title: Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors.
abstract: Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.
authors.affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, U.K. GBCA32@UDCF.GLA.AC.UK
authors.forename: G
authors.initials: G
authors.lastname: Milligan
authors.forename: D J
authors.initials: DJ
authors.lastname: MacEwan
authors.forename: M
authors.initials: M
authors.lastname: Mercouris
authors.forename: I
authors.initials: I
authors.lastname: Mullaney
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000378:metabolism*; D000818:Animals; D015784:Betaxolol / Q000378:metabolism*; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D051379:Mice; D017465:Receptors, Opioid, delta / Q000037:antagonists & inhibitors* / Q000235:genetics; D013015:Sotalol / Q000378:metabolism*
chemical_list: D000319:Adrenergic beta-Antagonists; D017465:Receptors, Opioid, delta; D004743:Enkephalin, Leucine; C040643:N,N-diallyl-tyrosyl-alpha-aminoisobutyric acid-phenylalanyl-leucine; D013015:Sotalol; D015784:Betaxolol