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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 379 records · Page 21Linked to original sources
PubMed1984

The mammalian beta 2-adrenergic receptor: reconstitution of functional interactions between pure receptor and pure stimulatory nucleotide binding protein of the adenylate cyclase system.

Pure beta-adrenergic receptors (beta-AR) isolated from guinea pig lung and pure guanine nucleotide binding regulatory protein (NS) of adenylate cyclase isolated from human erythrocytes have been inserted into phospholipid vesicles, resulting in the functional coupling of these two components. The reconstitution of receptor and NS interactions results in the establishment of a guanine nucleotide sensitive state of the receptor that binds agonists with high affinity. Competition curves of isoproterenol for labeled antagonist binding to vesicles containing both beta-AR and NS are biphasic and reveal two affinity states, one of high (approximately 2 nM) and the other of low affinity (approximately 300 nM). In the presence of guanine nucleotides, the competition curves become monophasic and are shifted to a single low-affinity state for the agonist similar to the situation observed in membrane preparations. In addition, the interactions of the receptor and NS lead to the induction of a GTPase activity in NS. The GTPase activity can be stimulated by beta-adrenergic agonists such as isoproterenol (2-5-fold) and is completely blocked by antagonists such as alprenolol in a stereoselective manner. The established hormone responsive activity retains the beta 2-adrenergic specificity conferred by the pure receptor, and similar extents of stimulation (up to 4-fold) are observed with pure receptor from frog erythrocytes, indicating a similar efficiency of coupling between receptors from different species and NS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

Reconstitution of a hormone-sensitive adenylate cyclase system. The pure beta-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit.

A hormone responsive adenylate cyclase has been reconstituted in phosphatidylcholine vesicles from its isolated protein components. The proteins used were the affinity chromatography purified (500-2000-fold) or pure Mr = 64,000 beta-adrenergic receptors (beta AR) isolated from hamster and guinea pig lung membranes, the pure heterotrimeric (Mr: alpha = 42,000; beta = 35,000; gamma approximately equal to 5,000) guanine nucleotide regulatory protein (Ns) isolated from human erythrocyte membranes; and the catalytic unit of the adenylate cyclase (C) solubilized from bovine brain caudate nucleus and resolved from beta AR and Ns by gel filtration. Adenylate cyclase activity in vesicles containing C alone was stimulated by forskolin but not by guanine nucleotides or by the beta-adrenergic agonist isoproterenol. Reconstitution of Ns and C interactions in the lipid vesicles resulted in guanine nucleotide but not beta-adrenergic agonist sensitivity. When beta AR was inserted together with Ns and C into lipid vesicles, the catalytic unit became responsive to beta-adrenergic agonists as well and this stimulation was blocked in a stereoselective manner by the beta-adrenergic antagonist alprenolol. Regulation of adenylate cyclase activity in the reconstituted system by beta-adrenergic agonists, guanine nucleotides, and Mg2+ showed properties similar to those observed in native membranes. The interactions of the various protein components in the reconstituted system were also monitored by GTPase activity. Such activity was observed to occur primarily as a result of receptor-Ns interactions. The results described in this report document the feasibility of studying hormone-responsive adenylate cyclase in a totally reconstituted system which retains the major regulatory properties of the enzyme in its native membrane-bound environment.

Adenylyl Cyclases↗
PubMed1984

Direct demonstration of impaired functionality of a purified desensitized beta-adrenergic receptor in a reconstituted system.

Long-term exposure of various cell types to beta-adrenergic agonists such as isoproterenol leads to an attenuated responsiveness ("desensitization") of the adenylate cyclase system to further challenge with these agonists. The turkey erythrocyte model system was used earlier to show that a covalent modification of the receptor (phosphorylation) is associated with this process. The functionality of the "desensitized" beta-adrenergic receptor was assessed by implanting purified beta-adrenergic receptor preparations from control and desensitized turkey erythrocytes into phospholipid mixtures and then fusing them with receptor-deficient cells (Xenopus laevis erythrocytes). Desensitized beta-adrenergic receptors showed a 40 to 50 percent reduction in their ability to couple to the heterologous adenylate cyclase system, comparable to the reduction in their functionality observed in their original membrane environment. These results demonstrate the utility of recently developed receptor reconstitution techniques for assessing the functionality of purified receptors and show a direct link between a covalent modification of a membrane-bound receptor and its impaired functionality in a reconstituted system.

Adenylyl Cyclases↗
PubMed1984

Desensitization of turkey erythrocyte adenylate cyclase. Beta-adrenergic receptor phosphorylation is correlated with attenuation of adenylate cyclase activity.

Preincubation of turkey erythrocytes with beta-adrenergic agonists leads to an attenuation of the responsiveness of adenylate cyclase to subsequent hormonal stimulation. Recently, our laboratory has shown (Stadel, J. M., Nambi, P., Shorr, R. G. L., Sawyer, D. D., Caron, M. G., and Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3173-3177) using 32Pi incorporation that phosphorylation of the beta-adrenergic receptor accompanies this desensitization process. We now report that, as determined from intracellular [gamma-32P] ATP specific activity measurements, this phosphorylation reaction occurs in a stoichiometric fashion. Under basal conditions there exists 0.75 +/- 0.1 mol of phosphate per mol of receptor whereas under maximally desensitized conditions this ratio increases to 2.34 +/- 0.13 mol/mol. This phosphorylation of the receptor is dose-dependent with respect to isoproterenol and exhibits a dose-response curve coincidental with that for isoproterenol-induced desensitization of adenylate cyclase. The time courses for receptor phosphorylation and adenylate cyclase desensitization are identical. In addition, the rate of resensitization of adenylate cyclase activity is comparable to the rate of return of the phosphate/receptor stoichiometries to control levels. Both the phosphorylation and desensitization reactions are pharmacologically specific as indicated by the high degree of stereoselectivity, rank order of catecholamines, and blockade by the specific beta-adrenergic antagonist, propranolol. Incubation of turkey erythrocytes with cAMP and cAMP analogs maximally activates cAMP-dependent protein kinase but only partially mimics isoproterenol in promoting phosphorylation of the receptor in concordance with their partial effects in inducing desensitization. Conversely, activators or inhibitors of Ca2+/calmodulin kinase or protein kinase C do not affect the isoproterenol-induced desensitization. These results indicate that desensitization of turkey erythrocyte adenylate cyclase is highly correlated with phosphorylation of the beta-adrenergic receptor and that these events are mediated, at least partially, by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains.

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Affinity Labels↗
PubMed1984

Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes.

Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4 alpha-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. The catecholamine isoproterenol induced a 51% desensitization. Incubation of 32Pi labeled erythrocytes with TPA promoted a 3- to 4-fold increase in phosphorylation of the beta-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating beta-adrenergic receptor function.

Adenylyl Cyclase Inhibitors↗
PubMed1984

Effect of pertussis toxin on alpha 2-adrenoceptors: decreased formation of the high-affinity state for agonists.

Administration of pertussis toxin to hamsters abolishes the alpha 2-adrenergic inhibition of adenylate cyclase in their adipocytes. The toxin did not modify the number of adipocyte alpha 2-adrenoceptors or their affinity for antagonists. In contrast, the affinity for agonists was significantly diminished in adipocyte membranes obtained from pertussis toxin-treated hamsters as compared to the controls. This decrease in affinity was due to a significant diminution in the proportion of sites displaying the high-affinity state for agonists. It is concluded that pertussis toxin induces a shift in the proportion of sites in high- and low-affinity states. for agonists towards the low-affinity conformation.

Adenylate Cyclase Toxin↗
PubMed1984

Identification of the subunit-binding site of alpha 2-adrenergic receptors using [3H]phenoxybenzamine.

alpha 2-Adrenergic receptors are members of an important class of membrane-bound receptors which appear to mediate physiologic responses by decreasing the activity of the regulatory enzyme adenylate cyclase. This report describes the first direct indentification of the subunit-binding site of alpha 2-adrenergic receptors. alpha 2-Adrenergic receptors from human platelets were solubilized with 1% digitonin and were purified approximately 600-fold by repetitive affinity chromatography. In saturation and competition binding studies using [3H]yohimbine the original alpha 2-adrenergic characteristics were retained by the partially purified receptor, i.e. the following potency series (based on Ki values) was obtained: phentolamine approximately equal to yohimbine much greater than prazosin and (-)epinephrine greater than (+)epinephrine. Phenoxybenzamine was found to have a Ki for the partially purified alpha 2-adrenergic receptor of 108 nM. As judged by the loss of specific [3H]yohimbine binding, phenoxybenzamine (a known alkylating agent) was found to bind irreversibly to the partially purified alpha 2-adrenergic receptor. Using [3H]phenoxybenzamine, covalent labeling of proteins in the partially purified receptor preparation was obtained. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, a specifically labeled peptide with a relative molecular mass of 61,000 was visualized. Irreversible labeling of this peptide by [3H]phenoxybenzamine could be prevented with either phentolamine or (-)epinephrine, but not with prazosin or (+)epinephrine, suggesting that this peptide of Mr = 61,000 represents the major subunit binding site of the human platelet alpha 2-adrenergic receptor.

Binding, Competitive↗
PubMed1984

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization.

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.

Adenylyl Cyclases↗
PubMed1984

Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors.

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Photoaffinity labeling of turkey erythrocyte beta-adrenergic receptors: degradation of the Mr = 49,000 protein explains apparent heterogeneity.

The potent photoaffinity probe [125I]p-azidobenzylcarazolol was used to identify beta-adrenergic receptors from turkey erythrocytes. Two peptides were specifically labeled with apparent Mr = 39,000 and 49,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The ratio of labeling of these peptides was found to be dependent on the method of tissue preparation. Thus, the Mr = 39,000/Mr = 49,000 ratio was 1:2 when labeling was done on intact cells, 1:1 for labeling of crude membranes, and 4:1 for purified membrane preparations. Moreover, incubation of intact cells or either membrane preparation at 37 degrees C led to a diminution of the Mr = 49,000 labeled peptide which was associated with a stoichiometric increase in the Mr = 39,000 form of the receptor. These results suggest that the turkey erythrocyte beta-adrenergic receptor is a protein of Mr = 49,000 and that the commonly observed Mr = 39,000 peptide is derived from this protein.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of mammalian alpha 1-adrenergic receptors. Identification of the ligand binding subunit with a high affinity radioiodinated probe.

We have synthesized and characterized a novel high affinity radioiodinated alpha 1-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido - 3 - [125I]iodophenyl) pentanoyl] - 1 - piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (KD = 130 pM) in a reversible and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an alpha 1-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical alpha 1-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at Mr = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the Mr = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the alpha 1-adrenergic receptor.

Affinity Labels↗
PubMed1984

Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes.

We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20-50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.

Animals↗
PubMed1984

Development of an affinity ligand for purification of alpha 2-adrenoceptors from human platelet membranes.

Human platelets contain alpha 2-adrenoceptors which are negatively coupled to the enzyme adenylate cyclase. In order to better understand the interaction of this subtype of alpha receptor with this key enzyme, we have initiated a program to isolate and characterize the alpha 2-adrenoceptor. This report describes the synthesis and biological characterization of a series of molecules that were prepared as affinity ligands for this purpose. The best of these is 9-(allyloxy)-6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 101253). This compound is an alpha 2-adrenoceptor antagonist, which was obtained by synthetic modification of 6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 86466), a novel antagonist with high affinity for the alpha 2-receptor.

Affinity Labels↗
PubMed1984

Cardiac adrenergic receptors.

Recently developed pharmacological and biochemical techniques have brought new insights about the structure, function, and regulation of beta-adrenergic receptors. This chapter focuses on the cardiac beta-adrenergic receptor and the possible clinical and physiological implications of this new information.

Adenylyl Cyclases↗