Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Cherksey, Bruce D.; Zadunaisky, J. A.; Murphy, Randall B.

doi:10.1073/pnas.77.11.6401

Cited by 42 publications

(24 citation statements)

References 26 publications

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“…We recently found an association between a subpopulation of this receptor and digitoning shells of rat erythrocytes (unpublished data). Moreover, agonists, but not antagonists, seem to release the receptor from the cytoskeletal constraints, suggesting that this reaction may be required for hormone action (29). Thus, activation of both the N-protein and the f3adrenergic receptor may involve the dissociation of each of these components from its cytoskeletal binding site.…”

Section: Discussionmentioning

confidence: 99%

Molecular complexes involved in the regulation of adenylate cyclase.

Sahyoun¹,

LeVine²,

Davis³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Selective extraction of the adenylate cyclase regulatory protein (N-protein) from pigeon erythrocyte plasma membranes provided evidence for its cytoskeletal association. Cholate, but not Triton X-100 or digitonin, was effective in solubilizing the ADP-ribosylated N-protein. The labeled protein complex or components thereof that were associated with the Triton-insoluble cytoskeleton (shells) could be partly released by 0.1 mM EDTA; 1 M KCI in the presence of Triton X-100 achieved complete solubilization. 5'-Guanylyl imidodiphosphate (p[NH]ppG) and NaF, activators of adenylate cyclase, promoted the release of the regulatory protein from the cytoskeleton but MnC12, an "uncoupler" of the adenylate cyclase system, had the opposite effect. The solubilized, labeled N-protein was able to bind specificially to rat eryth-ocyte inside-out vesicles in the presence of divalent cations. A proteolytic product of inside-out vesicles inhibited the binding of the N-protein to fresh vesicles. Three molecular species which contained the Mr 45,000 polypeptide component of the N-protein were identified by gel permeation chromatography and by sucrose density gradient velocity sedimentation. p[NH]ppG appeared to convert the two larger molecular complexes to a smaller molecular entity. Such a molecular dissociation might be relevant to the effects of guanyl nucleotides on the activity of adenylate cyclase and on the affinity of hormone receptors.Activation ofthe adenylate cyclase system by hormones or GTP analogs appears to involve changes in the molecular associations among the receptor, the regulatory nucleotide-binding protein (N-protein), and the catalytic component. Gel permeation chromatography (1), sucrose density gradient sedimentation (2, 3), and target-irradiation analysis (4) have provided evidence for these molecular changes. Furthermore, selective extraction (5) and reconstitution (6) experiments suggested that constituents of the adenylate cyclase complex were bound to the cytoskeleton in a manner that depended on the state of activation of the enzyme (6). This report focuses on several molecular interactions of the N-protein and on their regulation by agents that activate adenylate cyclase. We have also attempted to solubilize, separate, and characterize the putative molecular complexes that result from these interactions. This approach may help to elucidate further the mechanism of activation of the enzyme as well as the mechanism of the regulation ofthe number and the affinity of hormone receptors that act on adenylate cyclase.Our (125-150 g) and from White Carneau pigeons. Rat reticulocytes were produced by the phenylhydrazine injection method (1). Pigeon erythrocytes were lysed by freeze-thawing, and the plasma membrane fraction was separated as reported (9). Rat reticulocyte and erythrocyte ghosts were made by hypotonic lysis (10) and they were resealed in the presence of MgCl2 (11). Inside-out vesicles were prepared by incubation of the erythrocyte ghosts in 0.1 mM sodium phosphate buffer (pH 7.6) at 37°C ...

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Section: Discussionmentioning

confidence: 99%

Molecular complexes involved in the regulation of adenylate cyclase.

Sahyoun¹,

LeVine²,

Davis³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Five-minute preincubation with isoproterenol (16 uM, 37°C) did not affect the mobility or the distribution ofAlp-NBD on the cells (data not shown).-No change was observed for an additional 15 min at 23°C (Table 3). However, after [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] min at 23°C, the fluorescence became more diffuse, and the Rf increased gradually ( Table 3).…”

Section: Receptorsmentioning

confidence: 99%

Lateral motion of beta receptors in membranes of cultured liver cells.

Henis¹,

Hekman²,

Elson³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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We have studied the lateral mobility and distribution of fi receptors on Chang human liver cells by fluorescence photobleaching recovery and video intensification microscopy.The ,3 receptors were labeled with the fluorescent antagonist 7-(2-allylphenoxy)-, 2 -dimethyl-6 -hydroxy-1 -(4 -nitrobenzo -2 -oxa-1,3-diazolyl)-1,4-diazaheptane (Alp-NBD). Sixty to 75% of the staining was specific (displaceable by unlabeled antagonists). Most of the antagonist-occupied I8 receptors were immobile, because only 15-25% of their fluorescence recovered on the experimental time scale at 23TC. This immobility correlates with the clustered distribution of Alp-NB)-/-receptor complexes at 40C and 37C. The .8 receptors appear to be aggregated prior to antagonist binding, because visible patches were observed immediately after labeling for 30 sec at 4C. Preincubation at 37°C with (-)-isoproterenol, a .8 agonist, prior to Alp-NBD labeling induced a time-dependent release of the /3 receptors to a more homogeneous distribution and increased the mobile fraction to 70-80% (lateral diffusion coefficient = 1.4 X 10-9 cm2/sec at 23°C). This is not due to an effect on membrane fluidity, because the diffusion coefficient of a lipid probe was not altered. The time course of agonist-induced fl-receptor mobilization correlates with receptor loss and adenylate cyclase desensitization but is much slower than adenylate cyclase activation. This indicates that adenylate cyclase activation by (3 receptors does not require macroscopic lateral mobility of the majority of the (3 receptors.Recent models of adenylate cyclase activation by /8 receptors stress the role of diffusional encounters between hormonereceptor complexes in the plasma membrane and other components of the cyclase system (1). The hybridization experiments of Schramm et al. (2) and the kinetic analyses of Levitzki et aL (3) are usually cited to support these models based on the floating receptor hypothesis (4). Lateral mobility of hormone-receptor complexes relative to the other cyclase components is supposed to be necessary for their effective interaction, although the receptor complexes themselves need not be mobile if the other components are. Recent studies of reconstituted systems (5) have, however, shown that the encounter of the hormonereceptor complex with the guanyl nucleotide binding protein (G protein) (6, 7) is much faster than the rate ofcyclase activation and so cannot be rate-limiting in the activation process. This result must still be tested in native membranes. Further consideration of the kinetic role of receptor-G protein encounters requires direct measurement of the lateral mobility and distribution of both the receptors and the G proteins in living cells. Here we report measurements of the distribution and lateral diffusion coefficients of / receptors labeled with a fluorescent antagonist on Chang human liver cells, in which the influence of membrane structure on ,B-adrenergic stimulation of adenylate cyclase has previously been studied (8). The experiments u...

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“…In yeasts, similar diploid meiotic products have resulted due to the interruption of sporulation (9) and as a consequence of cdc5 and cdc14 (8) The data in the literature on the action of these drugs in mammals and in yeasts may be some help in a preliminary interpretation of our data. The ,-blockers (propranolol, in particular, was used) are able to lower the intracellular cAMP level by acting on the regulatory adenylate cyclase subunit (2). Data have also been reported on the ability of propranolol to antagonize calmodulin and the calmodulin-dependent activity of cyclic nucleotide phosphodiesterase (10).…”

Section: Discussionmentioning

confidence: 77%

Propranolol, atenolol, and trifluoperazine reduce the spontaneous occurrence of meiotic diploid products in Saccharomyces cerevisiae.

Sora¹,

Bianchi²

1982

Mol. Cell. Biol.

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The effect of atenolol, propranolol, trifluoperazine, and caffeine on the occurrence of meiotic diploid and disomic products in Saccharomyces cerevisiae was investigated. We demonstrated that atenolol, propranolol, and trifluoperazine reduce the occurrence of meiotic diploid products and that propranolol also slightly decreases the spontaneous frequency of disomics. On the other hand, caffeine appears to be a powerful inducer of diploid meiotic products, but also shows a lesser effect on disomic induction. Since spontaneous or caffeine-induced diploids arise from a failure of the second meiotic division, it appears that the target of these drugs is at the beginning of the second meiotic division. The only common effect of trifluoperazine and propranolol, mainly investigated in mammals, was an inhibition of calmodulin activity via direct interaction. We tend, therefore, to believe that calmodulin activity must be a crucial point for the second meiotic division to begin. The increased induction of diploids, due to caffeine, may be interpreted as a consequence of an increased cyclic AMP level.The study of spontaneous and induced "nondisjunction," using the Saccharomyces cerevisiae strain DIS13 (8a), provided evidence of the occurrence of two kinds of anomalous meiotic products: aneuploids (n+l) and diploids. Aneuploids spontaneously arose during both meiotic divisions, whereas diploids appeared to originate from a failure of the second meiotic division. Very often, chemical treatments during meiosis result in an increase of meiotic diploid progeny. For example, bleomycin and mitomycin C induce, almost exclusively, diploids, which originate from a failure of the second meiotic division (S. Sora, M. Crippa, and G. Lucchini, Mutat. Res. in press).The spontaneous or induced failure to enter into the second meiotic division is similar to the effects of some spo and cdc mutants on meiosis (5,8). It is possible that compounds able to induce the failure of meiosis II are able to interfere with structures or signals responsible for the coordination of the sequence of the meiotic process.In this study, we report the effect of some drugs on the induction of diploid meiotic products. The tested compounds are known to interact with tubulinic structures and with the regulation of the level of cyclic AMP (cAMP). Trifluoperazine is known to interact with calmodulin, which is involved in the regulation of several cell activities. Of interest is its role in the modulation of cAMP and in the disassembly of tubulins (3). The ,-adrenergic receptor system is involved in adenylate cyclase regulation and is located on microtubulinic structures (7). For this reason, we assayed the two P-adrenergic receptor antagonists, propranolol and atenolol. Caffeine was also investigated because of its action on the cAMP level (6).Our findings show caffeine to have an action opposite to that of atenolol, propranolol, or trifluoperazine. The majority of effects were on diploid induction. Caffeine increased the total amount of meiotic diploid progeny, wh...

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Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Cited by 42 publications

References 26 publications

Molecular complexes involved in the regulation of adenylate cyclase.

Molecular complexes involved in the regulation of adenylate cyclase.

Lateral motion of beta receptors in membranes of cultured liver cells.

Propranolol, atenolol, and trifluoperazine reduce the spontaneous occurrence of meiotic diploid products in Saccharomyces cerevisiae.

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