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′-(γ-[ 35 S ]thio)-triphosphate ([ 35 S ]GTPγS) 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 [ 35 S ]GTPγS, to (RG) occupied by GDP. The abundance of (RG) ∗ is favored by agonists and by the absence of GDP.