title: Modulation of agonist responses at the A(1) adenosine receptor by an irreversible antagonist, receptor-G protein uncoupling and by the G protein activation state.
abstract: Potency and intrinsic activity of agonists depend on ligand structure, but are also regulated by receptor-G protein stoichiometry. A potential functional reserve in adenosine A(1) receptor-mediated G protein activation was investigated by stimulation of guanosine-5'-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding by the full agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) and the partial agonist 5'-deoxy-5'-methylthioadenosine (MeSA). Pretreatment of rat brain membranes with the irreversible antagonist 1-propyl-3-[3-[[4-(fluorosulfonyl)benzoyl]oxy]-propyl]-8-cyclopentylxanthine revealed no classical receptor reserve for either agonist. The functional significance of the G protein coupling state of the receptor and occupancy of G proteins by guanine nucleotides was assessed after partial uncoupling of receptor-G protein complexes with N-ethylmaleimide and in the presence of increasing GDP concentrations. Agonist EC(50) values in G protein activation were increased after NEM pretreatment and at higher GDP concentrations, and a decrease in the relative intrinsic activity of MeSA was observed. The shift of agonist concentration-response curves to the right, the decrease in maximal effects and the decrease in relative intrinsic activity of the partial agonist point to a functional reserve which has to be attributed to GDP-free receptor-G protein complexes. The mechanisms of action of FSCPX, NEM and GDP were fully consistent with the two-state model of receptor activation. The apparent reserve revealed by GDP reflects a shift from spontaneously active GDP-free receptor-G protein complexes (RG)(*), which can bind [35S]GTPgammaS, to (RG) occupied by GDP. The abundance of (RG)(*) is favored by agonists and by the absence of GDP.
authors.affiliation: Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. anna.lorenzen@urz.uni-heidelberg.de
authors.forename: Anna
authors.initials: A
authors.lastname: Lorenzen
authors.forename: Margot W
authors.initials: MW
authors.lastname: Beukers
authors.forename: Piet Hein
authors.initials: PH
authors.lastname: van der Graaf
authors.forename: Heidrun
authors.initials: H
authors.lastname: Lang
authors.forename: Jacqueline
authors.initials: J
authors.lastname: van Muijlwijk-Koezen
authors.forename: Miriam
authors.initials: M
authors.lastname: de Groote
authors.forename: Wiro
authors.initials: W
authors.lastname: Menge
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwabe
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D008024:Ligands; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
chemical_list: D006150:Guanine Nucleotides; D008024:Ligands; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D013439:Sulfhydryl Reagents; D014970:Xanthines; C100640:8-cyclopentyl-3-(3-((4-(fluorosulfonyl)benzoyl)oxy)propyl)-1-propylxanthine; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide