Roles of GABAB receptor subtypes in presynaptic auto- and heteroreceptor function regulating GABA and glutamate release

Γ-Aminobutyric acid B (GABAB) receptors are heterodimers composed of two subunits GABAB(1) and GABAB(2), the former existing in two isoforms GABAB(1a) and GABAB(1b). The contributions of individual receptor subunits and isoforms to GABAB auto- and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABAB autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABAB heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [3H]GABA release in slices from GABAB(1)−/− mice. The amount of [3H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABAB(1)−/− and GABAB(2)−/− demonstrating a loss of GABAB autoreceptor function in these knockout animals. The GABAB receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABAB(2)−/− mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABAB(1) isoforms expressed in brain, GABAB(1a) and GABAB(1b). In cortical, hippocampal and striatal slices from both GABAB(1a)−/− and GABAB(1b)−/− mice, the frequency dependence of [3H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABAB autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABAB(1a)−/−, but essentially unaltered in GABAB(1b)−/− mice. Our data suggest that functional GABAB heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABAB(1a) and GABAB(2) subunit

Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses

The nuclear hormone receptor retinoic acid receptor-related-orphan-receptor-gamma t (RORγt) is the key transcription factor required for Th17 cell differentiation and for production of IL-17 family cytokines by innate and adaptive immune cells. Dysregulated Th17 immune responses have been associated with the pathogenesis of several inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. In this article, we describe the in vitro pharmacology of a potent and selective low molecular weight RORγt inhibitor identified after a structure-based hit-to-lead optimization effort. The compound interfered with co-activator binding to the RORγt ligand binding domain and impaired the transcriptional activity of RORγt as evidenced by blocked IL-17A secretion and RORE-mediated transactivation of a luciferase reporter gene. The inhibitor effectively reduced IL-17A production by human naive and memory T-cells and attenuated transcription of pro-inflammatory Th17 signature genes, such as IL17F, IL22, IL26, IL23R, and CCR6. The compound selectively suppressed the Th17/IL-17 pathway and did not interfere with polarization of other T helper cell lineages. Furthermore, the inhibitor was selective for RORγt and did not modify the transcriptional activity of the closely related family members RORα and RORβ. Using human keratinocytes cultured with supernatants from compound treated Th17 cells we showed that pharmacological inhibition of RORγt translated to suppressed IL-17-regulated gene expression in keratinocyte cell cultures. Furthermore, in ex vivo immersion skin cultures our RORγt inhibitor suppressed IL-17A production by Th17-skewed skin resident cells which correlated with reduced human β defensin 2 expression in the skin. Our data suggests that inhibiting RORγt transcriptional activity by a low molecular weight inhibitor may hold utility for the treatment of Th17/IL-17-mediated skin pathologies

GABAB receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Functional GABAB receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on GABAB receptors [11, 72]) are formed from the heterodimerization of two similar 7TM subunits termed GABAB1 and GABAB2 [11, 71, 28, 72, 85]. GABAB receptors are widespread in the CNS and regulate both pre- and postsynaptic activity. The GABAB1 subunit, when expressed alone, binds both antagonists and agonists, but the affinity of the latter is generally 10-100-fold less than for the native receptor. Co-expression of GABAB1 and GABAB2 subunits allows transport of GABAB1 to the cell surface and generates a functional receptor that can couple to signal transduction pathways such as high-voltage-activated Ca2+ channels (Cav2.1, Cav2.2), or inwardly rectifying potassium channels (Kir3) [12, 11, 5]. The GABAB1 subunit harbours the GABA (orthosteric)-binding site within an extracellular domain (ECD) venus flytrap module (VTM), whereas the GABAB2 subunit mediates G protein-coupled signalling [11, 71, 40, 39]. The two subunits interact by direct allosteric coupling [63], such that GABAB2 increases the affinity of GABAB1 for agonists and reciprocally GABAB1 facilitates the coupling of GABAB2 to G proteins [71, 54, 39]. GABAB1 and GABAB2 subunits assemble in a 1:1 stoichiometry by means of a coiled-coil interaction between α-helices within their carboxy-termini that masks an endoplasmic reticulum retention motif (RXRR) within the GABAB1 subunit but other domains of the proteins also contribute to their heteromerization [5, 71, 15]. Recent evidence indicates that higher order assemblies of GABAB receptor comprising dimers of heterodimers occur in recombinant expression systems and in vivo and that such complexes exhibit negative functional cooperativity between heterodimers [70, 22]. Adding further complexity, KCTD (potassium channel tetramerization proteins) 8, 12, 12b and 16 associate as tetramers with the carboxy terminus of the GABAB2 subunit to impart altered signalling kinetics and agonist potency to the receptor complex [84, 3, 79] and are reviewed by [73]. The molecular complexity of GABAB receptors is further increased through association with trafficking and effector proteins [Schwenk et al., 2016, Nature Neuroscience 19(2): 233-42] and reviewed by [69]. Four isoforms of the human GABAB1 subunit have been cloned. The predominant GABAB1a and GABAB1b isoforms, which are most prevalent in neonatal and adult brain tissue respectively, differ in their ECD sequences as a result of the use of alternative transcription initiation sites. GABAB1a-containing heterodimers localise to distal axons and mediate inhibition of glutamate release in the CA3-CA1 terminals, and GABA release onto the layer 5 pyramidal neurons, whereas GABAB1b-containing receptors occur within dendritic spines and mediate slow postsynaptic inhibition [75, 89]. Only the 1a and 1b variants are identified as components of native receptors [11]. Additional GABAB1 subunit isoforms have been described in rodents and humans [55] and reviewed by [5]

GABAB receptors in GtoPdb v.2021.2

Functional GABAB receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on GABAB receptors [11, 71]) are formed from the heterodimerization of two similar 7TM subunits termed GABAB1 and GABAB2 [11, 70, 28, 71, 87]. GABAB receptors are widespread in the CNS and regulate both pre- and postsynaptic activity. The GABAB1 subunit, when expressed alone, binds both antagonists and agonists, but the affinity of the latter is generally 10-100-fold less than for the native receptor. Co-expression of GABAB1 and GABAB2 subunits allows transport of GABAB1 to the cell surface and generates a functional receptor that can couple to signal transduction pathways such as high-voltage-activated Ca2+ channels (Cav2.1, Cav2.2), or inwardly rectifying potassium channels (Kir3) [12, 11, 5]. The GABAB1 subunit harbours the GABA (orthosteric)-binding site within an extracellular domain (ECD) venus flytrap module (VTM), whereas the GABAB2 subunit mediates G protein-coupled signalling [11, 70, 40, 39]. The cryo-electron microscopy structures of the human full-length GABAB1-GABAB2 heterodimer have been solved in the inactive apo state, two intermediate agonist-bound forms and an active state in which the heterodimer is bound to an agonist and a positive allosteric modulator [81]. The positive allosteric modulator binds to the transmembrane dimerization interface and stabilizes the active state. Recent evidence indicates that higher order assemblies of GABAB receptor comprising dimers of heterodimers occur in recombinant expression systems and in vivo and that such complexes exhibit negative functional cooperativity between heterodimers [69, 22]. Adding further complexity, KCTD (potassium channel tetramerization proteins) 8, 12, 12b and 16 associate as tetramers with the carboxy terminus of the GABAB2 subunit to impart altered signalling kinetics and agonist potency to the receptor complex [86, 3, 79] and are reviewed by [72]. The molecular complexity of GABAB receptors is further increased through association with trafficking and effector proteins [80] and reviewed by [68]. The predominant GABAB1a and GABAB1b isoforms, which are most prevalent in neonatal and adult brain tissue respectively, differ in their ECD sequences as a result of the use of alternative transcription initiation sites. GABAB1a-containing heterodimers localise to distal axons and mediate inhibition of glutamate release in the CA3-CA1 terminals, and GABA release onto the layer 5 pyramidal neurons, whereas GABAB1b-containing receptors occur within dendritic spines and mediate slow postsynaptic inhibition [74, 91]. Amyloid precursor protein (APP) and soluble APP (sAPP) bind to the N- terminal sushi domain of the GABAB1a isoform to regulate axonal trafficking of GABAB receptors and release of neurotransmitters [76]

Stochastic models and dynamic measures for the characterization of bistable circuits

During the last few years, a great deal of interest has risen concerning the applications of stochastic methods to several biochemical and biological phenomena. Phenomena like gene expression, cellular memory, bet-hedging strategy in bacterial growth and many others, cannot be described by continuous stochastic models due to their intrinsic discreteness and randomness. In this thesis I have used the Chemical Master Equation (CME) technique to modelize some feedback cycles and analyzing their properties, including experimental data. In the first part of this work, the effect of stochastic stability is discussed on a toy model of the genetic switch that triggers the cellular division, which malfunctioning is known to be one of the hallmarks of cancer. The second system I have worked on is the so-called futile cycle, a closed cycle of two enzymatic reactions that adds and removes a chemical compound, called phosphate group, to a specific substrate. I have thus investigated how adding noise to the enzyme (that is usually in the order of few hundred molecules) modifies the probability of observing a specific number of phosphorylated substrate molecules, and confirmed theoretical predictions with numerical simulations. In the third part the results of the study of a chain of multiple phosphorylation-dephosphorylation cycles will be presented. We will discuss an approximation method for the exact solution in the bidimensional case and the relationship that this method has with the thermodynamic properties of the system, which is an open system far from equilibrium.In the last section the agreement between the theoretical prediction of the total protein quantity in a mouse cells population and the observed quantity will be shown, measured via fluorescence microscopy

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

THE CONCISE GUIDE TO PHARMACOLOGY 2019/20 : G protein- coupled receptors

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14748. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.Peer reviewe

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate