Shaun Conway

Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-gamma and RelA.

The human gut microflora is important in regulating host inflammatory responses and in maintaining immune homeostasis. The cellular and molecular bases of these actions are unknown. Here we describe a unique anti-inflammatory mechanism, activated by nonpathogenic bacteria, that selectively antagonizes transcription factor NF-κB. Bacteroides thetaiotaomicron targets transcriptionally active NF-κB subunit RelA, enhancing its nuclear export through a mechanism independent of nuclear export receptor Crm-1. Peroxisome proliferator activated receptor-γ (PPAR-γ), in complex with nuclear RelA, also undergoes nucleocytoplasmic redistribution in response to B. thetaiotaomicron. A decrease in PPAR-γ abolishes both the nuclear export of RelA and the anti-inflammatory activity of B. thetaiotaomicron. This PPAR-γ-dependent anti-inflammatory mechanism defines new cellular targets for therapeutic drug design and interventions for the treatment of chronic inflammation.

Digging deep--structure-function relationships in the melatonin receptor family.

Abstract: The melatonin receptor family is a small group of receptors within the G protein‐coupled receptor (GPCR) superfamily. The group comprises of three subtypes which bind melatonin and one member, the melatonin related receptor (MRR), that shares >40% sequence identity with the other melatonin receptors but does not bind melatonin. Identification of two subtypes expressed in the mouse suprachiasmatic nucleus, one of which (MT1) inhibits neuronal firing and the other (MT2) mediating the phase advancing properties of melatonin has given renewed interest to the development of subtype specific compounds for each of the mammalian melatonin receptors. Towards this goal site‐directed and chimaeric receptor mutagenesis studies have been performed which have provided some insight into the structure–function relationships of the melatonin receptors. Furthermore, these studies may lead to the identification of the ligand for the orphan MRR.

Cloning and functional analysis of a polymorphic variant of the ovine Mel 1a melatonin receptor.

We have isolated a novel variant of the Mel 1a melatonin receptor from an ovine PT cDNA library. Relative to the reported sequence for the Mel 1a melatonin receptor there are 8 changes in the DNA sequence. Only 3 of these result in amino acid substitutions, one in extracellular loop 3 and two in the carboxy-terminal tail. We have designated the novel variant of the sheep Mel 1a receptor Mel 1a(beta), and correspondingly the previously reported variant Mel 1a(alpha). As minor changes in the primary amino acid sequence of G-protein-coupled receptors can influence their functional characteristics we have accordingly characterized this novel variant of the Mel 1a melatonin receptor. This melatonin receptor displays high affinity binding and inhibits the cAMP second messenger pathway in transfected L-cells demonstrating that this receptor is fully functional. PCR analysis shows Mel 1a(beta) is present in several breeds of sheep and suggests that the Mel 1a(beta) receptor was established early in the evolution of the sheep species.

The Ovine Melatonin-Related Receptor: Cloning and Preliminary Distribution and Binding Studies

A melatonin‐related receptor was cloned from an ovine genomic library. The sequenced gene has a similar structure to that of the melatonin receptor gene family and consists of two exons separated by an intron of approximately 3 kb. Exon 1 and exon 2 of the ovine melatonin‐related receptor encode a protein of 575 amino acids which is 73.8% homologous to the human melatonin‐related receptor and shows 40.9% homology with the ovine Mel1a melatonin receptor. COS‐7 cells transiently expressing ovine melatonin‐related receptors did not bind 2‐[125I]iodomelatonin or 3H‐melatonin. Reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization studies revealed expression of the ovine melatonin‐related receptor in the hypothalamus, pituitary, retina and retinal pigment epithelium. Furthermore, expression of the ovine melatonin‐related receptor is shown to be coincident with Mel1a and 2‐[125I]iodomelatonin binding in the pituitary and serotonin N‐acetyl transferase (arylalkylamine N‐acetyl transferase, AANAT) expression in the retina. Expression patterns and similarity with the melatonin receptor gene family suggest a role for this novel G protein‐coupled receptor in control and regulation of endocrine function and retinal physiology.

Characterisation of human melatonin mt1 and MT2 receptors by CRE-luciferase reporter assay

A cyclic AMP response element (CRE)-luciferase reporter gene assay was used to characterise the functional responses of human melatonin mt(1) and human melatonin MT(2) receptors, stably expressed in the human embryonic kidney cell line HEK293, to a series of six naphthalenic analogues of melatonin. By comparison to the observed melatonin-mediated inhibition of stimulated luciferase levels the naphthalenic series was identified as comprising agonists, partial agonists and one antagonist of melatonin mt(1) and melatonin MT(2) receptor function. Three of the agonist/partial agonist members of this series were also identified as displaying a functional selectivity for the melatonin MT(2) receptor. Competitive displacement of 2-[125I]iodomelatonin binding to the ovine pars tuberalis melatonin ML(1) receptor demonstrated a close correlation to the observed functional luciferase responses of the human melatonin mt(1) receptor. We conclude that the CRE-luciferase reporter gene assay provides an effective functional screening method for the pharmacological characterisation of human melatonin receptor subtypes.

Chimeric Melatonin mt1 and Melatonin-Related Receptors: Identification of Domains and Residues Participating in Ligand Binding and Receptor Activation of the Melatonin mt1 Receptor

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt1 receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[125I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[125I]iodomelatonin binding. The subsequent testing of a “reverse” chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt1receptor sequences identified specific 2-[125I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt1 receptor. This identified Gly258 (Gly6.55) as a critical residue required for high affinity ligand binding and receptor function.

Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein‐coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels

Binding assays using 2‐[125I]iodomelatonin revealed high‐affinity, guanosine 5′‐O‐(3‐thiotriphosphate) sensitive, melatonin binding sites (B max 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT‐PCR techniques identified these sites as human Mel1a melatonin receptors. Challenge of HEK293 cells with 1 μM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over‐express the human Mel1a melatonin receptor (B max>400 fmol/mg protein) melatonin dose‐dependently inhibited stimulated cyclic AMP levels (IC50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein‐coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.

Differential coupling of the extreme C-terminus of G protein α subunits to the G protein-coupled melatonin receptors

Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Galphas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Galpha subunits. The Galphas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT(1) or MT(2) melatonin receptors with Galphas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Galpha C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Galpha chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Galphas chimaeras coupled. Recognition of the C-terminal five amino acids of the Galpha subunit is a requisite for coupling to a receptor, but it is not the sole determinant.