guanylyl-imidodiphosphate and hydroxybenzylisoproterenol

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Anura; Cell Membrane; Cells, Cultured; Colforsin; Dihydroalprenolol; Epinephrine; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; Iodocyanopindolol; Isoproterenol; Pindolol; Receptors, Adrenergic, beta; Sodium Fluoride; Yohimbine

P815, a murine mastocytoma cell line, possesses beta-adrenergic binding sites as assessed by using [3H]dihydroalprenolol (antagonist) and [3H]hydroxybenzylisoproterenol (agonist). The number of binding sites per cell was 29 000 for the agonist and 75 000 for the antagonist, as determined by direct binding assays and inhibition experiments on intact cells. On membrane preparations from the same cells, binding of alprenolol was only displaceable by antagonists, while stereospecific binding of hydroxybenzylisoproterenol was only displaceable by agonists. The P815 membranes also possessed an adenylate cyclase stimulated by Gpp(NH)p and NaF but not by 1-isoproterenol. The intracellular cAMP level of intact cells was not modulated by 1-isoproterenol or by 1-epinephrine, but was increased by forskolin. These results suggest that the beta-adrenergic receptor of P815 mastocytoma cells is non-functional. This may explain the failure of agonists to stimulate adenylate cyclase activity in these cells.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Colforsin; Cyclic AMP; Dihydroalprenolol; Diterpenes; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Neoplasm Proteins; Receptors, Adrenergic, beta; Sarcoma, Experimental; Sodium Fluoride

We report the development and application of a novel assay for high affinity binding of the agonist [3H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [32P]GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min-1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 microM and 0.1 microM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [32P]GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [3H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. 3H-agonist binding and agonist-promoted [32P]GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.

Topics: Adenylyl Cyclases; Animals; Anura; Dose-Response Relationship, Drug; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Guanine Nucleotides; Guanosine Diphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Receptors, Adrenergic; Receptors, Adrenergic, beta

Topics: Adrenergic beta-Agonists; Animals; Binding, Competitive; Dihydroalprenolol; Erythrocyte Membrane; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Adrenergic; Receptors, Adrenergic, beta; Reticulocytes; Stereoisomerism; Temperature

Recent evidence suggests that the molecular interactions of agonists with beta-adrenergic receptors differ from those of antagonists. Most of this evidence has come from studies of agonist inhibition of radiolabeled antagonist binding. We have examined agonist binding directly in rat lung membranes using radiolabeled hydroxybenzylisoproterenol (3H-HBI). Specific binding of 3H-HBI was stereoselective and was inhibited by catecholamines with a potency order characteristic of beta 2-adrenergic receptors. Gpp(NH)p increased the rates of association and dissociation of 3H-HBI from the receptor. In the absence of Gpp(NH)p, Scatchard plots were curvilinear suggesting a complex interaction of the agonist with the receptor. The total number of 3H-HBI binding sites was similar to that of 125I-IHYP binding sites. In the presence of increasing concentrations of Gpp(NH)p, the affinity of 3H-HBI was decreased and Scatchard plots became linear. Sodium chloride mimicked the effect of Gpp(NH)p in lowering the affinity of the receptor for 3H-HBI. Magnesium chloride had the opposite effect in that it promoted high affinity binding. The effect of sodium chloride was largely overcome by the presence of magnesium chloride.

Topics: Adrenergic beta-Agonists; Animals; Catecholamines; Cell Membrane; Erythrocyte Membrane; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lung; Male; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta; Reticulocytes