PubMed Health⌕ Search

Biomedical subjects

R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 433 records · Page 24Linked to original sources
PubMed1982

Molecular characterization of the beta-adrenergic receptor of frog erythrocytes.

The beta-adrenergic receptor of the frog erythrocyte has been solubilized in an active form with digitonin and purified by affinity chromatography and high performance liquid chromatography. Purified preparations contain a single band of iodinated protein of apparent Mr = 58,000. This peptide appears to represent the ligand binding subunit of the receptor since purified preparations bind ligands with the same beta-adrenergic specificity as the solubilized or membrane-bound receptor, display the same isoelectric point and similar sedimentation characteristics in sucrose density gradients. The same ligand binding subunit can also be identified in partially purified receptor preparations or in membranes by photoaffinity labelling or photodependent crosslinking of two radiolabelled beta-adrenergic antagonists, p-azidobenzylcarazolol and p-aminobenzylcarazolol.

Affinity Labels↗
PubMed1982

Affinity chromatography of human platelet alpha 2-adrenergic receptors.

Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving alpha(2)-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with alpha(2)-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing alpha(2)-adrenergic receptors, can be specifically labeled with [(3)H]yohimbine and can be solubilized with digitonin without loss of their alpha(2)-adrenergic binding characteristics. Chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in the adsorption of 70-80% of the initial [(3)H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both alpha-adrenergic antagonists (phentolamine >/= yohimbine > prazosin) and by alpha-adrenergic agonists [p-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [(3)H]yohimbine binding activity from the affinity gel is effected with the aforementioned agonists and antagonists in the same order of potency. Other drugs that do not interact appreciably with alpha-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [(3)H]yohimbine binding activity from the affinity gel. In addition to the specificity of the interaction, chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in a 40-50% overall yield and an approximately 200-fold increase in the specific binding activity for [(3)H]yohimbine. The results indicate that the SKF 101253-Sepharose CL-4B affinity adsorbent should provide a powerful tool for the purification of the adenylate cyclase-inhibitory alpha(2)-adrenergic receptor of human platelets.

Adrenergic alpha-Agonists↗
PubMed1982

Reciprocal modulation of agonist and antagonist binding to muscarinic cholinergic receptor by guanine nucleotide.

The ability of guanine nucleotide to decrease the binding affinity of agonists but not antagonists has been documented in a number of hormone and neurotransmitter receptor systems. By contrast, recent reports indicate that both agonist and antagonist binding to the muscarinic cholinergic receptors appear to be regulated in a reciprocal fashion by guanine nucleotide. We document two forms of the muscarinic cholinergic receptor in frog heart, which are present in approximately equal proportions and which display high-agonist/low antagonist and low-agonist/high-antagonist affinities, respectively. Guanine nucleotide appears to convert the former type of site into the latter type. These observations can be interpreted in terms of a model for two interconvertible forms of the muscarinic cholinergic receptor reciprocally favored by agonists and antagonists. This model has implications both for the understanding of neurotransmitter-receptor interactions generally and for the nature of the biological effects of receptor antagonists.

Animals↗
PubMed1982

The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.

New procedures for the rapid purification and covalent labeling of the beta-adrenergic receptors have been developed that should greatly accelerate progress in the study of these widely distributed adenylate cyclase-coupled receptors. Chromatography of solubilized receptor preparations on a Sepharose-alprenolol affinity gel followed by HPLC on steric exclusion columns lead to rapid (2 days) and high yield (approximately 30%) purification of the receptors from frog erythrocytes. The receptor obtained by these rapid procedures appears to be composed entirely of 58,000 Mr subunit(s) and to be identical to that previously purified by much lengthier procedures [Shorr, R. G. L., Lefkowitz, R. J. & Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826]. A novel, very high affinity, specific beta-adrenergic antagonist, p-aminobenzylcarazolol, has also been synthesized. It can be radioiodinated to theoretical specific radioactivity with 125I (2,200 Ci/mmol). This radioligand, which possesses an arylamine moiety, may then be covalently incorporated into the receptor binding subunit (58,000 Mr peptide) of the frog erythrocyte membranes by the use of the bifunctional photoactive crosslinker N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (SANAH). Covalent incorporation is blocked by various drugs with a strict beta-adrenergic specificity. This suggests that the photoaffinity crosslinking approach may be useful for labeling a variety of small molecule and neurotransmitter receptors when appropriate ligands can be synthesized.

Affinity Labels↗
PubMed1982

Agonist interactions with alpha-adrenergic receptors.

alpha-Adrenergic receptors have been grouped into two major subtypes, termed alpha 1- and alpha 2-receptors. Radioligand binding techniques have been utilized to measure the number of alpha 1- and alpha 2-receptors in a variety of tissues. [3H]Dihydroergocryptine labels the entire alpha-receptor population; the alpha-receptor subtypes may be delineated by constricting competition curves with unlabeled selective antagonists and analyzing the data with computer modeling techniques. Alternatively, alpha 1- and alpha 2-receptors may be directly identified with selective radioligands such as [3H]prazosin and [3H]yohimbine, respectively. For example, rat liver membranes have been shown to contain alpha 1- (80%) and alpha 2- (20%) receptors; the alpha 1-receptors activate glycogen phosphorylase. Radioligands have also been used to probe the mechanism by which alpha 2-receptors may inhibit adenylate cyclase activity. Agonist competition curves with [3H]dihydroergocryptine at eht human platelet's alpha 2-receptor may be resolved into two affinity components, interconvertible by guanine nucleotides. These data suggest the agonist-promoted association of the alpha 2-receptor with an additional membrane component. More direct evidence in favor of this possibility was indicated by the increase in sedimentation velocity of solubilized agonist-labeled receptor on sucrose density gradients.

Adrenergic alpha-Agonists↗
PubMed1982

Clinical physiology of adrenergic receptor regulation.

Radioligand binding studies now permit the direct investigation of the alpha- and beta-adrenergic receptors. The concentration of the receptors in cell membranes is modulated by a wide variety of physiological and pathophysiological variables and may be either increased or decreased in different conditions. Ligand binding techniques have also shed light on the ways in which the adrenergic receptors are coupled to other components of the hormone-sensitive adenylate cyclase system. This "coupling" is also a highly regulated step, control of which importantly influences catecholamine and sensitivity of tissues.

Adenylyl Cyclases↗
PubMed1982

Interactions of agonists with platelet alpha 2-adrenergic receptors.

Epinephrine induces human platelet aggregation by interacting with alpha-adrenergic receptors. These sites were demonstrated by radioligand-binding techniques using the new antagonist ligand, [3H]yohimbine. The sites labeled by [3H]yohimbine had the specificity of alpha 2-receptors with the affinity of yohimbine much greater than prazosin. Epinephrine-mediated inhibition of prostaglandin E1-stimulated adenylate cyclase activity in human platelet lysates was also found to have an alpha 2-receptor specificity. Competition curves of antagonists with [3H]yohimbine indicated a homogeneous population of alpha 2-receptors. In contrast, competition curves of a series of full and partial agonists with [3H]yohimbine were resolved into two distinct affinity states; the ratio of the dissociation constants of agonists for the low and high affinity states was positively correlated with the agonist's intrinsic activity for inhibition of adenylate cyclase. Guanine nucleotides were found to destabilize the high affinity form of the alpha 2-receptors. At high nucleotide concentrations, all high affinity states of the receptor were converted to the low affinity form. The formation of the high affinity agonist-binding state may reflect an interaction between the agonist-receptor complex and an additional membrane component, and probably reflects events involved in alpha 2-receptor-adenylate cyclase coupling.

Adenylyl Cyclases↗
PubMed1982

Mechanisms of hormone receptor-effector coupling: the beta-adrenergic receptor and adenylate cyclase.

The beta-adrenergic receptors that are coupled to adenylate cyclase have provided a model system for studying the mechanisms by which a plasma membrane receptor is coupled to a well-defined biochemical effector. The beta 2-adrenergic receptors from frog erythrocyte membranes have been purified to homogeneity and the ligand-binding subunit has been identified as a glycoprotein with an approximate molecular weight of 58,000. This subunit has also been identified with the use of newly developed photoaffinity reagents. Under the influence of agonist hormones (H), the receptors (R) form transient complexes with another component of this system, termed the nucleotide regulatory protein (N). Formation of this ternary complex, HRN, leads to the dissociation of GDP from N and the interaction of stimulatory GTP with N. N charged with GTP appears to activate the catalytic moiety of the adenylate cyclase enzyme. Although some striking analogies have been found for the mechanisms by which inhibitory receptors interact with adenylate cyclase, much less is known about the molecular properties of the components involved and the ways in which they interact to dampen adenylate cyclase activity in the plasma membrane.

Adenylyl Cyclases↗
PubMed1981

Effect of thyroid status on alpha- and beta-catecholamine responsiveness of hamster adipocytes.

It has been suggested that part of the increased beta-catecholamine responsiveness in hyperthyroid animals is due to a decrease in alpha-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the alpha 2-adrenergic inhibition of adenylate cyclase or the alpha 1-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamsters. No effect of hyperthyroidism was found on the Kd for binding of [3H]dihydroergocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to beta-catecholamines was enhanced in adipocytes from hyperthyroid hamsters, as was lipolysis. However, in adipocytes from hyperthyroid hamsters the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on alpha-catecholamine action by enhances the activation of lipolysis by beta-catecholamine agonists.

Adipose Tissue↗
PubMed1981

Photoaffinity labeling of the beta-adrenergic receptor.

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.

Affinity Labels↗
PubMed1981

Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity.

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.

Adenosine Diphosphate Ribose↗