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Biomedical subjects

R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 487 records · Page 27Linked to original sources
PubMed1980

Agonist-promoted coupling of the beta-adrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system.

Binding of the beta-adrenergic agonist [3H]hydroxybenzylisoproterenol to the beta-adrenergic receptor of rat reticulocyte membranes results in the coupling of the receptor to the guanine nucleotide regulatory protein associated with the adenylate cyclase system. This regulatory component, referred to as the G-protein, was identified by its specific [32P]-ADP-ribosylation catalyzed by cholera toxin. Incubation of [32P]ADP-ribosylated rat reticulocyte membranes with the [3H]hydroxybenzylisoproterenol agonist prior to membrane solubilization and gel exclusion chromatography resulted in the coelution of the 42,000 Mr [32P]ADP-ribosylated G-proteins with the agonist-occupied beta-adrenergic receptors. The receptor-G-protein complex was not formed when receptors were unoccupied or occupied with antagonists at the time of solubilization. Incubation of rat reticulocyte membranes with [3H]hydroxybenzylisoproterenol in the presence of guanine nucleotides reversed or prevented the formation of this receptor-G-protein complex. These data provide direct evidence for the molecular interactions promoted by agonist occupancy of beta-adrenergic receptors. It is probable that the formation of a receptor-G-protein complex is crucial for catecholamine stimulation of the adenylate cyclase enzyme and, hence, transmembrane information transfer.

Adenosine Diphosphate Ribose↗
PubMed1980

Macromolecular beta-adrenergic antagonists discriminating between receptor and antibody.

The beta-adrenergic antagonist, alprenolol, was attached in an irreversible manner to macromolecular dextran via side arms that differed in length. The ability of these macromolecules to bind to the beta-adrenergic receptor of frog erythrocytes and to catecholamine-binding antibodies raised against partially purified receptors was studied. Compared to the parent drug the potency of binding of macromolecular alprenolol to the receptor decreased about 1/10, 1/600, and 1/8000 when the length of the arm separating alprenolol from the dextran moiety was 13, 8, and 4 atoms, respectively. In contrast, the binding potencies of the parent drug and of all its macromolecular derivatives for the antibody were within the same order of magnitude. Thus, conversion of a drug to a macromolecular form may not only sustain its binding activity but may also lead in a higher selectivity. The macromolecular derivatives described here may be suitable probes for investigation of the location and of the molecular properties of the binding sites for beta-adrenergic drugs.

Alprenolol↗
PubMed1980

Agonist versus antagonist binding to alpha-adrenergic receptors.

The binding properties of two alpha-adrenergic radioligands, [3H]epinephrine (an agonist) and [3H]dihydroergocryptine (an antagonist), were compared in two model systems--membranes derived from human platelets and membranes from rat liver. The platelet contains exclusively alpha 2 and the liver mostly (approximately 80%) alpha 1 receptors. Agonists induce the formation of a guanine nucleotide-sensitive high-affinity state of alpha 2 but not alpha 1 receptors. [3H]Dihydroergocryptine labels all the alpha receptors, whereas [3H]epinephrine at low concentrations labels predominantly the high-affinity form of the alpha 2 receptor in both platelet and liver. However, in the liver, alpha-adrenergic effects such as glycogen phosphorylase activation are shown to be mediated via alpha 1 receptors. Thus, in liver membranes the endogenous "physiological" agonist may not label the physiologically relevant alpha 1 receptors in typical radioligand binding assays using low concentrations of [3H]epinephrine.

Adrenergic alpha-Agonists↗
PubMed1980

The human placenta--a rich source of beta-adrenergic receptors: characterization of the receptors in particulate and solubilized preparations.

A crude particulate fraction of human placenta possesses a high concentration of beta-adrenergic receptors, as determined by (-)-[3H]dihydroalprenolol binding (approximately 240 fmol/mg protein; Kd approximately 2 nM). The sites display all the typical characteristics of beta-adrenergic receptors, including rapid and reversible kinetics, saturability, and appropriate specificity and stereospecificity. Computer modelling of ligand binding data indicate that the binding of (-)-[3H]dihydroalprenolol to these sites conforms closely to the pattern anticipated for interactions of the ligand with a homogeneous class of receptors according to the law of mass action. The rejeptors are readily solubilized with digitonin, retaining their typical beta-adrenergic characteristics. The human placenta is likely to be a particularly useful source of beta-adrenergic receptors for purification because of its high receptor content and its ready availability in substantial quantities.

Adrenergic beta-Antagonists↗
PubMed1980

Corticosteroid-induced differential regulation of beta-adrenergic receptors in circulating human polymorphonuclear leukocytes and mononuclear leukocytes.

A method of reproducibility measuring human leukocyte beta-adrenergic receptor density and affinity has been developed and applied to the study of receptor regulation in man. The method has the advantages of using a membrane preparation which binds highly specifically and employing techniques such as using low concentrations of [3H]dihydroalprenol, analyzing the data by computer modelling techniques, and providing data from both granulocytes and lymphocytes in the same individual to minimize measurement errors. Using this methodology, human beta-adrenergic receptor regulation is examined. Cortisone acetate was found to induce an acute rise in granulocyte beta-adrenergic receptor density and adenylate cyclase activity and an acute fall in lymphocyte beta-adrenergic receptor density. This potentially differential regulation of a single receptor subtype in two lines of leukocytes has important implications for the study of receptor regulation in man using leukocyte models.

Adenylyl Cyclases↗
PubMed1979

Biochemical characterization of the beta-adrenergic receptor of the frog erythrocyte.

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.

Adenylyl Cyclases↗
PubMed1979

Multiple effects of guanine nucleotides on human platelet adenylated cyclase.

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occurring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed1979

Differential effects of GTP on the coupling of beta-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes. Application of new methods for the analysis of receptor-effector coupling.

A detailed comparison of the interaction of beta-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.

Adenylyl Cyclases↗