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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 595 records · Page 33Linked to original sources
PubMed1972

Identification of the cardiac beta-adrenergic receptor protein: solubilization and purification by affinity chromatography.

A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified beta-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 x 10(5) liters/mol (as compared to two association constants, 10(7) and 10(6) liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for beta-adrenergic drugs and antagonists.

Amination↗
PubMed1971

A fraction of the ventricular myocardium that has the specificity of the cardiac beta-adrenergic receptor.

The 78,000 x g microsomal fraction of canine ventricular myocardium effected a 20-fold concentration of [(3)H]norepinephrine from a 10(-9) M solution. The [(3)H]norepinephrine bound was displaced by physiologic concentrations of the beta-adrenergic catecholamines isoproterenol, epinephrine, and norepinephrine, in that order, which is the order of effectiveness of their actions on the force and rate of cardiac contraction. Alpha-adrenergic compounds did not displace [(3)H]norepinephrine until concentrations four orders of magnitude greater were reached. The beta-adrenergic blocker propranolol displaced at 10(-6) M, whereas the alpha-adrenergic blocker phentolamine was inactive. Metabolites of the catecholamines did not compete for this binding site. It is concluded that, on the basis of specificity and affinity of binding, these microsomal particles are likely to contain the beta-adrenergic receptor.

Adrenergic alpha-Antagonists↗
PubMed1970

Radioreceptor assay of adrenocorticotropic hormone: new approach to assay of polypeptide hormones in plasma.

Biologically active iodine-125-labeled adrenocorticotropic hormone (ACTH) binds specifically to ACTH receptors extracted from adrenals. Unlabeled ACTH at 1 picogram per milliliter significantly displaces labeled ACTH from these receptors. This system, which appears to be applicable to all polypeptide hormones, provides a rapid and sensitive method for measurements of biologically active ACTH in dilute whole plasma.

Adrenal Glands↗
PubMed1970

ACTH receptors in the adrenal: specific binding of ACTH-125I and its relation to adenyl cyclase.

Pure monoiodo ACTH-(125)I was prepared that was biologically active and free of unlabeled ACTH. Extracts of adrenal cortex that contained ACTH-sensitive adenyl cyclase, bound ACTH-(125)I; extracts that lacked the ACTH-sensitive cyclase did not bind ACTH-(125)I. Unlabeled ACTH inhibited the binding of ACTH-(125)I. Five ACTH derivatives which varied widely in biological activity were tested. All inhibited the binding of ACTH-(125)I in direct proportion to their biological activity. Albumin, insulin, and four unrelated iodinated hormones were inert. The addition of excess hormone or acetic acid produced rapid dissociation of bound ACTH-(125)I. This study demonstrates directly the binding of ACTH to its biologically significant site.

Adenylyl Cyclases↗
PubMedDate not supplied

Beta-adrenergic receptor-coupled adenylate cyclase. Biochemical mechanisms of regulation.

Beta-adrenergic receptor-coupled adenylate cyclase is regulated by both amplification and desensitization processes. Desensitization of adenylate cyclase is divided into two major categories. Homologous desensitization is initiated by phosphorylation of the receptors by a beta-adrenergic receptor kinase. This reaction serves to functionally uncouple the receptors and trigger their sequestration away from the cell surface. These sequestered receptors can rapidly recycle to the cell surface or, with time, become down regulated, being destroyed within the cell. Dephosphorylation of the receptors is accomplished in the sequestered compartment of the cell, which may functionally regenerate the receptors and allow their return to the cell surface. In heterologous desensitization, receptor function is also regulated by phosphorylation, but in the absence of receptor sequestration or down regulation. In this case, phosphorylation serves only to functionally uncouple the receptors, that is, to impair their interactions with the guanine nucleotide regulatory protein Ns. Several protein kinases are capable of promoting this phosphorylation, including the cAMP-dependent kinase and protein kinase C. In addition to the receptor phosphorylation, heterologous desensitization is associated with modifications at the level of the nucleotide regulatory protein Ns and perhaps Ni. Adenylate cyclase systems are also subject to amplification that involves a protein kinase C-mediated phosphorylation of the catalytic unit of the enzyme. Phosphorylation of the catalytic unit enhances its catalytic activity and results in amplified stimulation by the regulatory protein Ns. Other receptor/effector systems exhibit qualitatively similar regulatory phenomena, suggesting that covalent modification (phosphorylation) may represent a general mechanism for regulating receptor function.

Adenylyl Cyclases↗
PubMedDate not supplied

Pure beta-adrenergic receptor: the single polypeptide confers catecholamine responsiveness to adenylate cyclase.

The beta-adrenergic receptor binding subunits from frog erythrocytes, hamster lung and guinea pig lung have been purified to apparent homogeneity and in all cases reside on a single polypeptide. Insertion of the pure receptors into phospholipid vesicles and subsequent fusion of these vesicles with a receptor-deficient cell conveys beta-adrenergic responsiveness to the adenylate cyclase system of the acceptor cell. Such responsiveness is linearly dependent on the amount of receptor used in the fusion experiments and is independent of the receptor source. Moreover, this responsiveness displays appropriate beta-adrenergic specificity. These results indicate that the beta-adrenergic receptor polypeptide contains both the ligand binding site and the site responsible for mediating stimulation of adenylate cyclase activity, presumably via interaction with the guanine nucleotide regulatory protein.

Adenylyl Cyclases↗
PubMedDate not supplied

Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model.

Desensitization, the tendency of biological responses to wane over time despite the continuous presence of a stimulus of constant intensity, is observed in organisms as diverse as bacteria and mammals. Recently, new insights into the molecular mechanisms underlying these phenomena have emerged from the study of the receptors coupled to the ubiquitous second messenger-generating system adenylate cyclase. These mechanisms involve sequestration or down-regulation of the receptors from the cell surface as well as functionally significant covalent modifications of the receptors and/or guanine nucleotide regulatory proteins.

Adaptation, Physiological↗
PubMedDate not supplied

A role for Ni in the hormonal stimulation of adenylate cyclase.

The best understood system for transduction of extracellular messages into intracellular signals is the hormone receptor-coupled adenylate cyclase. In such systems receptors are functionally coupled to the enzyme by two special proteins, termed the stimulatory and inhibitory guanine nucleotide regulatory proteins (Ns and Ni, respectively). These proteins, thought to mediate, respectively, stimulatory and inhibitory influences on the adenylate cyclase, are members of a larger class of heterotrimeric guanine nucleotide regulatory proteins involved in membrane signal transduction. We have studied the interactions of the various components of the adenylate cyclase system by co-reconstituting pure beta-adrenergic receptors, pure Ns and Ni, and functionally resolved preparations of the catalyst in phospholipid vesicles. In the absence of Ni, beta-adrenergic receptor/Ns-mediated catecholamine stimulation of the enzyme is relatively modest (approximately 1.3-fold). Surprisingly, however, when Ni is also present, stimulation increases dramatically (up to 7-8-fold) because of a greater suppression of basal relative to agonist-stimulated enzyme activity. Thus, Ni may actually be required for maximal agonist stimulation as well as for inhibition of the adenylate cyclase.

Adenylyl Cyclases↗