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  • Title: Agonist-induced changes in the properties of beta-adrenergic receptors on intact S49 lymphoma cells. Time-dependent changes in the affinity of the receptor for agonists.
    Author: Hoyer D, Reynolds EE, Molinoff PB.
    Journal: Mol Pharmacol; 1984 Mar; 25(2):209-18. PubMed ID: 6142414.
    Abstract:
    The binding of the antagonist [125I]iodopindolol to beta-adrenergic receptors on intact wild-type S49 mouse lymphoma cells and mutants that have impaired abilities to generate cyclic AMP in response to catecholamines was studied. The binding of [125I]iodopindolol is of high affinity (KD = 35 pM), rapid, stable over 90 min, and rapidly reversible (t1/2 = 8 to 11 min). Nonspecific binding was very low (less than 5% of total binding at the KD). Kinetic and competition experiments performed under steady-state and non-equilibrium conditions revealed that the binding characteristics for agonists were very different in intact cells and in membranes. The interactions of antagonists, on the other hand, appeared to be identical in studies carried out with intact cells and membranes. In intact cells, the affinity of the receptor for agonists was observed to decrease rapidly within the first 5 min of exposure of the cells to an agonist. Competition experiments revealed that at least 80% of the receptor-agonist complex was in a high-affinity state when studies were carried out using short incubation times (0.5-1 min). Under equilibrium conditions, about 80% of the complex in wild-type, uncoupled, and kinase-deficient cells was of a low affinity. At equilibrium, only low-affinity binding was seen with coupling protein-deficient cells. This rapid, time-dependent decrease in the affinity of receptors for agonists was seen with most agonists although not with zinterol. The phenomenon was not due to differences in the kinetics of the interactions of agonists and [125I]iodopindolol with the receptor, and it is likely that the receptor undergoes a conformational change upon exposure to agonists. This effect was not observed in membranes and was not related to the presence of a functional guanine nucleotide-binding protein or to the production of cyclic AMP. Furthermore, hydrophilic agonists and antagonists, under short-term incubation conditions, did not fully compete for the binding sites labeled with the lipophilic radioligand [125I]iodopindolol, although this binding was fully and stereospecifically competed for by lipophilic antagonists. This suggests that in untreated cells a small but significant fraction of the receptors is sequestered in an environment not accessible to hydrophilic ligands.
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