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Showing papers by "Robert J. Lefkowitz published in 1984"



Journal ArticleDOI
TL;DR: Hormones and drugs initiate their biologic actions by binding to specific cellular recognition sites, termed receptors, and are followed by alterations of cellular metabol...
Abstract: VIRTUALLY all hormones and drugs initiate their biologic actions by binding to specific cellular recognition sites, termed receptors. Receptor binding is followed by alterations of cellular metabol...

326 citations


Journal ArticleDOI
TL;DR: The established hormone responsive activity retains the beta 2-adrenergic specificity conferred by the pure receptor, and similar extents of stimulation are observed with pure receptor from frog erythrocytes, indicating a similar efficiency of coupling between receptors from different species and NS.
Abstract: 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)

240 citations



Journal ArticleDOI
TL;DR: Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.
Abstract: The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.

195 citations


Journal ArticleDOI
TL;DR: 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.

151 citations


Journal ArticleDOI
TL;DR: 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.

113 citations


Journal ArticleDOI
TL;DR: The carbohydrate composition of the mammalian beta receptor is examined through the use of specific exo- and endoglycosidases and lectin affinity chromatography to 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.

110 citations


Journal ArticleDOI
TL;DR: A novel high affinity radioiodinated alpha 1-adrenergic receptor photoaffinity probe is synthesized and characterized, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido - 3 - [125I]iodophenyl) pentanoyl] - 1 - piperazinyl] quinazoline, which incorporates irreversibly into plasma membranes prepared from several mammalian tissues.

71 citations


Journal ArticleDOI
TL;DR: An in vivo mammalian model system, the rat lung, is established, to study the cellular and biochemical basis for beta-agonist induced desensitization, and indicates that the in vivo desensItization is itself a receptor-mediated event.
Abstract: Beta-adrenergic agonists and antagonists are widely used in many clinical situations to regulate beta-adrenergic stimulation. However, responsiveness to beta-stimulation may be reduced by the process of desensitization. We have established an in vivo mammalian model system, the rat lung, to study the cellular and biochemical basis for beta-agonist induced desensitization. After in vivo administration of a beta-agonist [(-)isoproterenol] the adenylate cyclase becomes rapidly insensitive to further stimulation by beta-agonists with no change in basal or NaF-stimulated activity. The in vivo desensitization can be blocked by the simultaneous administration of a beta-antagonist [(+/-)propranolol] and the process displays the pharmacological characteristics typifying the beta 2 receptor of rat lung. This indicates that the in vivo desensitization is itself a receptor-mediated event. The processes of de- and resensitization are very rapid with onset within 5 min, maximal effect at 10 min, and complete reversal by 2-3 h. The change of adenylate cyclase sensitivity is paralleled by a translocation of approximately 40% of the beta-receptors from the plasma membrane fraction to a light membrane fraction, which has very low activities of plasma membrane marker enzymes. The receptors translocated to the light membrane fraction as well as those remaining in the plasma membranes are uncoupled with loss of their ability to form the high affinity, nucleotide sensitive, physiologically active state of the receptor. During resensitization the receptors in the plasma membrane fraction are recoupled before all the translocated receptors have returned. This suggests that translocation and uncoupling of the receptors are two distinct, probably independent processes. During the entire process of de- and resensitization no structural change of the receptor protein residing in the plasma membranes or light membrane fraction can be demonstrated as visualized by photoaffinity labeling.

70 citations


Journal ArticleDOI
24 Aug 1984-Science
TL;DR: The utility of recently developed receptor reconstitution techniques for assessing the functionality of purified receptors are demonstrated and a direct link between a covalent modification of a membrane-bound receptor and its impaired functionality in a reconstituted system is shown.
Abstract: 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.

Journal ArticleDOI
TL;DR: Data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.

Journal ArticleDOI
TL;DR: This chapter focuses on the cardiac beta-adrenergic receptor and the possible clinical and physiological implications of this new information.
Abstract: 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.

Journal ArticleDOI
TL;DR: The synthesis and biological characterization of a series of molecules that were prepared as affinity ligands for the alpha 2-adrenoceptor isolation and characterization are described, including 9-(allyloxy)-6-chloro-3-methyl-2,3,4,5-tetrahydro-1H- 3-benzazepine (SK&F 101253).
Abstract: 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.

Journal ArticleDOI
TL;DR: The first direct indentification of the subunit-binding site of alpha 2-adrenergic receptors is described, as judged by the loss of specific [3H]yohimbine binding, and phenoxybenzamine (a known alkylating agent) was found to bind irreversibly to the partially purifiedalpha 2- adrenergic receptor.

Journal ArticleDOI
TL;DR: Data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization, and the availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.

Journal ArticleDOI
TL;DR: In vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP are consistent with the notion that N is a bifunctional protein interacting with both R and the adanine nucleotide regulatory protein.
Abstract: Decreased activity ofthe guanine nu- cleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes ofsome patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity ofN (PHP-Ib). Low N activity in PHP- Ia results in a decrease in hormone (H)-stimulatable ad- enylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared f3-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Com- petition curves that were generated by increasing con- centrations ofthe (3-agonist isoproterenol competing with ('251)pindolol were shallow (slope factors < 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist com- petition curves from the PHP-Ia patients were shifted significantly (P < 0.02) to the right as a result of a sig- nificant (P< 0.01) decrease in the percent offl-adrenergic receptors in the high affinity state from 64±22% in PHP- Ib and 56±5% in controls to 10±8% in PHP-Ia. The

Journal ArticleDOI
TL;DR: The results suggest that endogenous proteolysis does not directly impair the ability of beta-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein, however, they imply that endogenous proteinolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide Regulatory protein.

Journal ArticleDOI
TL;DR: The 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.

Journal ArticleDOI
TL;DR: The results demonstrate the applicability of using para-azidobenzylcarazolol to covalently label mammalian β-adrenergic receptors and suggest that mammalian β 1 and β 2 receptor binding sites primarily reside on peptides of M r 62,000–65,000 and that smaller ligand binding fragments may arise by proteolysis.

Journal Article
TL;DR: This new high-affinity radioiodinated probe has features which should make it useful for the molecular characterization of alpha 1-adrenergic receptors in tissues.
Abstract: We have synthesized and characterized a high-affinity alpha 1-adrenergic receptor probe, 4-amino-6,7-dimethoxy-2[4'- [5"(3"'-125I-iodo-4"'-aminophenyl)pentanoyl]-1'-piperazinyl] quinazoline (125I-A55453). This ligand binds reversibly to rat hepatic plasma membranes with high affinity (KD = 77 +/- 6 pM), and it labels the same number of "specific" prazosin-competable sites as the alpha 1-adrenergic receptor-selective radioligand [125I] iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone. Specific binding is stereoselective and competed for by alpha-adrenergic agents with an alpha 1-adrenergic receptor specificity. 125I-A55453 can be covalently photoincorporated into peptides of rat hepatic and splenic membranes using the bifunctional photoactive cross-linker, N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. Following photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled hepatic membranes reveals a major "specifically" labeled peptide of Mr = 82,000 (+/- 1,000) with minor peptides at Mr = 50,000 (+/- 500), and 40,000 (+/- 300). Covalent incorporation of 125I-A55453 into the Mr = 82,000 peptide is inhibited by adrenergic drugs with an alpha 1-adrenergic receptor specificity. Labeled splenic membranes demonstrate a broad band of photoincorporated radioactivity centered at Mr = 82,000, and covalent incorporation into this peptide is also attenuated with an alpha 1-adrenergic receptor specificity. This new high-affinity radioiodinated probe has features which should make it useful for the molecular characterization of alpha 1-adrenergic receptors in tissues.



Journal ArticleDOI
TL;DR: The mechanism of the observed sensitization of turkey erythrocyte adenylate cyclase by beta-adrenergic antagonists is receptor mediated and likely involves facilitation of N interaction with the catalytic subunit of the enzyme.

Journal ArticleDOI
TL;DR: The results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes of frog and turkey, and may serve as an indication of morphologic differentiation.

Journal ArticleDOI
TL;DR: Using immunocytochemical techniques with antibodies raised to the frog erythrocyte, beta 2-adrenergic receptor and beta-receptor immunoreactivity was found throughout dendritic processes with local accumulations at certain postsynaptic sites, consistent with the idea that the density of the receptors might be significantly increased at post Synaptic junctions of adrenergic neurons.
Abstract: Recent developments in the characterization of the adrenergic receptors have led to the identification and purification of the binding subunits of the various catecholamine receptors. beta-Adrenergic receptors have been identified in a wide variety of tissues by photoaffinity labeling with the antagonist [125I]p-azidobenzylcrazolol and have been purified to apparent homogeneity from several of these tissues. Thus, beta 1- and beta 2-adrenergic receptor binding sites appear to reside on peptides with molecular weights of 60,000 to 65,000. The alpha 1-adrenergic receptor binding subunit has been identified in several peripheral tissues by photoaffinity labeling with a newly developed probe (4-amino-6,7-dimethoxy-2[4(5(3-[125I]-iodo-4-azidophenyl) pentanoyl)-1-piperazinyl]-quinazoline, or [125I]APDQ). This binding site resides on a peptide with a molecular weight of 80,000. These techniques have been applied to the elucidation of the binding subunit structure of these receptors in the rat central nervous system with the result that beta 1-, beta 2-, and alpha 1-adrenergic binding sites appear to reside on peptides of similar molecular weight to those identified in peripheral tissues (i.e., 60,000-65,000 and 80,000). Using immunocytochemical techniques with antibodies raised to the frog erythrocyte, beta 2-adrenergic receptor, beta-adrenergic receptors were identified at the light microscopic level in regions of the rat and frog brain previously found by ligand binding and autoradiography to be richest in beta-adrenergic receptors. At the electron microscopic level, beta-receptor immunoreactivity was found throughout dendritic processes with local accumulations at certain postsynaptic sites. This finding is consistent with the idea that the density of the receptors might be significantly increased at postsynaptic junctions of adrenergic neurons.


01 Jan 1984
TL;DR: Data indicate that &adrenergic agonists promote a CAMPmediated process which leads to receptor alterations and desensitization, and the availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.
Abstract: Conditions have been developed for desensitizing the P-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 “C. Maximally effective concentrations of isoproterenol produce a 41.0 2 1.55% loss of iosproterenol-stimulated and a 15.0 2 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 2 1.5% fall in isoproterenol-stimulated and a 21.5 f 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the &adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factorb) 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 B-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 &adrenergic agonists promote a CAMPmediated 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.

Book ChapterDOI
01 Jan 1984
TL;DR: Although numerous hormone receptors are coupled in a stimulatory or inhibitory fashion to the adenylate cyclase, to date only the β-adrenergic receptors, which respond to catecholamines such as adrenaline and noradrenaline, have been characterized in any significant detail.
Abstract: Of all the receptor-coupled effector systems found in mammalian cells none has received more attention than the adenylate cyclase system (Lefkowitz et al., 1983; Smigel et al., 1984). Its ubiquity and the diversity of hormones and drugs which stimulate it has made it a premier model system for the study of the mechanisms whereby drugs and hormones activate their physiological effectors. Although numerous hormone receptors are coupled in a stimulatory or inhibitory fashion to the adenylate cyclase, to date only the β-adrenergic receptors, which respond to catecholamines such as adrenaline and noradrenaline, have been characterized in any significant detail. They are the only adenylate cyclase-coupled receptors which have been purified (Lefkowitz et al., 1983; Stiles et al., 1984a).