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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 181 records · Page 10Linked to original sources
PubMed1994

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Adenylate Cyclase Toxin↗
PubMed1994

Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor.

Transgenic mice were created with cardiac-specific overexpression of the beta 2-adrenergic receptor. This resulted in increased basal myocardial adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo; these parameters at baseline in the transgenic animals were equal to those observed in control animals maximally stimulated with isoproterenol. These results illustrate a useful approach for studying the effect of gene expression on cardiac contractility. Because chronic heart failure in humans is accompanied by a reduction in the number of myocardial beta-adrenergic receptors and in inotropic responsiveness, these results suggest a potential gene therapy approach to this disease state.

Adenylyl Cyclases↗
PubMed1994

Binding of G protein beta gamma-subunits to pleckstrin homology domains.

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.

Amino Acid Sequence↗
PubMed1994

Differential desensitization and phosphorylation of three cloned and transfected alpha 2-adrenergic receptor subtypes.

Genes encoding 3 distinct subtypes of human alpha 2-adrenergic receptor are known and are found, respectively, on chromosome 10, 4, and 2 (alpha 2-C10, alpha 2-C4, and alpha 2-C2 adrenergic receptors). All 3 receptors inhibit adenylyl cyclase via Gi proteins. To study and compare their regulatory properties we assessed the ability of each to undergo agonist-promoted desensitization and phosphorylation. When Chinese hamster ovary cells stably expressing each of the three receptor genes were incubated with epinephrine for 20 min, a marked decrease in sensitivity to subsequent agonist-mediated inhibition of adenylyl cyclase was observed for the alpha 2-C10 and alpha 2-C2 receptors but not for the alpha 2-C4 receptors. When similar incubations were performed with 32Pi-labeled cells and the receptors were immunoprecipitated with specific antibodies, alpha 2-C10 and alpha 2-C2 receptors were found to undergo an approximately 3-fold increase in receptor phosphorylation after epinephrine exposure. When transfected into COS cells epinephrine also stimulated phosphorylation of alpha 2-C10 and alpha 2-C2 receptors while having only a slight effect on alpha 2-C4 receptors. Cotransfection of the cells with the cDNA encoding the beta-adrenergic receptor kinase further increased receptor phosphorylation for alpha 2-C10 and alpha 2-C2 receptors while having little or no effect on alpha 2-C4 receptors. Moreover purified and reconstituted recombinant alpha 2-C10 receptors could be phosphorylated in an agonist-dependent fashion whereas alpha 2-C4 receptors could not. These observations suggest receptor subtype-specific differences in susceptibility to regulatory phosphorylation and desensitization.

Adenylyl Cyclase Inhibitors↗
PubMed1994

A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated.

The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.

Adrenergic beta-Agonists↗
PubMed1994

Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases.

A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner and phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function.

Amino Acid Sequence↗
PubMed1994

Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling.

The beta gamma subunits (G beta gamma) of heterotrimeric G proteins modulate the activity of several signal-transducing effector molecules including G protein-coupled receptor kinases. G beta gamma binds to the carboxyl terminus of the beta-adrenergic receptor kinase (beta ARK) and regulates its activity. To investigate the effect of such a G beta gamma-binding domain on heterologous G beta gamma interactions, various receptors that can stimulate phospholipase C and/or type II adenylate cyclase were coexpressed in COS-7 cells with the carboxyl terminus of beta ARK1. Phosphoinositol hydrolysis in response to activation of receptors that stimulate phospholipase C via Gi beta gamma (alpha 2-adrenergic and M2-muscarinic cholinergic receptors) was markedly inhibited by the coexpressed beta ARK1 polypeptide, whereas that mediated by Gq alpha subunits (alpha 1-adrenergic and M1-muscarinic cholinergic receptors) was unaffected. Increased cellular cAMP levels due to stimulation of receptors and coexpressed adenylate cyclase II displayed marked inhibition in the presence of the beta ARK1 polypeptide. Moreover, inhibition of adenylate cyclase produced by alpha 2-adrenergic receptor stimulation (a Gi alpha-mediated process) was unaffected, indicating that the beta ARK1 polypeptide provides a useful tool for distinguishing between G alpha and G beta gamma pathways.

Adenylyl Cyclases↗
PubMed1994

A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2-adrenergic receptor sequestration.

An aromatic residue, tyrosine 326 in the prototypical human beta 2-adrenergic receptor, exists in a highly conserved sequence motif in virtually all members of the G protein-coupled receptor family. The potential role of this conserved aromatic amino acid residue in the cellular processes of sequestration (a rapid internalization of the surface receptor) and down-regulation (a slower loss of total cellular receptors) associated with agonist-mediated desensitization of the beta 2-adrenergic receptor was assessed by replacing tyrosine residue 326 with an alanine residue (beta 2AR-Y326A). This mutation completely abolishes agonist-mediated receptor sequestration without affecting the ability of the receptor to activate maximally adenylyl cyclase, to undergo rapid desensitization, and to down-regulate in response to agonist. The only other major change associated with the mutated receptor is a complete loss of the ability to resensitize following rapid desensitization. These results imply that this tyrosine residue, which is part of a highly conserved sequence motif in G protein-coupled receptors, may be responsible for their agonist-mediated sequestration and that sequestration and down-regulation of the receptor are dissociable phenomena. The lack of resensitization in the sequestration-defective beta 2-adrenergic receptor mutant strongly suggests that the sequestration pathway is an important mechanism by which cells re-establish the normal responsiveness of G protein-coupled receptors following the removal of agonist.

Alanine↗
PubMed1994

Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases.

The thrombin receptor, a member of the seven membrane-spanning superfamily of G-protein coupled receptors, is activated by an irreversible proteolytic mechanism, but signaling by activated thrombin receptors shuts off soon after receptor activation. This shut-off mechanism is thought to be required for concentration-dependent responses to thrombin and an important determinant of the cell's sensitivity to thrombin. We report that the thrombin receptor is rapidly phosphorylated upon activation, consistent with the action of a G-protein-coupled receptor kinase. Moreover, the G-protein coupled receptor kinase BARK2 (beta-adrenergic receptor kinase 2) blocked signaling by thrombin receptors coexpressed in Xenopus oocytes. In this system, rhodopsin kinase was inactive and BARK1 was markedly less effective than BARK2. Thrombin receptor mutants which lacked potential serine and threonine phosphorylation sites in the receptor's cytoplasmic tail were insensitive to inhibition by exogenous BARK2 but did confer concentration-dependent responses to thrombin. Our studies demonstrate that a G-protein coupled receptor kinase can shut off thrombin receptor signaling but that additional mechanism(s) for terminating signaling exist. These studies also reveal functional specificity among G-protein coupled receptor kinases in a novel in vivo reconstitution system and show that heterologous expression of these kinases can be used to manipulate cellular responsiveness.

Amino Acid Sequence↗
PubMed1994

Olfactory desensitization requires membrane targeting of receptor kinase mediated by beta gamma-subunits of heterotrimeric G proteins.

Olfaction is mediated by G protein-coupled receptors. In isolated rat olfactory cilia, odorants such as citralva stimulate a burst of cAMP, which peaks in 50 ms and returns almost to base-line level within 150 ms in the continuing presence of odorant. This desensitization is mediated by the cAMP dependent protein kinase and a specialized G protein-coupled receptor kinase originally termed beta ARK2 (GRK3). In vitro experiments suggest that the prenylated beta gamma-subunits of heterotrimeric G proteins target the cytosolic beta ARK1 (GRK2) enzyme to its membrane bound receptor substrate by binding to sites in its carboxyl terminus. Here we demonstrate that odorants stimulate translocation of GRK3 from cytosol to membranes in isolated rat olfactory cilia. We introduced a glutathione S-transferase-GRK3ct fusion protein, containing the carboxyl-terminal 222 amino acid residues of GRK3, which includes the beta gamma binding site, or a 28-amino acid peptide derived therefrom, into permeabilized cilia preparations. These reagents block odorant-mediated enzyme translocation and desensitization while markedly attenuating odorant-stimulated phosphorylation of olfactory proteins. These findings suggest that beta gamma-subunits may physiologically regulate a G protein-coupled receptor kinase and that enzyme translocation may be a general and required feature of the activity of some members of this enzyme family.

Animals↗
PubMed1994

Distribution of alpha 2-adrenergic receptor subtype gene expression in rat brain.

alpha 2-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. alpha 2-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective alpha 2-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B, and alpha 2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with 35S-labeled RNA probes to map the distribution of alpha 2-adrenoceptor subtype gene expression in rat brain. alpha 2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. alpha 2B mRNA was observed only in the thalamus. alpha 2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the alpha 2-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of alpha 2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of alpha 2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the alpha 2-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.

Animals↗
PubMed1994

Negative antagonists promote an inactive conformation of the beta 2-adrenergic receptor.

The beta 2-adrenergic receptor undergoes isomerization between an inactive conformation (R) and an active conformation (R*). The formation of the active conformation of the receptor molecule can be promoted by adrenergic agonists or by mutations in the third cytoplasmic domain that constitutively activate the receptor. Here we show that, of several beta-adrenergic receptor-blocking drugs tested, only two, ICI 118551 and betaxolol, inhibit the basal signaling activity of the beta 2-adrenergic receptor, thus acting as negative antagonists. We document the molecular properties of the more efficacious ICI 118551; (i) it shows higher affinity for the inactive form of the receptor and (ii) it inhibits the spontaneous formation of a beta-adrenergic receptor kinase substrate by the receptor. These properties are opposite those of adrenergic agonists, indicating that, in a fashion reciprocal to that of agonists, negative antagonists promote the formation of an inactive conformation of the receptor.

Adrenergic beta-Antagonists↗
PubMed1994

Localization of mRNA for three distinct alpha 1-adrenergic receptor subtypes in human tissues: implications for human alpha-adrenergic physiology.

alpha 1-Adrenergic receptors (alpha 1ARs) are virtually ubiquitous in human tissues and mediate important physiological functions as diverse as smooth muscle contraction, glycogenolysis, and myocardial inotropy. At least three alpha 1AR subtypes (alpha 1A/D, alpha 1B, and alpha 1C) have been described using molecular and pharmacological techniques. The identification of species heterogeneity (rat versus rabbit) in alpha 1AR subtype distribution has made it imperative to determine the distribution of alpha 1AR subtypes in human tissues. Accordingly, RNA extracted from human tissues was analyzed using RNase protection assays to determine alpha 1AR subtype expression. Of the cloned alpha 1ARs, alpha 1CAR mRNA predominates in many human tissues (heart, liver, cerebellum, and cerebral cortex), in contrast to its restricted distribution in both rats and rabbits. alpha (1B)AR mRNA is present in highest concentrations in human spleen, kidney, and fetal brain. alpha 1A/DAR mRNA is present in highest concentrations in human aorta and cerebral cortex. Hence, alpha 1AR subtype mRNA distribution is tissue selective and differs from that reported for rats and rabbits. These results have potentially significant implications for understanding human adrenergic physiology and are important for the rational development of alpha 1AR subtype-selective drugs.

Adrenal Glands↗