Showing papers by "Robert J. Lefkowitz published in 1979"

Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding: application of a computerized model fitting technique.

Abstract: Frog myocardium appears to possess both beta1 and beta2 receptors, based on the potency order of several adrenergic agonists to compete for [3H]dihydroalprenolo1 binding. Selective beta blocking agents are able to distinguish two receptor subtypes in frog myocardium, but only one site in rat ventricle. Computer modeling using a PDP 11/45 indicates that all rat beta receptors are beta1, whereas only 15%-25% of frog ventricular beta receptors are of the beta1 subtype. Computerized curve fitting can provide a more accurate estimate of receptor parameters than currently available graphical methods of analysis.

Agonist-specific effects of guanine nucleotides on alpha-adrenergic receptors in human platelets.

Abstract: The effect of GTP on the binding affinity of alpha-adrenergic receptors for alpha-adrenergic agents was studied in human platelet lysates using direct ligand binding methods with [3H]dihydroergocryptine. GTP at a concentration of 0.1 mM markedly decreased the binding affinity of the agonist (-)epinephrine for the receptors (more than 10-fold) but had no effect on the binding of antagonists. The half maximal effect of GTP on epinephrine binding occurred at a concentration of 4 µM. Gpp(NH)p was as effective as GTP at the same concentration, whereas GDP was only 80% as effective. Other nucleotides such as ATP and ITP were less effective. The extent of the GTP-induced reduction in the affinity of alpha-adrenergic agents for the receptors was directly related to the intrinsic activity of these agents for inhibition of PGE1-stimulated adenylate cyclse. The effect of GTP appears to depend on the concurrent presence of Mg++. In the absence of Mg++, GTP caused only a slight decrease in the agonist binding affinity. When Mg++ was present without GTP, the binding affinity of the agonist (-)epinephrine was increased by 5-fold. GTP in the presence of Mg++ induces a state of diminished affinity of the receptor for the agonist which is lower than that induced by the nucleotide in the absence of MgCl2. The relationship between GTP and MgCl2 in the regulation of platelet alpha-adrenergic receptors which are inhibitory to adenylate cyclase activity appears to be analogous to their role in regulating beta-adrenergic receptors which are stimulatory for the enzyme in other tissues.

Direct Binding Studies of Adrenergic Receptors: Biochemical, Physiologic, and Clinical Implications

Abstract: Recently developed radioligand binding techniques permit direct investigation of the alpha- and beta-adrenergic receptors for catecholamines in a wide variety of tissues. These techniques ...

Selective Alteration in High Affinity Agonist Binding: A Mechanism of Beta-Adrenergic Receptor Desensitization

Abstract: Beta-adrenergic receptor desensitization of intact frog erythrocytes results in a greater percent fall in agonist ((±)[3H]hydroxybenzylisoproterenol) than in antagonist ((-)[3H]-dihydroalprenolol) binding. We now report the results of detailed studies on the alterations in agonist binding associated with beta-adrenergic receptor desensitization. Competition binding experiments revealed a lower overall affinity of agonist binding after desensitization. The affinity of antagonist ([3H]dihydroalprenolol) binding appeared unchanged after desensitization. Three reagents (N-ethylmaleimide, EDTA, and Gpp(NH)p) that decrease high affinity agonist binding were shown to have less effect on [3H]hydroxybenzylisoproterenol binding after desensitization. Two such agents (N-ethylmaleimide and dicyclohexyl carbodiimide) were also shown to block desensitization of intact cells. Guanine nucleotides failed to restore beta-adrenergic antagonist ([3H]dihydroalprenolol) binding sites after desensitization. Adenylate cyclase catalytic activity was not impaired by desensitization and remained normally responsive to guanine nucleotides. These findings are consistent with a model of beta-adrenergic receptor desensitization in which desensitized receptors arise from receptors in the high affinity state, with chronic occupancy by agonist. The process of desensitization thus appears to result in selective loss or inactivation of these high affinity agonist binding sites.

Multiple reactive sulfhydryl groups modulate the function of adenylate cyclase coupled beta-adrenergic receptors.

Abstract: The beta -adrenergic receptor adenylate cyclase complex in the frog erythrocyte contains at least two reactive sulfhydryl groups, which have been identified by their different sensitivities to N-ethylmaleimide (NEM). Adenylate cyclase catalytic activity is completely inhibited by [NEM] = 1 mM. In contrast, ability of beta -adrenergic agonists to form a high affinity guanine nucleotide sensitive state is reduced by NEM only at concentrations ≥ 1 mM. The inhibition of cyclase activity did not affect high affinity agonist binding nor perturb the ability of guanine nucleotides to reduce agonist affinity as determined in direct radioligand binding assays utilizing the beta -adrenergic agonist [ 3 H]hydroxybenzylisoproterenol and (-)isoproterenol competition for antagonist [ 3 H]dihydroalprenolol binding. Preincubation of frog erythrocyte membranes with antagonists or guanine nucleotide did not alter the effect of NEM on agonist affinity. However, once formed by preincubation of membranes with agonist, the high affinity state is resistant to the effects of NEM. The data suggest that the sulfhydryl group modulating agonist affinity 1) is located on the receptor complex but distal to the hormonal binding site and 2) may be associated with guanine nucleotide regulation of agonist affinity. The widely different sensitivities of adenylate cyclase activity and agonist binding affinity toward NEM have been exploited to investigate the mechanism of desensitization in frog erythrocytes. Incubation of intact erythrocytes with NEM under appropriate conditions inactivates the cyclase enzyme without altering the ability of membranes prepared from these cells to bind agonist with high affinity. This NEM treatment prior to prolonged exposure to (-)isoproterenol prevents receptor desensitization in frog erythrocytes, suggesting that agonist occupancy of a nucleotide-sensitive receptor is insufficient, by itself, to initiate the receptor regulation process.

Thyroxine and Propylthiouracil Effects on Alpha- and Beta-adrenergic Receptor Number, Atpase Activities, and Sialic Acid Content of Rat Cardiac Membrane Vesicles

Abstract: Summary: Membrane vesicle preparations from the hearts of euthyroid, hyperthyroid, and hypothyroid rats were analyzed in an attempt to identify biochemical changes which might correlate with known functional changes occurring in these thyroid states. Several sarcolemmal constituents which have been shown to influence myocardial contractility were measured in membrane vesicle preparations from the hearts of animals in the three thyroid states. These constituents included the apparent number of alpha- and beta-adrenergic receptors (judged from specific binding of radiolabeled adrenergic antagonists) and Na-, K--ATPase activity. As a control for the recovery of sarcolemma in the preparations, the sialic acid content was measured in all preparations. The activity of K-, Ca2--ATPase, a sarcoplasmic reticulum enzyme which regulates intracellular ionized Ca2- concentration, was also measured. Membrane vesicles of the thyroxine-treated hyperthyroid rats showed a decrease (41%, p

Thyroid hormone regulation of alpha-adrenergic receptors: studies in rat myocardium.

Abstract: The effects of alterations in thyroid state on cardiac alpha-adrenergic receptors were investigated by the binding of [3H]dihydroergocryptine (DHE), a potent alpha-adrenergic antagonist. In seven experiments, cardiac membranes from euthyroid rats bound 47 +/- 9 fmoles DHE/mg protein (mean +/- SE) at saturation and demonstrated a dissociation constant (KD) of 2.5 +/- 0.4 nM. Hyperthyroidism, produced by parenteral injection of triiodothyronine, significantly reduced the binding of DHE at all concentrations studied. Scatchard analysis showed this reduction of binding to be due largely to a decreased affinity (KD = 4.0 +/- 0.8 nM, p less than 0.05), although possibly due to a decreased number of binding sites as well (29 +/- 7 fmoles/mg protein, p less than 0.10). Hypothyroidism, produced either by oral propylthiouracil or by surgical thyroidectomy, did not produce a significant change in either the number of binding sites for DHE (56 +/- 8 fmoles/mg protein, p less than 0.40) or in binding affinity (KD = 3.1 +/- 0.5 nM, p less than 0.40). Thus, in addition to the regulation of cardiac beta-receptors by thyroid hormone that has been described previously, thyroid hormone exerts a regulatory effect on the characteristics of cardiac alpha-receptors as well. These changes provide a possible molecular mechanism for the thyroid hormone-induced alterations in cardiac responsiveness to alpha-adrenergic stimulation that have been reported previously.

β-Adrenergic Receptors of Human Lymphocytes Are Unaltered by Hyperthyroidism*

Abstract: Lymphocytes from 12 patients with untreated hyperthyroidism were compared to lymphocytes from age- and sex-matched euthyroid control subjects to test the hypothesis that alterations in β-adrenergic response mechanisms occur in human hyperthyroidism. The binding of (-)[3H]dihydroalprenolol, a compound previously shown in these cells to label binding sites having the characteristics of β-adrenergic receptors,was assayed and no significant difference was found between the two groups. In addition, the accumulation of cAMP in response to isoproterenol was determined by RIA and, again, no difference was found. (J Clin Endocrinol Metab 48: 503, 1979)

Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes.

Abstract: Desensitization of catecholamine stimulated adenylate cyclase (AC) activity is demonstrated in membranes derived from turkey erythrocytes pre-treated with isoproterenol. Membranes from desensitized cells had a loss in maximal catecholamine stimulated adenylate cyclase activity of 104 +/- 13 (pmols/mg protein/10', p less than .001) compared with controls. When adenylate cyclase was maximally stimulated with NaF or Gpp(NH)p, the decrements were 84 +/- 19 (p less than .005) and 92 +/- 32 (p less than .05) pmol/mg protein/10' respectively. There was no change in beta-adrenergic receptor number in membranes derived from treated cells. While the molecular mechanism accounting for the desensitization is uncertain, the data is consistent with the hypothesis that there is a lesion distal to the beta-adrenergic receptor, possibly involving the nucleotide site or the catalytic subunit of adenylate cyclase, causing the desensitization in the isoproterenol treated cells.

β -Adrenoreceptors determine affinity but not intrinsic activity of adenylate cyclase stimulants

Abstract: SIGNIFICANT progress has recently been made toward defining and understanding the molecular basis of hormone sensitivity in the β-adrenoreceptor-adenylate cyclase system. Through the use of biochemical1,2 and genetic3,4 techniques it has become apparent that this system is comprised of at least three separate components: the β-adrenoreceptor, which is responsible for the binding of β-adrenergic agents; the catalytic unit of the enzyme adenylate cyclase, responsible for the conversion of ATP to cyclic AMP; and a nucleotide regulatory component or components which seems to possess a GTPase activity5 and to be required for coupling of the β-adrenoreceptor to the adenylate cyclase enzyme6. As has been demonstrated by Orly and Schramm7 and confirmed by Schwarzmeier and Oilman8 in cell fusion experiments, these components interact to produce the hormone-responsive adenylate cyclase system and this interaction occurs even when the separate functions are donated by heterologous cell types. Frog and turkey erythrocytes each possess a catecholamine-responsive adenylate cyclase. Although similar in some respects these two well-studied systems differ markedly with respect to their drug affinities and intrinsic activities. Intrinsic activity is defined as the maximal ability of a drug to stimulate a biological or biochemical response. To elucidate the role of the β-adrenoreceptor in the expression of these properties, we report here the use of cell fusion techniques to construct a hybrid which possesses the β-adrenoreceptor of the turkey erythrocyte and the adenylate cyclase of the frog erythrocyte. The characteristics of this hybrid indicate that although the receptor determines relative drug affinities (for example, β1 compared with β2 subtypes of receptor), it is a component distal to the receptor which determines relative drug intrinsic activities.

Biochemical characterization of the BETA-adrenergic receptor of the frog erythrocyte

Abstract: The beta-adrenergic receptor which is coupled to adenylate cyclase in the frog erythrocyte plasma membrane provides a convenient model system for probing the molecular characteristics of an adenylate cyclase coupled hormone receptor Direct radioligand binding studies with beta-adrenergic agonists and antagonists such as [3H]hydroxybenzylisoproterenol and [3H]dihydroalprenolol have shed new light on the biochemical properties of the receptor as well as on its mode of interaction with other components of the adenylate cyclase system Agonist binding to the receptor induces a high affinity state of the receptor which can be selectively reverted to a low agonist affinity state by guanyl nucleotides This agonist-induced high affinity state of the receptor appears to correspond to a receptor moiety which has larger apparent molecular weight and which is probably a complex of the beta-adrenergic receptor and nucleotide regulatory binding protein Antagonists do not appear capable of inducing or stabilizing the formation of this high affinity receptor-nucleotide site complex The beta-adrenergic receptors have been solubilized using the plant glycoside digitonin as the detergent and have been highly purified by biospecific affinity chromatography on an alprenolol-agarose affinity support These highly purified receptor preparations retain all of the binding characteristics observed in the unpurified soluble receptor preparations Remarkably, antibodies raised in rabbits against affinity chromatography purified preparations of the receptor, themselves bind beta-adrenergic ligands with typical beta-adrenergic specificity Such antibodies which possess binding sites similar to those of physiological receptors provide useful model systems for further probing the molecular characteristics of beta-adrenergic binding sites

Antibodies raised against purified β-adrenergic receptors specifically bind β-adrenergic ligands

Abstract: Antibodies raised against purified beta-adrenergic receptors themselves specifically bind beta-adrenergic ligands. Digitonin-solubilized frog (Rana pipiens) erythrocyte beta-adrenergic receptors, purified 100- to 200-fold by adsorption to an alprenolol-agarose affinity support and specifically eluted from the affinity resin by 1-100 mM (+/-)-isoproterenol, were used to immunize six rabbits. All immune sera, in contrast to preimmune sera, bound the beta-adrenergic antagonist [(3)H]Dihydroalprenolol binding activity was due to immunoglobulins. By competition studies, antibody [(3)H]dihydroalprenolol binding was found to display a specificity and stereoselectivity resembling that of the beta-adrenergic receptor, [i.e., (-)-isoproterenol > (-)-epinephrine > (-)-norepinephrine; alprenolol approximately propranolol >> phentolamine = aloperidol; and (-) isomers of both agonists and antagonists 10-100 times more potent than (+) isomers]. A portion of the [(3)H]dihydroalprenolol binding antibodies could be specifically adsorbed onto purified frog erythrocyte membranes, whereas Xenopus and human erythrocyte membranes, both of which are almost devoid of beta-adrenergic receptors, were ineffective in adsorbing [(3)H]dihydroalprenolol binding antibodies. We suggest that the likely immunogen was a beta-adrenergic receptor-isoproterenol complex and that immunization with drugs noncovalently bound to their receptors might be a means of raising antibodies to biologically active otherwise nonimmunogenic small molecules. Such antibodies, whose specificity mimics that of a receptor, should also provide useful models for the study of the structure of the receptor binding sites.

The molecular size of adenylate cyclase in the absence and presence of nucleotide and hormone effectors.

Abstract: The molecular size of adenylate cyclase solubilized from frog erythrocyte membranes by digitonin extraction has been determined by chromatography on Sepharose 6B. Regardless of whether the membranes are exposed to catecholamines, GPP(NH)P, NaF or no effector prior to solubilization, the apparent molecular size of the adenylate cyclase enzyme is the same. Furthermore, a similar elution profile for the enzyme is observed when the catalytic activity in the eluates is measured in the presence of Mn++, rather than Mg++. Since it is generally assumed that the persistent activation of adenylate cyclase by GPP(NH)P requires interaction of the catalytic moiety with the guanine nucleotide regulatory site, it appears that the adenylate cyclase activity detected in the column eluates represents an intact catalytic-regulatory site complex. The adenylate cyclase activity derived from catecholamine pretreated frog erythrocyte membranes does not co-elute with catecholamine-occupied beta-adrenergic receptors, indicating that the agonist-promoted increase in apparent receptor size observed here and in earlier studies does not represent a physical coupling of the receptor and the adenylate cyclase enzyme.

The beta-adrenergic receptor of frog erythrocyte: purification by biospecific affinity chromatography

Abstract: Beta-adrenergic receptors of frog erythrocytes, solubilized by treatment with 1% digitonin, can be extensively purified by chromatography on a new alprenolol-agarose affinity support. Adsorption and elution of receptor activity displayed properties of a typical beta-adrenergic process. Thus, beta-adrenergic agonists and antagonists competed for receptor adsorption and eluted receptor activity with a typical stereospecific beta-adrenergic order of potency. Adsorption of soluble receptor activity to the affinity support was in excess of 90% while more than 90–95% of total protein was unretarded by the gel. Biospecific elution of receptor activity yielded 30–60% of the adsorbed activity with a purification of 1500 fold over the starting soluble preparations. Binding affinities of the highly purified preparations were essentially identical to those of unpurified soluble preparations.