Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells.

Using the ligands [125I]iodohydroxybenzylpindolol and [3H]prostaglandin E1 ([3H]PGE1), we have studied the relationship of receptors for beta-adrenergic agents and for PGE1 to adenylate cyclase in membranes of parental, hybrid, and variant mammalian cell lines. Fusion of parental clones responsive to beta-adrenergic agonists (beta+) with unresponsive clones (beta-) produced hybrid clones with a greatly diminished beta-adrenergic response; beta+ X beta leads to beta-. Binding studies with [125I]iodohydroxybenzylpindolol showed a decreased concentration of beta receptors in six such hybrid clones. Thus, paucity of beta-adrenergic receptors is probably a sufficient, albeit not necessarily complete, explanation for the decreased beta-adrenergic responsiveness of the hybrid clones. When a clone with beta receptor but without apparent adenylate cyclase activity (HC-1) was hybridized with a beta- clone that has adenylate cyclase (B82), a responsive hybrid clone was obtained. In nine cell hybrids produced by the fusion of clones responsive (PGE1+) and unresponsive (PGE1-) to PGE1, high affinity binding sites for [3H]PGE1 were expressed in the same manner as was PGE1-sensitive adenylate cyclase: PGE1+ X PGE1 leads to PGE1+. The chemical specificities and affinities of the parental receptors and responsive adenylate cyclases were faithfully reproduced in the hybrid clones. Activation by PGE1 was proportional to the occupation of the high affinity receptors. In a wild type lymphoma clone (24.3.2), the concentration dependences for binding of [3H]PGE1 and for activation of adenyalte cyclase by PGE1 were identical. In a variant lymphoma clone (94.15.1) lacking adenylate cyclase activity, no high affinity receptors for PGE1 were detected, whereas beta-adrenergic receptors have been demonstrated in this variant clone (Insel, P.A., Maguire, M.E., Gilman, A.G., Coffino, P., Bourne, H., and Melmon, K. (1976) Mol. Pharmacol. 12, 1062-1069). Hybrid cells formed by the fusion of 94.15.1 with cell line RAG (PGE1-) responded to PGE1. Clone 94.15.1 may have receptors for PGE1 of reduced affinity or in low concentration. Alternatively, RAG and 94.15.1 may have complementary genetic defects such that the RAG X 94.15.1 hybrid cells express a hormonally responsive receptor-adenylate cyclase system.     

Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation

Using site-directed mutagenesis of the human beta 2-adrenergic receptor and continuous expression in B-82 cells, the role of 3 conserved cysteines in transmembrane domains and 2 conserved cysteines in the third extracellular domain in receptor function was examined. Cysteine was replaced with serine in each mutant receptor as this amino acid is similar to cysteine in size but it cannot form disulfide linkages. Replacement of cysteine residues 77 and 327, in the second and seventh transmembrane-spanning domains, respectively, had no effect on ligand binding or the ability of the receptor to mediate isoproterenol stimulation of adenylate cyclase. Substitution of cysteine 285, in the sixth transmembrane domain of the receptor, produced a mutant receptor with normal ligand-binding properties but a significantly attenuated ability to mediate stimulation of adenylate cyclase. Mutation of cysteine residues 190 and 191, in the third extracellular loop of the beta 2 receptor, had qualitatively similar effects on ligand binding and isoproterenol-mediated stimulation of adenylate cyclase. Replacement of either of these residues with serine produced mutant receptors that displayed a marked loss in affinity for both beta-adrenergic agonists and antagonists. Replacement of both cysteine 190 and 191 with serine had an even greater effect on the ability of the receptor to bind ligands. Consistent with the loss of Ser190 and/or Ser191 mutant receptor affinity for agonists was a corresponding shift to the right in the dose-response curve for isoproterenol-induced increases in intracellular cyclic AMP concentrations in cells expressing the mutant receptors. These data implicate one of the conserved transmembrane cysteine residues in the human beta 2-adrenergic receptor in receptor activation by agonists and also suggest that conserved cysteine residues in an extracellular domain of the receptor may be involved in ligand binding.     

Functional integrity of desensitized beta-adrenergic receptors

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.     

Somatostatin induces translocation of the beta-adrenergic receptor kinase and desensitizes somatostatin receptors in S49 lymphoma cells

The beta-adrenergic receptor kinase is a cytosolic enzyme that specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta AR). Beta AR kinase appears to be translocated from the cytosol to the plasma membrane when kin- S49 lymphoma cells are incubated with either beta-adrenergic agonists or prostaglandin E1, both of which act through receptors which stimulate adenylate cyclase. We report here that brief (approximately 20 min) exposure of wild type S49 lymphoma cells to somatostatin (which inhibits adenylate cyclase) promotes the translocation of beta AR kinase to an extent comparable to that observed in the presence of the beta agonist isoproterenol or prostaglandin E1. Beta AR kinase activity can be measured using either beta AR or rhodopsin, the retinal receptor for light, as a substrate. The translocation process triggered by somatostatin is rapid, reversible, and is associated with somatostatin receptor desensitization. The latter is apparent as an attenuation of the inhibition by somatostatin of forskolin-stimulated adenylate cyclase activity in membranes of S49 cells preincubated in the presence of the peptide. These results strongly suggest that beta AR kinase is able to phosphorylate and desensitize both stimulatory and inhibitory adenylate cyclase-coupled receptors, thus emerging as a general kinase that regulates the function of different receptors in an agonist-specific fashion.     

Effect of prostaglandin E1 on the cyclic adenosine-3',5'--monophosphate system and radiosensitivity of B-82 cells cultured in vitro

The cyclic AMP (cAMP) system of mice fibroblasts L, clone B-82 has been studied. B-82 cells possess receptors to prostaglandin E1 (PGE1) and are deficient in beta-adrenoreceptors. It has been shown that 10 mM NaF, 1.10(-4) M GTP and 1.10(-5) M PGE1 stimulate adenylate cyclase of B-82 cells. PGE1 causes increase in the intracellular content of cAMP and protects B-82 cells against irradiation. Specifict beta-agonist isoproterenol fails to modulate adenylate cyclase activity and does not change cAMP content in B-82 cells. Isoproterenol has no effect on the B-82 cells radiosensitivity, although it is known to be a potent radioprotector. It is presumed that the stimulation of cAMP system by radioprotector is necessary for the performance of radioprotective effect.     

Agonist-dependent phosphorylation of the alpha 2-adrenergic receptor by the beta-adrenergic receptor kinase

Desensitization of the beta-adrenergic receptor, a receptor which is coupled to the stimulation of adenylate cyclase, may be regulated via phosphorylation by a unique protein kinase. This recently discovered enzyme, known as the beta-adrenergic receptor kinase, only phosphorylates the agonist-occupied form of the beta-adrenergic receptor. To assess whether receptors coupled to the inhibition of adenylate cyclase might also be substrates, we examined the effects of beta-adrenergic receptor kinase on the partially purified human platelet alpha 2-adrenergic receptor. Phosphorylation of the reconstituted alpha 2-adrenergic receptor was dependent on agonist occupancy and was completely blocked by coincubation with alpha 2-antagonists. The time course of phosphorylation of the alpha 2-adrenergic receptor was virtually identical to that observed with the beta-adrenergic receptor with maximum stoichiometries of 7-8 mol of phosphate/mol of receptor in each case. In contrast, the alpha 1-adrenergic receptor, which is coupled to stimulation of phosphatidylinositol hydrolysis, is not a substrate for the beta-adrenergic receptor kinase. These results suggest that receptors coupled to either stimulation or inhibition of adenylate cyclase may be regulated by an agonist-dependent phosphorylation mediated by the beta-adrenergic receptor kinase.     

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.     

Evidence for two types of beta-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to beta-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [3H]adenine labeling and was found to be coupled to a beta 1-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same medium with [3H]ATP was stimulated via beta 2-adrenergic receptors.     

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.     

Homologous desensitization of adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor

We recently demonstrated that heterologous desensitization of adenylate cyclase in turkey erythrocytes is highly correlated with phosphorylation of the beta-adrenergic receptor. In contrast, little is known of the biochemical mechanisms underlying the homologous form of beta-adrenergic receptor desensitization, which is agonist-specific and not cAMP-mediated. Accordingly, the present studies were undertaken to examine if phosphorylation of the beta-adrenergic receptor is also associated with this form of desensitization in a well studied model system, the frog erythrocyte. Preincubation of these cells with the beta-adrenergic agonist isoproterenol leads to a 45% decline in isoproterenol-stimulated adenylate cyclase activity without significant changes in basal, prostaglandin E1-, NaF-, guanyl-5'-yl-imidodiphosphate-, forskolin-, or MnCl2-stimulated enzyme activities. There is also a 48% decline in [125I]iodocyanopindolol membrane binding sites. Conversely, preincubation of the cells with prostaglandin E1 attenuates only the prostaglandin E1-stimulated enzyme activity and does not affect [125I]iodocyanopindolol binding. Phosphorylation of the beta-adrenergic receptor was assessed by preincubating the cells with 32Pi and desensitizing them, and subsequently purifying the receptors by affinity chromatography. Under basal conditions there is about 0.62 mol of phosphate/mol of receptor whereas after desensitization with isoproterenol this increases to 1.9 mol/mol. This isoproterenol-induced receptor phosphorylation exhibits stereospecificity and is blocked by the beta-adrenergic antagonist propranolol. In addition, preincubation with prostaglandin E1 does not promote beta-adrenergic receptor phosphorylation. These data suggest that receptor phosphorylation is involved in homologous as well as heterologous forms of desensitization and may provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.     

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.     

Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [3H]prostaglandin E1 to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E1, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E1 binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E1 and have reduced prostaglandin E1 binding Exposure of cells to prostaglandin E1 results both in decreased prostaglandin E1 responsiveness and reduced prostaglandin E1 binding. Activation of adenylate cyclase is correlated to binding of prostaglandin E1 to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes

The catecholamine-sensitive adenylate cyclase system appears to be comprised of at least three components; the beta-adrenergic receptor (R component), the catalytic unit of adenylate cyclase (C component) and a nucleotide regulatory protein (N component), responsible for mediating the effects of guanine nucleotides on the system. Cell fusion techniques were used to investigate the role of these three components in the process of homologous desensitization in the frog erythrocyte. Dicyclohexylcarbodiimide (DCCD) was used to inhibit beta-receptor function in one population of frog erythrocytes, whilst phenyl glyoxal was employed to inactivate the N and C components in a second population of frog erythrocytes. Using Sendai virus to fuse the two types of modified cell, heterologous beta-adrenergic receptor-adenylate cyclase systems were constructed which contained components from each cell type. When beta receptors from cells previously desensitized to catecholamines were coupled to N-C components derived from fresh erythrocytes, the resulting hybrid exhibited a densitized response to isoproterenol. By contrast, when beta-adrenergic receptors from fresh cells were coupled to N-C components derived from desensitized erythrocytes, no decreased responsiveness to isoproterenol was apparent in the hybrid. That this resensitization was the result of the addition of fresh beta-adrenergic receptors was demonstrated in a control experiment. Frog erythrocytes were desensitized simultaneously to catecholamines and prostaglandin E1 and modified with DCCD which inactivates the beta-adrenergic receptor but not the prostaglandin receptor. When fresh beta-adrenergic receptors were supplied by cell fusion to these doubly desensitized erythrocytes, only the beta-adrenergic response was restored to control levels. The response to prostaglandin remained desensitized in the hybrids, indicating that the observed resensitization of catecholamine-stimulated adenylate cyclase activity was specific and was due to the addition of fresh beta-adrenergic receptors. These data suggest that in the frog erythrocyte, homologous desensitization is primarily the result of receptor-related alterations.     

Isolation and characterization of 9-hydroxy-10-trans,12-cis-octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae

We have identified and characterized a fatty acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as 'lierre terrestre', 'ground ivy' or 'creeping Charlie'; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10-20 microM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9-HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5'-[beta,gamma-imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (alpha 2-adrenergic receptor). Studies of the interaction of 9-HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin E1 (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9-HODE alone. From these results, we conclude that 9-HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant-derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.     

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.     

Indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins in human A431 cells.

Polyclonal antibodies directed against (i) rodent lung beta 2-adrenergic receptor, (ii) a synthetic fragment of an extracellular domain of the receptor, and (iii) human placenta G-protein beta-subunits, were used to localize these antigens in situ in intact and permeabilized human epidermoid carcinoma A431 cells. Antibodies directed against beta 2-adrenergic receptors showed a punctate immunofluorescence staining throughout the cell surface of fixed intact cells. Punctate staining was also observed in clones of Chinese hamster ovary cells transfected with an expression vector harbouring the gene for the hamster beta 2-adrenergic receptor. The immunofluorescence observed with anti-receptor antibodies paralleled the level of receptor expression. In contrast, the beta-subunits common to G-proteins were not stained in fixed intact cells, presumably reflecting their intracellular localization. In detergent-permeabilized fixed cells, strong punctate staining of G beta-subunits was observed throughout the cytoplasm. This is the first indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins. Punctate immunofluorescence staining suggests that both antigens are distributed in clusters.     

Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.