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.
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.
Lorenzen
Anna
A
Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. anna.lorenzen@urz.uni-heidelberg.de
Beukers
Margot W
MW
van der Graaf
Piet Hein
PH
Lang
Heidrun
H
van Muijlwijk-Koezen
Jacqueline
J
de Groote
Miriam
M
Menge
Wiro
W
Schwabe
Ulrich
U
IJzerman
Adriaan P
AP
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
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