric antagonist. A receptor homology model was built and docking studies were performed to analyze ligand–receptor interactions. Compound 64 (PSB-1699, sodium 1-amino-4-[4-(3-pyridin-3-ylmethylthio)phenylamino]-9,10-di...
- Published
- 2017
15. Phosphorylation of the phytosulfokine peptide receptor PSKR1 controls receptor activity
- Author
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Michael Motzkus, Margret Sauter, and Christine Kaufmann
- Subjects
0301 basic medicine ,calmodulin ,Calmodulin ,Physiology ,Arabidopsis ,Receptors, Cell Surface ,Plant Science ,growth regulation ,peptide signaling ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme-linked receptor ,Phosphorylation ,Kinase activity ,calcium ,pull-down ,receptor phosphorylation ,biology ,Arabidopsis Proteins ,Kinase ,Phytosulfokine ,Autophosphorylation ,Cell biology ,receptor-like kinase ,phytosulfokine receptor ,030104 developmental biology ,Protein kinase domain ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Relaxin/insulin-like family peptide receptor 2 ,Research Paper - Abstract
Phosphorylation of the phytosulfokine peptide receptor PSKR1 differentially affects auto- and transphosphorylation activity, binding to Ca2+-calmodulin, and has differential effects on shoot and root growth., The phytosulfokine peptide receptor PSKR1 is modified by phosphorylation of its cytoplasmic kinase domain. We analyzed defined phosphorylation sites by site-directed mutagenesis with regard to kinase activity in vitro and receptor activity in planta. S696 and S698 in the juxtamembrane (JM) domain are phosphorylated in planta. The phosphomimetic S696D/S698D replacements resulted in reduced transphosphorylation activity of PSKR1 kinase in vitro but did not reduce autophosphorylation activity. Growth-promoting activity of the PSKR1(S696D/S698D) receptor isoform was impaired in the shoot but not in the root. The JM domain thus seems to be important for phosphorylation of a target protein required for shoot growth promotion. The phosphomimetic replacement T998D at the C-terminus (CT) abolished kinase activity in vitro but not receptor function in planta, indicating that additional levels of regulation exist in planta. A possible mode of receptor regulation is the interaction with regulatory proteins such as the calcium sensor calmodulin (CaM). We show that the previously reported binding of CaM2 to PSKR1 is calcium-dependent, occurs predominately to the hypophosphorylated soluble PSKR1 kinase, and does not significantly change PSKR1 kinase activity. In conclusion, our results show that peptide signaling of growth by PSKR1 is regulated by differential phosphorylation of the juxtamembrane and C-terminal domains of the intracellular receptor part and suggest that interaction of PSKR1 with CaM serves a function other than the regulation of kinase activity.
- Published
- 2017
16. Development of spiroguanidine-derived α7 neuronal nicotinic receptor partial agonists
- Author
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Lizbeth Gallagher, Yulia Benitex, Matthew D. Hill, Richard E. Olson, Nicholas J. Lodge, Francine L. Healy, Debra J. Post-Munson, John E. Macor, Sivarao V. Digavalli, Daniel G. Morgan, JoAnne E Natale, Linda J. Bristow, Ping Chen, and Haiquan Fang
- Subjects
0301 basic medicine ,Quinuclidines ,alpha7 Nicotinic Acetylcholine Receptor ,Clinical Biochemistry ,Pharmaceutical Science ,Pharmacology ,Biochemistry ,Partial agonist ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Ganglion type nicotinic receptor ,Drug Discovery ,Enzyme-linked receptor ,Humans ,Spiro Compounds ,Receptor ,Molecular Biology ,Guanidine ,Arc (protein) ,Dose-Response Relationship, Drug ,Molecular Structure ,Chemistry ,Organic Chemistry ,Nicotinic acetylcholine receptor ,030104 developmental biology ,Nicotinic agonist ,Molecular Medicine ,Alpha-4 beta-2 nicotinic receptor ,030217 neurology & neurosurgery - Abstract
We describe the synthesis of quinuclidine-containing spiroguanidines and their utility as α7 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists. The convergent synthetic route developed for this study allowed for rapid SAR investigation and provided access to a structurally diverse set of analogs. A potent and selective α7 nAChR partial agonist, N‐(6‐methyl‐1,3‐benzoxazol‐2‐yl)‐3′,5′‐dihydro‐4‐azaspiro[bicyclo[2.2.2]octane‐2,4′‐imidazole]‐2′‐amine (BMS-910731, 16), was identified. This compound induced immediate early genes c-fos and Arc in a preclinical rodent model of α7 nAChR-derived cellular activation and plasticity. Importantly, the ability to incorporate selectivity for the α7 nACh receptor over the 5-HT3A receptor in this series suggested a significant difference in steric requirements between the two receptors.
- Published
- 2017
17. C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment
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Karin Mörl, Kerstin Burkert, Stefanie Babilon, Vsevolod V. Gurevich, Lizzy Wanka, and Annette G. Beck-Sickinger
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0301 basic medicine ,Neuropeptide Y receptor Y1 ,Neuropeptide Y receptor Y2 ,media_common.quotation_subject ,Cell Biology ,Biology ,Neuropeptide Y receptor ,Endocytosis ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Arrestin ,Enzyme-linked receptor ,Internalization ,media_common ,G protein-coupled receptor - Abstract
The human neuropeptide Y4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hY4R desensitization and endocytosis. The role of distinct segments of the hY4R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY4R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif 7.78EESEHLPLSTVHTEVSKGS7.96 was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis.
- Published
- 2017
18. Deubiquitylating enzymes in receptor endocytosis and trafficking
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Aidan P. McCann, Christopher J. Scott, Sandra Van Schaeybroeck, and James F. Burrows
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0301 basic medicine ,Endocytosis ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,Lysosome ,medicine ,Enzyme-linked receptor ,Animals ,Humans ,Molecular Biology ,Insulin-like growth factor 1 receptor ,biology ,Cell Membrane ,Cell Biology ,LRP1 ,Cell biology ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,ROR1 ,biology.protein ,Estrogen-related receptor gamma ,Lysosomes ,030217 neurology & neurosurgery - Abstract
In recent times, our knowledge of the roles ubiquitin plays in multiple cellular processes has expanded exponentially, with one example being the role of ubiquitin in receptor endocytosis and trafficking. This has prompted a multitude of studies examining how the different machinery involved in the addition and removal of ubiquitin can influence this process. Multiple deubiquitylating enzymes (DUBs) have been implicated either in facilitating receptor endocytosis and lysosomal degradation or in rescuing receptor levels by preventing endocytosis and/or promoting recycling to the plasma membrane. In this review, we will discuss in detail what is currently known about the role of DUBs in regulating the endocytosis of various transmembrane receptors and ion channels. We will also expand upon the role DUBs play in receptor sorting at the multivesicular body to determine whether a receptor is recycled or trafficked to the lysosome for degradation. Finally, we will briefly discuss how the DUBs implicated in these processes may contribute to the pathogenesis of a range of diseases, and thus the potential these have as therapeutic targets.
- Published
- 2016
19. Receptor for Advanced Glycation End Products Regulates Leukotriene B4Receptor 1 Signaling
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Takako Ichiki, Takehiko Yokomizo, and Tomoaki Koga
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0301 basic medicine ,Leukotriene B4 ,Leukotriene B4 Receptor 1 ,Cell Biology ,General Medicine ,respiratory system ,Biology ,RAGE (receptor) ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Biochemistry ,chemistry ,Interleukin-21 receptor ,Genetics ,Enzyme-linked receptor ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Coagulation factor II receptor ,Protease-activated receptor 2 ,G protein-coupled receptor - Abstract
Leukotriene B4 receptor 1 (BLT1), a high-affinity G protein-coupled receptor (GPCR) for leukotriene B4 (LTB4), plays important roles in inflammatory and immune reactions. Although the LTB4–BLT1 axis is known to promote inflammation, the binding proteins that modulate LTB4–BLT1 signaling have not been identified. Recently, we discovered that receptor for advanced glycation end products (RAGE) interacts with BLT1 and modulates LTB4–BLT1 signaling. We propose RAGE as a new class of GPCR modulator and a new target of future GPCR studies.
- Published
- 2016
20. Synthetic non‐peptide low molecular weight agonists of the relaxin receptor 1
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Juan J. Marugan, Alexander I. Agoulnik, Xin Hu, and Irina U. Agoulnik
- Subjects
0301 basic medicine ,Pharmacology ,Peptide Metabolism ,Relaxin ,Receptors, Peptide ,Chemistry ,Themed Section: Review Articles ,Receptors, G-Protein-Coupled ,Molecular Weight ,03 medical and health sciences ,030104 developmental biology ,In vivo ,Enzyme-linked receptor ,Animals ,Humans ,Receptor ,hormones, hormone substitutes, and hormone antagonists ,Relaxin/insulin-like family peptide receptor 2 ,Relaxin receptor ,ADME - Abstract
Relaxin is a small heterodimeric peptide hormone of the insulin/relaxin superfamily produced mainly in female and male reproductive organs. It has potent antifibrotic, vasodilatory and angiogenic effects and regulates the normal function of various physiological systems. Preclinical studies and recent clinical trials have shown the promise of recombinant relaxin as a therapeutic agent in the treatment of cardiovascular and fibrotic diseases. However, there are the universal drawbacks of peptide-based pharmacology that apply to relaxin: a short half-life in vivo requires its continuous delivery, and there are high costs of production, storage and treatment, as well as the possibility of immune responses. All these issues can be resolved by the development of low non-peptide MW agonists of the relaxin receptors which are stable, bioavailable, easily synthesized and specific. In this review, we describe the discovery and characterization of the first series of such compounds. The lead compound, ML290, binds to an allosteric site of the relaxin GPCR, RXFP1. ML290 shows high activity and efficacy, measured by cAMP response, in cells expressing endogenous or transfected RXFP1. Relaxin-like effects of ML290 were shown in various functional cellular assays in vitro. ML290 has excellent absorption, distribution, metabolism and excretion properties and in vivo stability. The identified series of low MW agonists does not activate rodent RXFP1 receptors and thus, the production of a RXFP1 humanized mouse model is needed for preclinical studies. The future analysis and clinical perspectives of relaxin receptor agonists are discussed.This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
- Published
- 2016
21. Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling
- Author
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Cornelius Krasel, Diana Zindel, Moritz Bünemann, Andrew B. Tobin, Adrian J. Butcher, Jean-Philippe Pin, Andrew R. Bottrill, Laurent Prézeau, Sandra Engel, Butcher, Adrian [0000-0001-5723-8720], Apollo - University of Cambridge Repository, Institut de Génomique Fonctionnelle (IGF), and Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Bioluminescence Resonance Energy Transfer Techniques ,0301 basic medicine ,Cell signaling ,Arrestins ,[SDV]Life Sciences [q-bio] ,Molecular Sequence Data ,Enzyme-Linked Immunosorbent Assay ,Biology ,Biochemistry ,Mass Spectrometry ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,parathyroid hormone receptor 1 (PTH1R) ,Fluorescence Resonance Energy Transfer ,Arrestin ,Enzyme-linked receptor ,Humans ,Immunoprecipitation ,5-HT5A receptor ,Amino Acid Sequence ,Molecular Biology ,Research Articles ,Receptor, Parathyroid Hormone, Type 1 ,G protein-coupled receptor kinase ,Sequence Homology, Amino Acid ,arrestin ,phosphorylation ,Cell Biology ,Interleukin-13 receptor ,G-protein-coupled receptor (GPCR) ,HEK293 Cells ,030104 developmental biology ,Phosphorylation ,Signal transduction ,Research Article ,Signal Transduction - Abstract
International audience; The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.
- Published
- 2016
22. Phosphorylation of STAT3 by Human Serotonin 2C Receptor
- Author
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Musarrat Maisha, M.E. Curtis, L.S Fears, Zeljka Miletic Lanaghan, MT Ivy, and Hugh Fentress
- Subjects
biology ,Chemistry ,5-HT2A receptor ,Serotonin 2C Receptor ,Biochemistry ,Cell biology ,Genetics ,biology.protein ,5-HT6 receptor ,Enzyme-linked receptor ,Phosphorylation ,5-HT5A receptor ,STAT3 ,Molecular Biology ,Endogenous agonist ,Biotechnology - Published
- 2019
23. OptoGluNAM4.1, a Photoswitchable Allosteric Antagonist for Real-Time Control of mGlu 4 Receptor Activity
- Author
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Amadeu Llebaria, Jesús Giraldo, Pau Gorostiza, Xavier Rovira, Charleine Zussy, Ana Trapero, Silvia Pittolo, Chris Jopling, Adèle Faucherre, Cyril Goudet, Jean-Philippe Pin, European Research Council, Trapero, Ana [0000-0003-4526-7895], Llebaría, Amadeu [0000-0002-8200-4827], Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Institute for Bioengineering of Catalonia [Barcelona] (IBEC), Trapero, Ana, and Llebaría, Amadeu
- Subjects
0301 basic medicine ,Time Factors ,Light ,[SDV]Life Sciences [q-bio] ,Clinical Biochemistry ,Chronic pain ,Pharmacology ,Receptors, Metabotropic Glutamate ,Biochemistry ,Mice ,0302 clinical medicine ,Drug Discovery ,ComputingMilieux_MISCELLANEOUS ,Zebrafish ,Azobenzene ,Metabotropic glutamate receptor 5 ,Metabotropic glutamate receptor 4 ,mGlu receptor ,Metabotropic glutamate receptor 7 ,Metabotropic glutamate receptor 6 ,Dolor crònic ,Pyrrolidonecarboxylic Acid ,3. Good health ,Cell biology ,Molecular Medicine ,Metabotropic glutamate receptor 1 ,Metabotropic glutamate receptor 2 ,Nervous system ,Pain Threshold ,Allosteric modulator ,OptoGluNAM4.1 ,Biology ,Structure-Activity Relationship ,03 medical and health sciences ,Allosteric Regulation ,Enzyme-linked receptor ,Animals ,Humans ,Sistema nerviós ,Molecular Biology ,Dose-Response Relationship, Drug ,Mice, Inbred C57BL ,Disease Models, Animal ,HEK293 Cells ,030104 developmental biology ,Schizophrenia ,Azo Compounds ,030217 neurology & neurosurgery - Abstract
OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system. © 2016 Elsevier Ltd, We are grateful to C. Serra, L. Muñoz, J. Hernando, F. Malhaire, Y. Pérez, M. Izquierdo-Serra, F. Aguado, S. Laffray, E. Bourinet, F.Codony (GenIUL), and Y. Chomis (Viewpoint) for helpful discussions and technical support. A.F. is supported by a Fondation Lefoulon Delalande postdoctoral fellowship , C.J. by a grant from INSERM ATIP-AVENIR and Marie Curie CIG ( PCIG12-GA-2012-332772 ). A.F. and C.J. are members of the Laboratory of Excellence Ion Channel Science and Therapeutics supported by a grant from the ANR . J.-P.P. is a member of the Laboratory of Excellence Epingenmed. We acknowledge financial support from the European Union's Seventh Framework Program for research, technological development, and demonstration under grant agreements 270483 (Focus), 210355 (Opticalbullet), and 335011 (Theralight) to P.G.; the Federation of European Biochemical Societies ; the Catalan government ( 2012FI_B 01122 to S.P., 2014SGR-1251 to P.G., and 2014SGR-0109 to A. Llebaria); the Spanish Government ( SAF2014-58396-R to J.G., CTQ2014-57020-R to A.L., and CTQ2013-43892R to P.G.); the ERANET Neuron LIGHTPAIN project (to A.L., J.G., and J.-P.P.); the Ramón Areces Foundation , the ERANET SynBio Modulightor project (to P.G.); the HBP Wavescales project (to P.G.); the Fondation Recherche Médicale (FRM team DEQ20130326522 to J.P.P.); the Agence Nationale de la Recherche ( ANR-13-BSV1-006 to C.G.) and the Beatriu de Pinós program of Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) for the support of X.R.
- Published
- 2016
24. The polymorphic deleted-form of the human α2B-adrenergic receptor and its wild-type counterpart display post-receptor signaling pathway differences in LLC-PK1 cells
- Author
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Hervé Paris, Gregory Sivolapenko, Antonis S. Manolis, Dimosthenis Lykouras, George Panayiotakopoulos, Christodoulos S. Flordellis, Stéphane Schaak, Georgios Karkoulias, Pierre-Antoine Crassous, Nicholas Patsouras, and Orthodoxia Mastrogianni
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,lcsh:Diseases of the circulatory (Cardiovascular) system ,Adrenergic receptor ,Alpha-1B adrenergic receptor ,NF-κB ,03 medical and health sciences ,0302 clinical medicine ,Enzyme-linked receptor ,Medicine ,Alpha-1D adrenergic receptor ,Protein kinase B ,Medicine(all) ,β-arrestin ,urogenital system ,business.industry ,Akt ,Alpha-1A adrenergic receptor ,MAPK ,Cell biology ,α2B-adrenergic receptor ,030104 developmental biology ,lcsh:RC666-701 ,Arrestin beta 2 ,Cardiology and Cardiovascular Medicine ,business ,030217 neurology & neurosurgery - Published
- 2016
25. Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor
- Author
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Callum F. Lawrence, Brian J. Smith, Mai B. Margetts, Michael C. Lawrence, Colin W. Ward, John G. Menting, and Nicholas A. Smith
- Subjects
0301 basic medicine ,Amino Acid Motifs ,Biochemistry ,Mice ,03 medical and health sciences ,Growth factor receptor ,Biomimetic Materials ,Peptide Library ,Insulin receptor substrate ,Enzyme-linked receptor ,Animals ,Humans ,Insulin ,5-HT5A receptor ,Binding site ,Molecular Biology ,Glucagon-like peptide 1 receptor ,Binding Sites ,biology ,Cell Biology ,Receptor, Insulin ,IRS2 ,Insulin receptor ,030104 developmental biology ,Protein Structure and Folding ,biology.protein - Abstract
Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe(701) and Phe(705) The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor.
- Published
- 2016
26. Identification and characterization of heptapeptide modulators of the glycine receptor
- Author
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Natasha C. Pflanz, S. John Mihic, Garrett L. Cornelison, and Megan E. Tipps
- Subjects
0301 basic medicine ,Phage display ,Allosteric regulation ,Biology ,Article ,Xenopus laevis ,03 medical and health sciences ,Receptors, Glycine ,0302 clinical medicine ,Allosteric Regulation ,Consensus Sequence ,Enzyme-linked receptor ,Animals ,Humans ,5-HT5A receptor ,Amino Acid Sequence ,Receptor ,Glycine receptor ,Pharmacology ,HEK 293 cells ,Electrophysiological Phenomena ,Zinc ,HEK293 Cells ,030104 developmental biology ,Biochemistry ,GPR18 ,Oligopeptides ,030217 neurology & neurosurgery - Abstract
The glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. This involved a panning procedure in which the phage library initially underwent subtractive panning against Human Embryonic Kidney (HEK) 293 cells expressing alternative glycine receptor subtypes before panning the remaining library over HEK 293 cells expressing the target, the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β, compared to α2β, glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol's enhancing actions at the receptor in the absence of zinc. Comparisons of the amino acid sequences of heptapeptides capable of potentiating glycine receptor function revealed several consensus sequences that may be predictive of a peptide's enhancing ability.
- Published
- 2016
27. Cross-Talk in the Female Rat Mammary Gland: Influence of Aryl Hydrocarbon Receptor on Estrogen Receptor Signaling
- Author
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Günter Vollmer, Janina Helle, Sridar V. Chittur, Georg Kretzschmar, Annekathrin Martina Keiler, Martin Tenniswood, Oliver Zierau, and Manuela I. Bader
- Subjects
Transcriptional Activation ,0301 basic medicine ,medicine.medical_specialty ,Health, Toxicology and Mutagenesis ,Estrogen receptor ,Real-Time Polymerase Chain Reaction ,03 medical and health sciences ,Estrogen-related receptor alpha ,Mammary Glands, Animal ,0302 clinical medicine ,Internal medicine ,medicine ,Enzyme-linked receptor ,Animals ,Estrogen receptor beta ,Estradiol ,biology ,Chemistry ,Research ,Estrogen Receptor alpha ,Public Health, Environmental and Occupational Health ,Aryl hydrocarbon receptor ,Rats ,030104 developmental biology ,Endocrinology ,Receptors, Aryl Hydrocarbon ,Receptors, Estrogen ,030220 oncology & carcinogenesis ,biology.protein ,Female ,Estrogen-related receptor gamma ,Signal transduction ,Estrogen receptor alpha ,Signal Transduction - Abstract
Background: Cross-talk between the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER) plays a major role in signaling processes in female reproductive organs. Objectives: We investigated the influence of the AHR ligand 3-methylcholanthrene (3-MC) on ER-mediated signaling in mammary gland tissue of ovariectomized (ovx) rats. Methods: After 14 days of hormonal decline, ovx rats were treated for 3 days with 4 μg/kg 17β-estradiol (E2), 15 mg/kg 8-prenylnaringenin (8-PN), 15 mg/kg 3-MC, or a combination of these compounds (E2 + 3-MC, 8-PN + 3-MC). Whole-mount preparations of the mammary gland were used to count terminal end buds (TEBs). Protein expression studies (immunohistochemistry, immunofluorescence), a cDNA microarray, pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the interaction between AHR- and ER-mediated signaling pathways. Results: E2 treatment increased the number of TEBs and the levels of Ki-67 protein and progesterone receptor (PR); this treatment also changed the expression of 325 genes by more than 1.5-fold. Although 3-MC treatment alone had marginal impact on gene or protein expression, when rats were co-treated with 3-MC and E2, 3-MC strongly inhibited E2-induced TEB development, protein synthesis, and the expression of nearly half of E2-induced genes. This inhibitory effect of 3-MC was partially mirrored when 8-PN was used as an ER ligand. The anti-estrogenicity of ligand-activated AHR was at least partly due to decreased protein levels of ERα in ductal epithelial cells. Conclusion: Our data show transcriptome-wide anti-estrogenic properties of ligand-activated AHR on ER-mediated processes in the mammary gland, thereby contributing an explanation for the chemopreventive and endocrine-disrupting potential of AHR ligands. Citation: Helle J, Bader MI, Keiler AM, Zierau O, Vollmer G, Chittur SV, Tenniswood M, Kretzschmar G. 2016. Cross-talk in the female rat mammary gland: influence of aryl hydrocarbon receptor on estrogen receptor signaling. Environ Health Perspect 124:601–610; http://dx.doi.org/10.1289/ehp.1509680
- Published
- 2016
28. The complexity of signalling mediated by the glucagon-like peptide-1 receptor
- Author
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Arthur Christopoulos, Madeleine M. Fletcher, Michelle L. Halls, Patrick M. Sexton, and Denise Wootten
- Subjects
0301 basic medicine ,endocrine system ,medicine.medical_specialty ,Incretin ,Tropomyosin receptor kinase B ,Biology ,Ligands ,Biochemistry ,Glucagon-Like Peptide-1 Receptor ,03 medical and health sciences ,Internal medicine ,medicine ,Functional selectivity ,Enzyme-linked receptor ,Humans ,5-HT5A receptor ,Insulin-like growth factor 1 receptor ,digestive, oral, and skin physiology ,Endocytosis ,IRS2 ,Insulin receptor ,030104 developmental biology ,Endocrinology ,Diabetes Mellitus, Type 2 ,biology.protein ,Neuroscience ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR that is a major therapeutic target for the treatment of type 2 diabetes. The receptor is activated by the incretin peptide GLP-1 promoting a broad range of physiological effects including glucose-dependent insulin secretion and biosynthesis, improved insulin sensitivity of peripheral tissues, preservation of β-cell mass and weight loss, all of which are beneficial in the treatment of type 2 diabetes. Despite this, existing knowledge surrounding the underlying signalling mechanisms responsible for the physiological actions downstream of GLP-1R activation is limited. Here, we review the current understanding around GLP-1R-mediated signalling, in particular highlighting recent contributions to the field on biased agonism, the spatial and temporal aspects for the control of signalling and how these concepts may influence future drug development.
- Published
- 2016
29. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein–Coupled Receptor 120
- Author
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Ashley M. Miller, Andrew B. Tobin, Elisa Alvarez-Curto, Graeme Milligan, Brian D. Hudson, Rudi Prihandoko, Adrian J. Butcher, and Trond Ulven
- Subjects
Threonine ,0301 basic medicine ,Arrestins ,MAP Kinase Signaling System ,Recombinant Fusion Proteins ,CHO Cells ,Biology ,Receptors, G-Protein-Coupled ,Mice ,03 medical and health sciences ,Cricetulus ,Bacterial Proteins ,Membrane Transport Modulators ,Serine ,Arrestin ,Enzyme-linked receptor ,Animals ,Humans ,5-HT5A receptor ,Enzyme Inhibitors ,Phosphorylation ,Pharmacology ,Cell Membrane ,Interleukin-13 receptor ,Cell biology ,Enzyme Activation ,Luminescent Proteins ,Protein Transport ,HEK293 Cells ,030104 developmental biology ,Amino Acid Substitution ,Biochemistry ,Interleukin-21 receptor ,Mutation ,Free fatty acid receptor ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Molecular Medicine ,Arrestin beta 2 ,Protein Processing, Post-Translational ,Proto-Oncogene Proteins c-akt - Abstract
It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.
- Published
- 2016
30. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor
- Author
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Manabu Murakami, Yuichi Hattori, Toshio Sekiguchi, Danfeng Jiang, Sayaka Nagata, Johji Kato, Kenji Kuwasako, Kazuo Kitamura, and Hidetaka Hayashi
- Subjects
G-Protein-Coupled Receptor Kinase 5 ,0301 basic medicine ,G-Protein-Coupled Receptor Kinase 4 ,Biophysics ,Down-Regulation ,Biology ,Biochemistry ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,Enzyme-linked receptor ,Humans ,5-HT5A receptor ,Receptors, Adrenomedullin ,Calcitonin receptor ,Molecular Biology ,Nuclear receptor co-repressor 1 ,Cell Membrane ,Cell Biology ,CALCRL ,Molecular biology ,HEK293 Cells ,030104 developmental biology ,RAMP2 ,Interleukin-21 receptor ,Estrogen-related receptor gamma ,Signal Transduction - Abstract
Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [(125)I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449-453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM1 receptor and further determined the region of the CLR C-tail responsible for this GRK function.
- Published
- 2016
31. Efficacy of Hybrid Tetrahydrobenzo[d]thiazole Based Aryl Piperazines D-264 and D-301 at D2 and D3 Receptors
- Author
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Joanna C. Jacob, Tamara Antonio, David K. Grandy, Juan Zhen, Aloke K. Dutta, Dana E. Selley, and Maarten E. A. Reith
- Subjects
0301 basic medicine ,Intrinsic activity ,Receptor expression ,General Medicine ,Pharmacology ,Biology ,Biochemistry ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,030104 developmental biology ,0302 clinical medicine ,Dopamine receptor D1 ,Dopamine receptor ,Dopamine ,Dopamine receptor D2 ,medicine ,Enzyme-linked receptor ,Receptor ,030217 neurology & neurosurgery ,medicine.drug - Abstract
In elucidating the role of pharmacodynamic efficacy at D3 receptors in therapeutic effectiveness of dopamine receptor agonists, the influence of study system must be understood. Here two compounds with D3 over D2 selectivity developed in our earlier work, D-264 and D-301, are compared in dopamine receptor-mediated G-protein activation in striatal regions of wild-type and D2 receptor knockout mice and in CHO cells expressing D2 or D3 receptors. In caudate-putamen of D2 knockout mice, D-301 was ~3-fold more efficacious than D-264 in activating G-proteins as assessed by [(35)S]GTPγS binding; in nucleus accumbens, D-301 stimulated G-protein activation whereas D-264 did not. In contrast, the two ligands exerted similar efficacy in both regions of wild-type mice, suggesting both ligands activate D2 receptors with similar efficacy. In D2 and D3 receptor-expressing CHO cells, D-264 and D-301 appeared to act in the [(35)S]GTPγS assay as full agonists because they produced maximal stimulation equal to dopamine. Competition for [(3)H]spiperone binding was then performed to determine Ki/EC50 ratios as an index of receptor reserve for each ligand. Action of D-301, but not D-264, showed receptor reserve in D3 but not in D2 receptor-expressing cells, whereas dopamine showed receptor reserve in both cell lines. Gαo1 is highly expressed in brain and is important in D2-like receptor-G protein coupling. Transfection of Gαo1 in D3- but not D2-expressing CHO cells led to receptor reserve for D-264 without altering receptor expression levels. D-301 and dopamine exhibited receptor reserve in D3-expressing cells both with and without transfection of Gαo1. Altogether, these results indicate that D-301 has greater intrinsic efficacy to activate D3 receptors than D-264, whereas the two compounds act on D2 receptors with similar intrinsic efficacy. These findings also suggest caution in interpreting Emax values from functional assays in receptor-transfected cell models without accounting for receptor reserve.
- Published
- 2015
32. GPR55 – a putative 'type 3' cannabinoid receptor in inflammation
- Author
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Juan Zhou, Hyewon Yang, and Christian Lehmann
- Subjects
0301 basic medicine ,Cannabinoid receptor ,Physiology ,medicine.medical_treatment ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,Drug Discovery ,medicine ,Cannabinoid receptor type 2 ,Enzyme-linked receptor ,Animals ,Humans ,Receptors, Cannabinoid ,Inflammation ,Pharmacology ,Cannabinoids ,General Medicine ,Endocannabinoid system ,030104 developmental biology ,GPR55 ,Interleukin-21 receptor ,GPR18 ,lipids (amino acids, peptides, and proteins) ,Cannabinoid ,Neuroscience ,Signal Transduction - Abstract
G protein-coupled receptor 55 (GPR55) shares numerous cannabinoid ligands with CB1 and CB2 receptors despite low homology with those classical cannabinoid receptors. The pharmacology of GPR55 is not yet fully elucidated; however, GPR55 utilizes a different signaling system and downstream cascade associated with the receptor. Therefore, GPR55 has emerged as a putative “type 3” cannabinoid receptor, establishing a novel class of cannabinoid receptor. Furthermore, the recent evidence of GPR55-CB1 and GPR55-CB2 heteromerization along with its broad distribution from central nervous system to peripheries suggests the importance of GPR55 in various cellular processes and pathologies and as a potential therapeutic target in inflammation.
- Published
- 2015
33. Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket
- Author
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Ashley M. Deacon, Robert J. Fletterick, Elena P. Sablin, Hsiu-Ju Chiu, Rubatharshini Uthayaruban, Holly A. Ingraham, Raymond D. Blind, and Debanu Das
- Subjects
Models, Molecular ,Protein Structure ,medicine.medical_specialty ,Cytoplasmic and Nuclear ,NR5A2 ,Biophysics ,Receptors, Cytoplasmic and Nuclear ,Ligand ,Biology ,Crystallography, X-Ray ,Phosphatidylinositols ,Steroidogenic Factor 1 ,Article ,PIP3 ,Models ,Structural Biology ,Internal medicine ,Receptors ,Genetics ,Enzyme-linked receptor ,medicine ,Humans ,Receptor ,Transcription factor ,Crystallography ,Binding Sites ,DAX-1 Orphan Nuclear Receptor ,Crystal structure ,Liver Disease ,Liver receptor homolog-1 ,Phosphatidylinositol binding ,Molecular ,Liver Receptor Homolog-1 ,Ligand (biochemistry) ,Protein Structure, Tertiary ,Cell biology ,Endocrinology ,Nuclear receptor ,PIP(3) ,Hormone receptor ,X-Ray ,Biochemistry and Cell Biology ,Digestive Diseases ,Zoology ,Tertiary ,LRH-1 ,Protein Binding - Abstract
The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.
- Published
- 2015
34. Recent Developments in Androgen Receptor Antagonists
- Author
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Fansheng Ran, Daoguang Zhang, Yang Liu, Guisen Zhao, Pengzhan Li, and Hualu Xing
- Subjects
business.industry ,Pharmaceutical Science ,Ligand binding domain ,Pharmacology ,urologic and male genital diseases ,medicine.disease ,Androgen deprivation therapy ,Androgen receptor ,Prostate cancer ,Drug Discovery ,Coactivator ,Enzyme-linked receptor ,medicine ,Androgen Receptor Antagonists ,business ,Transcription factor - Abstract
The androgen receptor (AR), a ligand-dependent transcription factor that regulates the expression of a series of downstream target genes after the binding of androgens, has been a target for the discovery of drugs used to treat prostate cancer. Prostate cancer always progresses to castration-resistant prostate cancer after a period of androgen deprivation therapy. Thus, developing potent androgen receptor antagonists for the therapy of castration-resistant prostate cancer possesses great significance. This review summarizes the preclinical development of androgen receptor antagonists, conventional androgen receptor antagonists that competitively bind to the ligand binding domain of the androgen receptor and coactivator antagonists of the androgen receptor, including both activation function-2 antagonists and binding function-3 antagonists. We hope that this review can help other researchers find new scaffolds and sites for the treatment of prostate cancer.
- Published
- 2015
35. Suppressive effect of membrane-permeable peptides derived from autophosphorylation sites of the IGF-1 receptor on breast cancer cells
- Author
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Yoshihiro, Kuroda, Nahoko, Kato-Kogoe, Emi, Tasaki, Mayumi, Yuasa-Sunagawa, Koji, Yamanegi, Keiji, Nakasho, Keiji, Nakasyo, Ikuhiko, Nakase, Shiroh, Futaki, Yumi, Tohyama, and Munetaka, Hirose
- Subjects
Pharmacology ,chemistry.chemical_classification ,Cell Membrane Permeability ,Dose-Response Relationship, Drug ,biology ,Cell growth ,Autophosphorylation ,Breast Neoplasms ,Peptide ,Peptide Fragments ,Receptor, IGF Type 1 ,Insulin receptor ,chemistry ,MCF-7 Cells ,biology.protein ,Enzyme-linked receptor ,Cancer research ,Cell-penetrating peptide ,Humans ,Phosphorylation ,Female ,Receptor ,Cell Proliferation - Abstract
Insulin-like growth factor-1 (IGF-1) receptors play a crucial role in the biology of human cancer, making them an attractive target for anti-cancer agents. We previously designed oligopeptides containing the amino-acid sequences surrounding the autophosphorylation sites of the insulin receptor and found that two of them, namely, Ac-DIYET-NH2 and Ac-DYYRK-NH2, suppressed phosphorylation of purified insulin receptors in a non-ATP-competitive manner, whereas Ac-NIYQT-NH2 and Ac-NYYRK-NH2 suppressed in an ATP-competitive manner. Because the IGF-1 receptor is closely related to the insulin receptor, the aim of this study was to observe the effects of these peptides, which correspond to the amino-acid sequences of the autophosphorylation sites of the IGF-1 receptor, on the activity of the human breast cancer cell lines MCF-7, T47D, MDA-MB-231, and MDA-MB-453. To facilitate peptide delivery into breast cancer cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to these peptides. When breast cancer cells were treated with each of these synthetic Tat-conjugated peptides, the conjugated peptides penetrated into the cells and suppressed cell proliferation. An inhibitory effect of Tat-conjugated peptides against IGF-1-stimulated phosphorylation of IGF-1 receptors was observed. In addition, we found that combinations of these peptides suppressed phosphorylation of IGF-1 receptors to a greater extent than the peptides did individually. In conclusion, IGF-1 receptor autophosphorylation site-derived membrane-permeable peptides have the potential to suppress IGF-1 receptor function in breast cancer cells and to be developed into novel and useful agents for cancer therapy.
- Published
- 2015
36. Development and validation of an enzyme-linked receptor assay based on mutant protein I188K/S19C/G24C for 40 beta-lactams antibiotics detection in 13 food samples
- Author
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Zonghui Yuan, Jianan Ning, Saeed Ahmed, Ijaz Ahmad, Ting Chen, Muhammad Kashif Maan, Guyue Cheng, and Samah Attia Algharib
- Subjects
Detection limit ,Residue (complex analysis) ,Kidney ,medicine.drug_class ,Chemistry ,010401 analytical chemistry ,Antibiotics ,02 engineering and technology ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,Analytical Chemistry ,medicine.anatomical_structure ,Mutant protein ,medicine ,Enzyme-linked receptor ,Food science ,0210 nano-technology ,Receptor ,IC50 ,Spectroscopy - Abstract
Antibiotics play an important role in disease prevention and as a growth promoter in the veterinary field. Animals derived foods holding antibiotic residues above the maximum residue limits can contribute as a hazardous effect for human health. In the current study, a BlaR-CTD mutant protein I188K/S19C/G24C enzyme-linked receptor assay (ELRA) was developed for the screening of 40 beta-lactams antibiotics in 12 edible animal tissues and in the milk sample. The IC50 for 40 drugs were ranged from 0.13 to 307.62 μg.L−1. The limit of detection (LOD) in tissues and milk samples were ranged from 0.01 to 110.2 μg.kg−1, and 0.07 to 194.64 μg.L−1 respectively. The evaluated average recovery rates in muscle, liver, kidney, and milk were ranged from 68.09 to 117.61%, 67.58 to 117.84%, 64.0 to 117.03%, and 68.08 to 116.06% respectively. The recovery rate in the entire sample examined in this study ranged from 60 to 120%, with co-efficient of variations below 15%. The applied ELRA showed a good correlation with that of LC-MS/MS. This study for the first time offers a simple and high throughput assay for the screening of 40 beta-lactams in 12 animal tissues and in milk sample, this assay was based on mutant protein I188K/S19C/G24C, with a wide range of high affinity and stability to beta-lactams antibiotics, exhibiting the potential to be commercially used.
- Published
- 2020
37. Androgen Receptor Ligands: Agonists and Antagonists
- Author
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Iain J. McEwan, Amy E. Monaghan, and Folake A Orafidiya
- Subjects
medicine.medical_specialty ,Chemistry ,medicine.medical_treatment ,Androgen Excess ,Androgen receptor ,Steroid hormone ,Endocrinology ,Dihydrotestosterone ,Internal medicine ,medicine ,Enzyme-linked receptor ,Receptor ,Testosterone ,Tissue homeostasis ,medicine.drug - Abstract
The androgen receptor is a key regulator of physiological development in utero and tissue homeostasis in adult men and woman. The activity of the receptor is tightly regulated by the binding of the natural steroid hormone agonists, testosterone and dihydrotestosterone. Activation of the receptor leads to dimerization, nuclear translocation and activation of target genes. Clinically, synthetic agonists are used to treat conditions of hormone insufficiency. Conversely conditions arising from androgen excess or a dependence upon the androgen receptor can be treated with antagonists to blunt the response to natural hormones.
- Published
- 2018
38. Beta Receptor Antagonists
- Author
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Jeffrey Brent and Michael Levine
- Subjects
Adrenergic receptor ,business.industry ,Enzyme-linked receptor ,Medicine ,Pharmacology ,business ,Glucagon-like peptide 1 receptor - Published
- 2018
39. Selective and Specific Activation of Rab5 during Endocytosis of Receptor Tyrosine Kinases
- Author
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Ivan Jozic
- Subjects
Insulin receptor ,biology ,Chemistry ,Insulin receptor substrate ,ROR1 ,LDL receptor ,biology.protein ,Enzyme-linked receptor ,Tropomyosin receptor kinase C ,IRS2 ,Receptor tyrosine kinase ,Cell biology - Published
- 2017
40. Novel Insights into the Mechanisms of Regulation of Tyrosine Kinase Receptors by Ras Interference 1
- Author
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Adriana Galvis
- Subjects
Insulin receptor ,biology ,Chemistry ,Anti-apoptotic Ras signalling cascade ,ROR1 ,biology.protein ,Enzyme-linked receptor ,Tropomyosin receptor kinase B ,Tropomyosin receptor kinase A ,Tropomyosin receptor kinase C ,Receptor tyrosine kinase ,Cell biology - Published
- 2017
41. 20[beta]-Dihydrocortisol; a weak endogenous agonist of the glucocorticoid receptor but a potent agonist of the mineralocorticoid receptor
- Author
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Diana Melchers, Brian R. Walker, Ruth Morgan, Natalie Z.M. Homer, René Houtman, Katharina Beck, Onno C. Meijer, Patrick W. F. Hadoke, Alex Odermatt, Mark Nixon, and John Keen
- Subjects
Agonist ,medicine.medical_specialty ,Chemistry ,medicine.drug_class ,Partial agonist ,Mineralocorticoid receptor ,Glucocorticoid receptor ,Endocrinology ,Internal medicine ,medicine ,Enzyme-linked receptor ,Inverse agonist ,Beta (finance) ,Endogenous agonist - Published
- 2017
42. Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding
- Author
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Colleen A. Flanagan and Ashmeetha Manilall
- Subjects
0301 basic medicine ,G protein-coupled receptor kinase ,lcsh:RC648-665 ,receptor activation ,Endocrinology, Diabetes and Metabolism ,Receptor expression ,ligand binding ,Gonadotropin-releasing hormone ,Review ,gonadotropin-releasing hormone receptor ,Biology ,Cytoplasmic receptor ,lcsh:Diseases of the endocrine glands. Clinical endocrinology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Endocrinology ,Biochemistry ,Enzyme-linked receptor ,receptor structure ,5-HT5A receptor ,G protein-coupled receptor ,Gonadotropin-releasing hormone receptor ,hormones, hormone substitutes, and hormone antagonists - Abstract
Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on "conformationally constrained" GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function.
- Published
- 2017
43. Modulation of the expression of sphingosine 1-phosphate 2 receptors regulates the differentiation of pre-adipocytes
- Author
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Jae Kyo Jeong, Myung Hee Moon, and Sang-Youel Park
- Subjects
Cancer Research ,Gene Expression ,Peroxisome proliferator-activated receptor ,Biology ,Biochemistry ,Mice ,chemistry.chemical_compound ,Downregulation and upregulation ,Sphingosine ,3T3-L1 Cells ,Adipocytes ,Genetics ,Enzyme-linked receptor ,Animals ,Sphingosine-1-phosphate ,Receptor ,Molecular Biology ,chemistry.chemical_classification ,Adipogenesis ,Lipid Metabolism ,PPAR gamma ,Receptors, Lysosphingolipid ,Gene Expression Regulation ,Oncology ,chemistry ,CCAAT-Enhancer-Binding Proteins ,Cancer research ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,Adiponectin ,Lysophospholipids ,Signal transduction ,Signal Transduction - Abstract
Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator that regulates multiple signals through S1P receptors responsible for biological responses. In particular, the S1P2 receptor has distinct roles in the S1P‑mediated differentiation of certain cell types. The present study was the first, to the best of our knowledge, to report the role of the S1P2 receptor in the adipocyte differentiation of 3T3‑L1 pre‑adipocytes. In order to investigate the influence of S1P2 receptors in the anti‑adipogenic effects of S1P, S1P2 receptor silencing and overexpression of were used. S1P2 overexpression with adenoviral vectors inhibited adipogenesis and inhibited the expression of peroxisome proliferator‑activated receptor γ (PPARγ), adiponectin and CCAAT/enhancer binding protein‑α, which were upregulated following incubation in differentiation media. Furthermore, S1P completely lost its ability to impair adipogenic differentiation following silencing of S1P2. Silencing of the S1P2 receptor additionally blocked the downregulation of PPARγ protein and phospho‑c‑Jun N‑terminal kinase protein induced by S1P treatment. In conclusion, the present study demonstrated that the S1P2 receptor is a key signaling molecule in the S1P‑dependent inhibition of adipogenic differentiation and additionally suggested that selective targeting of S1P2 receptors may have clinical applications for the treatment of obesity.
- Published
- 2015
44. Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)
- Author
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Kerry Barkan, David J. Roberts, Gary B. Willars, Naichang Li, Jing Lu, Timothy M. Skerry, Simon J. Dowell, Christopher A. Reynolds, Meenakshi Pardamwar, Graham Ladds, Gareth O. Richards, David R. Poyner, and Cathryn Weston
- Subjects
endocrine system ,G protein-coupled receptor (GPCR) ,Pharmacology ,Biology ,Ligands ,Receptor Activity-Modifying Protein 2 ,Biochemistry ,Glucagon-Like Peptide-1 Receptor ,Enzyme-linked receptor ,Humans ,5-HT5A receptor ,signal bias ,Molecular Biology ,Coagulation factor II receptor ,Glucagon-like peptide 1 receptor ,Receptor activity-modifying protein ,digestive, oral, and skin physiology ,glucagon receptor ,Cell Biology ,Glucagon ,receptor activity-modifying proteins (RAMPs) ,3. Good health ,HEK293 Cells ,glucagon-like peptide-1 ,Estrogen-related receptor gamma ,type 2 diabetes ,pharmacology ,Glucagon receptor family ,Glucagon receptor ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
Background: The glucagon and glucagon-like peptide-1 (GLP-1) receptors are important targets for treating type 2 diabetes. Results: We describe novel glucagon receptor pharmacology, through interaction with the receptor activity-modifying protein-2 (RAMP2). Conclusion: RAMP2 regulates both ligand binding and G protein selectivity of the glucagon receptor. Significance: The effect of RAMP2 should be considered when designing anti-diabetic treatments., The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.
- Published
- 2015
45. Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2
- Author
-
Torsten Schöneberg, Lilian M. Demberg, Ines Liebscher, and Sven Rothemund
- Subjects
Male ,Agonist ,G protein ,medicine.drug_class ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Biophysics ,Biology ,Biochemistry ,Receptors, G-Protein-Coupled ,Mice ,Chlorocebus aethiops ,medicine ,Enzyme-linked receptor ,Animals ,5-HT5A receptor ,Amino Acid Sequence ,Receptor ,Molecular Biology ,G protein-coupled receptor ,Epididymis ,Orphan receptor ,Sequence Homology, Amino Acid ,Cell Biology ,Fertility ,COS Cells ,Peptides ,Oligopeptides ,Endogenous agonist ,Signal Transduction - Abstract
The epididymis-specific adhesion G protein-coupled receptor (aGPCR) GPR64/ADGRG2 has been shown to be a key-player in the male reproductive system. As its disruption leads to infertility, GPR64 has drawn attention as potential target for male fertility control or improvement. Like the majority of aGPCRs GPR64 is an orphan receptor regarding its endogenous agonist and signal transduction. In this study we examined the G protein-coupling abilities of GPR64 and showed that it is activated through a tethered agonist sequence, which we have previously identified as the Stachel sequence. Synthetic peptides derived from the Stachel region can activate the receptor, opening for the first time the possibility to externally manipulate the receptor activity.
- Published
- 2015
46. Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release
- Author
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Ling Hong Zhang, Xiao Cui Yuan, Rui Zhou, Xian Fang Meng, Fang Gao, Tang Feng Su, Jing Shi, Bo Tian, Man Li, Miao Peng, Lin Li, Ning Sun, Hui Lin Pan, and Cai Hua Wu
- Subjects
Male ,0301 basic medicine ,MAPK/ERK pathway ,Pro-Opiomelanocortin ,Cannabinoid receptor ,medicine.medical_treatment ,Neuroscience (miscellaneous) ,Pharmacology ,Rats, Sprague-Dawley ,Receptor, Cannabinoid, CB2 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,medicine ,Cannabinoid receptor type 2 ,Enzyme-linked receptor ,Animals ,Humans ,Glucagon-like peptide 1 receptor ,Analgesics ,Cannabinoids ,Chemistry ,beta-Endorphin ,Nociceptors ,Heterotrimeric GTP-Binding Proteins ,030104 developmental biology ,Neurology ,Type C Phospholipases ,Interleukin-21 receptor ,Calcium ,lipids (amino acids, peptides, and proteins) ,Cannabinoid ,Mitogen-Activated Protein Kinases ,Signal transduction ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists.
- Published
- 2015
47. The atypical antipsychotics clozapine and olanzapine promote down‐regulation and display functional selectivity at human 5‐<scp>HT</scp>7receptors
- Author
-
C H Brevik, Andrea Hembre Ulsund, Kurt A. Krobert, Kjetil Wessel Andressen, Finn Olav Levy, Peter Vanhoenacker, and Ornella Manfra
- Subjects
Agonist ,Serotonin ,Drug Inverse Agonism ,Arrestins ,medicine.drug_class ,media_common.quotation_subject ,Down-Regulation ,Pharmacology ,Biology ,Ligands ,Benzodiazepines ,Radioligand Assay ,Phenols ,medicine ,Functional selectivity ,Enzyme-linked receptor ,Humans ,Inverse agonist ,Ergolines ,Receptor ,Internalization ,Clozapine ,Cells, Cultured ,beta-Arrestins ,media_common ,G protein-coupled receptor ,Sulfonamides ,Chloroquine ,Research Papers ,Serotonin Receptor Agonists ,HEK293 Cells ,Olanzapine ,Receptors, Serotonin ,Serotonin Antagonists ,Signal transduction ,Lysosomes ,Antipsychotic Agents ,Signal Transduction - Abstract
Background and Purpose Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs-coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors. Experimental Approach HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β-arrestins and lysosomal trafficking were measured. Key Results The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of β-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants. Conclusions and Implications Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor.
- Published
- 2015
48. Development and characterization of monoclonal antibodies against Protease Activated Receptor 4 (PAR4)
- Author
-
Maria de la Fuente, Michele M. Mumaw, Marvin T. Nieman, Amal Arachiche, and James K. Wahl
- Subjects
Blood Platelets ,B-cell receptor ,Ligands ,Article ,Epitopes ,Mice ,Enzyme-linked receptor ,Animals ,Humans ,5-HT5A receptor ,Protease-activated receptor ,Protease-activated receptor 2 ,G protein-coupled receptor ,Chemistry ,Thrombin ,Antibodies, Monoclonal ,Hematology ,Protein Structure, Tertiary ,Mice, Inbred C57BL ,HEK293 Cells ,Biochemistry ,Interleukin-21 receptor ,ROR1 ,Receptors, Thrombin ,Epitope Mapping ,Signal Transduction - Abstract
Protease activated receptor 4 (PAR4) is a G protein coupled receptor (GPCR) which is activated by proteolytic cleavage of its N-terminal exodomain. This generates a tethered ligand that activates the receptor and triggers downstream signaling events. With the current focus in the development of anti-platelet therapies shifted towards PARs, new reagents are needed for expanding the field's knowledge on PAR4. Currently, there are no PAR4 reagents which are able to detect activation of the receptor.Monoclonal PAR4 antibodies were purified from hybridomas producing antibody that were generated by fusing splenocytes with NS-1 cells. Immunoblotting, immunofluorescence, and flow cytometry were utilized to detect the epitope for each antibody and to evaluate the interaction of the antibodies with cells.Here, we report the successful generation of three monoclonal antibodies to the N-terminal extracellular domain of PAR4: 14H6, 5F10, and 2D6. We mapped the epitope on PAR4 of 14H6, 5F10, and 2D6 antibodies to residues (48-53), (41-47), and (73-78), respectively. Two of the antibodies (14H6 and 5F10) interacted close to the thrombin cleavage and were sensitive to α-thrombin cleavage of PAR4. In addition, 5F10 was able to partially inhibit the cleavage of PAR4 expressed in HEK293 cells by α-thrombin.These new antibodies provide a means to monitor endogenous PAR4 expression and activation by proteases on cells.
- Published
- 2015
49. Molecular-Interaction and Signaling Profiles of AM3677, a Novel Covalent Agonist Selective for the Cannabinoid 1 Receptor
- Author
-
Nikolai Zvonok, James Wager-Miller, Ken Mackie, Alexandros Makriyannis, Patricia H. Reggio, Dow P. Hurst, Edward L. Stahl, Suma Yaddanapudi, Lei Zhou, Laura M. Bohn, David R. Janero, Vidyanand G. Shukla, and Kumar V. Subramanian
- Subjects
Agonist ,Physiology ,medicine.drug_class ,Cognitive Neuroscience ,Drug Evaluation, Preclinical ,Arachidonic Acids ,Transfection ,Hippocampus ,Biochemistry ,Article ,Mice ,Radioligand Assay ,Receptor, Cannabinoid, CB1 ,Isothiocyanates ,Cyclic AMP ,medicine ,Enzyme-linked receptor ,Animals ,Humans ,5-HT5A receptor ,Receptor ,G protein-coupled receptor ,Cannabinoid Receptor Agonists ,Dose-Response Relationship, Drug ,Molecular Structure ,Drug discovery ,Chemistry ,Cell Membrane ,Colforsin ,Hydrogen Bonding ,Cell Biology ,General Medicine ,Endocannabinoid system ,Endocytosis ,Molecular Docking Simulation ,HEK293 Cells ,Mutation ,Biophysics - Abstract
The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.
- Published
- 2015
50. Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex
- Author
-
Sukru Sadik Oner, Joe B. Blumer, Stephen M. Lanier, and William G. Robichaux
- Subjects
Bioluminescence Resonance Energy Transfer Techniques ,Models, Molecular ,Yellow fluorescent protein ,Agonist ,Protein Conformation ,G protein ,medicine.drug_class ,GTP-Binding Protein alpha Subunits, Gi-Go ,Pertussis toxin ,Receptors, G-Protein-Coupled ,GTP-Binding Proteins ,medicine ,Enzyme-linked receptor ,Animals ,Humans ,5-HT5A receptor ,Receptor ,Pharmacology ,biology ,Transmembrane protein ,Rats ,Cell biology ,Molecular Docking Simulation ,HEK293 Cells ,Pertussis Toxin ,Biochemistry ,Accelerated Communications ,Mutation ,biology.protein ,Molecular Medicine ,GTP-Binding Protein alpha Subunit, Gi2 - Abstract
Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gβγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαβγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oβγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.
- Published
- 2015
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