PubMed Health⌕ Search

Biomedical subjects

R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

606 records · Page 34Linked to original sources
PubMedDate not supplied

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin.

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.

Amino Acid Sequence↗
PubMedDate not supplied

Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase.

The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic receptor (beta AR) for catecholamines or the 'light receptor' rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase). Molecular cloning has revealed that the beta AR shares significant sequence and three-dimensional homology with rhodopsin. The function of the beta AR is diminished by exposure to stimulatory agonists, leading to desensitization. Similarly, 'light adaptation' involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin. An analogous enzyme (termed beta AR kinase or beta ARK) phosphorylates only the agonist-occupied beta AR. We report here that beta ARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied beta AR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar.

Animals↗
PubMedDate not supplied

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation.

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.

Adenylyl Cyclases↗
PubMedDate not supplied

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins.

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.

Amino Acid Sequence↗
PubMedDate not supplied

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

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.

Animals↗
PubMedDate not supplied

Subtype specificity of alpha-adrenergic receptors in rat heart.

Recent studies suggest that two subtypes of alpha-adrenergic receptors (alpha 1 and alpha 2) can be distinguished on the basis of the differential affinities of certain adrenergic ligands for each subtype. We have investigated the binding characteristics of several such ligands for alpha-adrenergic receptors in membrane preparations derived from rat heart. alpha-Adrenergic antagonists competed for binding sites identified by the nonsubtype selective alpha-adrenergic ligand [3H]dihydroergocryptine (DHE) in the order of potency expected for alpha 1-receptors, namely, prazosin (EC50 = 0.35 nM) greater than phentolamine (EC50 = 37 nM) greater than yohimbine (EC50 = 918 nM). Furthermore, the nonsubtype selective radioligand [3H]DHE identified a quantitatively similar number of specific binding sites in rat cardiac membranes as the alpha 1-selective radioligand [3H]prazosin (33 and 36 fmoles/mg protein, respectively), while the alpha 2-selective ligand [3H]clonidine at concentrations up to 20 nM demonstrated negligible specific binding. We conclude that the alpha-adrenergic receptors of rat heart homogenates demonstrate binding characteristics typical of alpha 1-receptors. While we cannot exclude the presence of small numbers of alpha-2 receptors, the similar number of binding sites identified at saturation for the alpha 1-specific ligand [3H]prazosin and for the nonsubtype selective ligand [3H]DHE supports the hypothesis that the alpha-receptors of rat heart are predominantly of the alpha 1-subtype.

Animals↗
PubMedDate not supplied

Myocardial G protein-coupled receptor kinases: implications for heart failure therapy.

The beta-adrenergic signaling cascade is an important regulator of myocardial function. Significant alterations of this pathway are associated with several cardiovascular diseases, including congestive heart failure (CHF). Included in these alterations is increased activity and expression of G protein-coupled receptor kinases (GRKs), such as the beta-adrenergic receptor kinase (beta ARK1), which phosphorylate and desensitize beta-adrenergic receptors (beta ARs). A body of evidence is accumulating that suggests that GRKs, in particular beta ARK1, are critical determinants of cardiac function under normal conditions and in disease states. Transgenic mice with myocardial-targeted alterations of GRK activity have shown profound changes in the in vivo functional performance of the heart. Included in these studies is the compelling finding that inhibition of beta ARK1 activity or expression significantly enhances cardiac function and potentiates beta AR signaling in failing cardiomyocytes. This article summarizes the advances made in the study of beta ARK1 in the heart and addresses its potential as a novel therapeutic target for CHF.

Animals↗
PubMedDate not supplied

Molecular biology of adrenergic receptors in the rat and frog central nervous system.

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.

Affinity Labels↗
PubMedDate not supplied

Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Adrenergic beta-Agonists↗
PubMedDate not supplied

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Animals↗
PubMedDate not supplied

Adrenergic receptors.

Explore the source record for details and available documents.

Amino Acid Sequence↗