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M J Lohse

Publications and source records attributed to M J Lohse.

At least 73 records · Page 4Linked to original sources
PubMed1996

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

Adenylyl Cyclases↗
PubMed1996

Mutations of Tyr326 in the beta 2-adrenoceptor disrupt multiple receptor functions.

A tyrosine residue at the cytoplasmic end of the seventh transmembrane helix is conserved in many G-protein-coupled receptors. In the human beta 2-adrenoceptor, this tyrosine (Tyr326) has been proposed to be a specific determinant for agonist-induced receptor sequestration. In order to probe its contribution to the sequestration process we have replaced this tyrosine by alanine (Y326A) or phenylalanine (Y326F). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells. Agonist-induced sequestration was essentially abolished in Y326A receptors and only slightly reduced in Y326F receptors. However, cells expressing Y326A receptors displayed a high percentage of internal receptors under basal conditions while cells expressing wild-type receptors did not. In addition, high-affinity agonist binding and the ability to activate adenylyl cyclase were markedly reduced in Y326A receptors and slightly reduced in Y326F receptors. We conclude that Tyr326 is required for the functional integrity of the beta 2-adrenoceptor and that it may be involved in multiple agonist-induced effects.

Adenylyl Cyclases↗
PubMed1996

Interactions of phosducin with defined G protein beta gamma-subunits.

Phosducin has recently been identified as a cytosolic protein that interacts with the beta gamma-subunits of G proteins and thereby may regulate transmembrane signaling. It is expressed predominantly in the retina but also in many other tissues, which raises the question of its potential specificity for retinal versus nonretinal beta gamma-subunits. We have therefore expressed and purified different combinations of beta- and gamma-subunits from Sf9 cells and have also purified transducin-beta gamma from bovine retina and a mixture of beta gamma complexes from bovine brain. Their interactions with phosducin were determined in a variety of assays for beta gamma function: support of ADP-ribosylation of alpha 0 by pertussis toxin, enhancement of the GTPase activity of alpha 0, and enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase 1 (betaARK1). There were only moderate differences in the effects of the various beta gamma complexes alone on alpha 0, but there were marked differences in their ability to support betaARK1 catalyzed rhodopsin phosphorylation. Phosducin inhibited all beta gamma-mediated effects and showed little specificity toward specific defined beta gamma complexes with the exception of transducin-beta gamma (beta1 gamma1), which was inhibited more efficiently than the other beta gamma combinations. In a direct binding assay, there was no apparent selectivity of phosducin for any beta gamma combination tested. Thus, in contrast to betaARK1, phosducin does not appear to discriminate strongly between different G protein beta- and gamma-subunits.

Adenosine Diphosphate Ribose↗
PubMed1996

Inhibition of G-protein betagamma-subunit functions by phosducin-like protein.

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.

Animals↗
PubMed1996

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.

Amino Acid Sequence↗
PubMed1996

Mechanisms of beta-adrenergic receptor desensitization: from molecular biology to heart failure.

beta-Adrenergic receptors are often studied as prototypes of the large family of G-protein-coupled receptors, which includes many other well-known members such as the muscarinic acetylcholine receptors, but also the receptors for light, taste and olfaction. These receptors are regulated by multiple mechanisms which can affect either their function or their expression to a rapidly changing environment. The most obvious changes are effected by receptor agonists, and this process is called receptor desensitization. On the functional level, the most intriguing and important mechanism of desensitization involves the phosphorylation of beta-adrenergic and homologous receptors by specific receptor kinases, termed the G-protein-coupled receptor kinases (GRKs). This phosphorylation is followed by binding of arrestins to the receptors, which causes uncoupling of receptors and G-proteins and thus results in a loss of receptor function. On the expression level, there appear to be two major pathways leading to a reduction of the receptor number: degradation of the receptors themselves, or reduced receptor synthesis brought about by reduced receptor mRNA levels. Heart failure is accompanied by a markedly reduced responsiveness of the beta-adrenergic receptor system, which in many ways resembles the phenomena seen in agonist-induced receptor desensitization. The levels of beta 1-adrenergic receptors are reduced, and this reduction is paralleled by similar decreases in the levels of the corresponding mRNA. At the same time, the activity and the mRNA levels of one of the GRK-isoforms, GRK2 (which is identical to the beta-adrenergic receptor kinase 1) are increased. These alterations may contribute to the loss of beta-adrenergic receptor responsiveness in heart failure and result in further impairment of cardiac function.

Animals↗
PubMed1996

Analysis of beta-adrenergic receptor mRNA levels in human ventricular biopsy specimens by quantitative polymerase chain reactions: progressive reduction of beta 1-adrenergic receptor mRNA in heart failure.

OBJECTIVES: This study investigated the relation between the severity of heart failure and the extent of the reduction of beta 1-adrenergic receptor messenger ribonucleic acid (mRNA) levels in biopsy specimens from the ventricular septum obtained during cardiac catheterization of patients with various degrees of heart failure. BACKGROUND: Heart failure is accompanied by desensitization of the beta-adrenergic receptor system, which is in part due to downregulation of beta 1-adrenergic receptors. Downregulation of beta 1-adrenergic receptors has been suggested to be caused by reductions in mRNA levels. METHODS: Because biopsy specimens were small and receptor mRNAs not abundant, mRNA levels were determined by quantitative reverse transcription/polymerase chain reactions. This method was validated by measuring synthetic ribonucleic acid (RNA) standards and samples from explanted hearts by solution hybridization assays. Both methods yielded similar results, but the polymerase chain reaction method was approximately 1,000-fold more sensitive. Sources of variations in the polymerase chain reaction were quantitated and found to be best controlled for by determination of the glyceraldehyde phosphate dehydrogenase mRNA as an endogenous control. RESULTS: Beta 1-adrenergic receptor mRNA levels in the biopsy specimens were decreased by 7% in mild (New York Heart Association functional class II), 26% in moderate (functional class III) and > 50% in severe heart failure (functional class IV). There was a good correlation between hemodynamic indicators of heart failure and beta 1-adrenergic receptor mRNA levels. In contrast, beta 2-adrenergic receptor mRNA levels were apparently unaffected by heart failure. CONCLUSIONS: Reduced beta 1-adrenergic receptor mRNA levels occur early in heart failure and can be detected in septal biopsy specimens during right heart catheterization. The reduction in beta 1-adrenergic receptor expression may contribute to further loss of cardiac function.

Adult↗
PubMed1996

G-protein-coupled receptor kinases.

beta-Adrenergic receptors are prototypes of the many G-protein-coupled receptors. Activation and inactivation of these receptors are regulated by multiple mechanisms which can affect either their function or their expression. The most obvious changes of such receptor systems are induced by activation of the receptors themselves by their respective agonists, and this process is called receptor desensitization. One of these mechanisms of desensitization is due to the actions of specific receptor kinases, termed the G-protein-coupled receptor kinases (GRKs). These kinases specifically phosphorylate only the agonist-occupied form of such receptors. This phosphorylation is then followed by binding of inhibitor proteins, called arrestins, to the receptors. Binding of arrestins results in displacement of the G-proteins from the receptors and hence causes uncoupling of receptors and G-proteins. Recent data indicate that the function and subcellular distribution of GRKs is itself subject to regulation. Various mechanisms have evolved to anchor the different GRKs to the plasma membrane. In addition, recent data indicate that GRKs can also associate with intracellular membranes where they may exert as yet unknown functions. A pathophysiological role for GRKs can be inferred from recent studies on heart failure as well as the observation that chronic treatment with various agonists or antagonists for G-protein-coupled receptors results in alterations of GRK expression.

Animals↗
PubMed1996

Activation of beta-adrenergic receptor kinase during myocardial ischemia.

During myocardial ischemia, a local release of noradrenaline coincides with an increased density of beta-adrenergic receptors. The functional activity of these receptors, however, is mainly determined by their state of phosphorylation. The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates and thereby inactivates beta-adrenergic receptors after stimulation by receptor agonists, facilitating the binding of the inhibitor protein beta-arrestin to the receptors. beta ARK activation involves a translocation of the enzyme to the membrane. In the present study, we investigated the density and the functional activity of beta-adrenergic receptors, the enzymatic activity of beta ARK in membranes and cytosol, the mRNA levels of beta ARK-1, and the expression of beta-arrestin during stop-flow and low-flow ischemia in the isolated perfused rat heart. After 60 minutes of stop-flow ischemia, beta-adrenergic receptor density was upregulated, but beta-agonist-mediated adenylate cyclase activity was blunted. Simultaneously, beta ARK activity in the particulate fraction was significantly induced. The increase in beta ARK activity was reversible after inhibition of ischemia-evoked noradrenaline release by desipramine. Also, exposure to externally given noradrenaline increased beta ARK activity in the particulate fraction. Cytosolic beta ARK activity remained largely unchanged during stop-flow or low-flow ischemia. The steady state concentration of beta ARK-1 mRNA increased after 20 minutes of stop-flow ischemia and then returned to baseline values after another 20 minutes. Cardiac ischemia did not alter beta-arrestin levels. During myocardial ischemia, an increase in the number of beta-adrenergic receptors is paralleled by increased membrane activity of the receptor kinase beta ARK. This increased membrane activity may contribute to enhanced receptor phosphorylation and inactivation.

Animals↗
PubMed1996

[Mechanisms of the regulation of adrenergic beta-receptors].

Adrenaline and noradrenaline exert their effects via specific adrenergic receptors. Of the nine known subtypes of these receptors, the beta 1-subtype mediates an increase in cardiac contractility, while the beta 2-subtype is responsible for vasodilatation. These receptors are not just simple signal transducers but components of a complex and highly regulated signalling machinery. A loss of sensitivity of this machinery may be induced by exogenous as well as endogenous stimuli (= desensitisation). Desensitisation of cardiac beta-adrenergic receptors may contribute to contractile dysfunction in chronic heart failure.

Animals↗
PubMed1995

Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors.

beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.

Adenylyl Cyclases↗
PubMed1995

Sequestration and recycling of beta 2-adrenergic receptors permit receptor resensitization.

Stimulation of beta 2-adrenergic receptors in intact cells causes, first, rapid functional uncoupling from Gs, which is triggered by receptor phosphorylation, and, second, somewhat slower sequestration of the receptors to an internal compartment. The present study addresses a possible role of sequestration in the resensitization of desensitized beta 2-adrenergic receptors in human A431 cells. Exposure of these cells to isoproterenol caused rapid phosphorylation, desensitization (as assessed in adenylyl cyclase assays), and sequestration of the receptors. Subsequent removal of the agonist led to recycling of the receptors to the cell surface, dephosphorylation, and restoration of receptor function. These effects occurred without any change in the total receptor number. The rate constant of agonist-induced sequestration was 0.03/min; the rate constant of receptor recycling was 0.06/min and was not markedly altered by the presence of agonist. Blockade of sequestration with concanavalin A or 0.6 M sucrose prevented receptor dephosphorylation as well as receptor resensitization. Inhibition of protein phosphatases with calyculin A caused a similar blockade of beta 2-adrenergic receptor resensitization; the effects of maximally effective concentrations of concanavalin A and calyculin A were not additive. Monensin impaired recycling of desensitized beta 2-adrenergic receptors to the cell surface and also prevented receptor resensitization. We conclude that sequestration of beta 2-adrenergic receptors, followed by dephosphorylation and recycling to the cell surface, may serve to restore the function of desensitized receptors.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1994

Phosducin inhibits receptor phosphorylation by the beta-adrenergic receptor kinase in a PKA-regulated manner.

Homologous or receptor-specific desensitization of beta-adrenergic receptors is thought to be triggered by receptor phosphorylation mediated by the beta-adrenergic receptor kinases (beta ARK). Upon receptor activation, cytosolic beta ARK translocates to the membrane, probably by binding to G-protein beta gamma-subunits. Using the purified proteins reconstituted into phospholipid vesicles we show here that this binding process can be inhibited by phosducin, a cytosolic protein that has recently been described as a regulator of G-protein-mediated signalling. Phosducin appears to complete very effectively with beta ARK for the G-protein beta gamma-subunits. These inhibitory effects of phosducin on receptor phosphorylation are antagonized following phosphorylation of phosducin by protein kinase A. It is proposed that phosducin may act as a regulator of homologous beta-adrenergic receptor desensitization.

Amino Acid Sequence↗
PubMed1994

A1 adenosine receptors expressed in CHO-cells couple to adenylyl cyclase and to phospholipase C.

A1 adenosine receptors are in general coupled to inhibition of adenylyl cyclase, but have more recently been reported to be capable of also activating phospholipase C. The present study was done in order to investigate whether these different effects can be elicited by a single A1 receptor, or whether A1 receptor subtypes have to be invoked. The cDNA of a rat brain A1 adenosine receptor was stably expressed in CHO-cells, resulting in clones with varying receptor densities; a clone expressing 1.9 pmol receptors/mg membrane protein was used for further characterization. The ligand binding properties of the expressed receptors were typical for the rat A1 adenosine receptor. A1 receptor agonists caused a concentration-dependent inhibition of adenylyl cyclase activity in the membranes, with maximal inhibition by 70%. A1 receptor stimulation also caused concentration-dependent stimulation of inositol phosphate generation in these cells, with maximal effects of 300%. Both adenylyl cyclase inhibition and enhancement of inositol phosphate generation were essentially abolished after pretreatment of the cells with pertussis toxin. These results indicate that a single A1 adenosine receptor can couple to two effector pathways, and that both effectors are activated via pertussis toxin sensitive G proteins.

Adenylate Cyclase Toxin↗
PubMed1994

Quantification of beta-adrenoceptors and beta-adrenoceptor kinase on protein and mRNA levels in heart failure.

The alterations of the beta-adrenoceptor adenylyl cyclase are reviewed. In failing myocardium, the down-regulation of beta 1-adrenoceptors is accompanied by a decrease in steady state mRNA levels, as studied with quantitative polymerase chain reactions in dilated and ischaemic cardiomyopathy. The density of beta 2-adrenoceptors and beta 2-adrenoceptor mRNA was unchanged in both pathological conditions compared to non-failing myocardium. In addition to down-regulation of beta 1-receptor protein and mRNA, an increased activity of the beta-adrenoceptor kinase (beta-ARK) was observed. Correspondingly, quantification of beta-ARK mRNA by a 5' and a middle portion PCR-product suggests that increased enzyme activity could be due to increased transcription. In summary, decreased steady state levels of beta 1-adrenoceptor mRNA could contribute to reduced beta-adrenoceptor density in failing myocardium. The known uncoupling of beta-adrenoceptors could be due to an increased mRNA expression and activity of beta-ARK.

Animals↗
PubMed1994

Expression of beta-arrestins and beta-adrenergic receptor kinases in the failing human heart.

The beta-adrenergic receptor system of the failing human heart is markedly desensitized. We have recently postulated that this desensitization may in part be caused by an increase in beta-adrenergic receptor kinase (beta ARK) expression. beta ARK is thought to effect desensitization by acting in concert with an inhibitor protein, called beta-arrestin. Two isoforms have been identified both for beta ARK and for beta-arrestin. In the present study, we have investigated the expression of the individual isoforms of beta-arrestin and of beta ARK in left ventricles from failing and control human hearts. mRNAs for all four proteins, beta-arrestin-1, beta-arrestin-2, beta ARK-1, and beta ARK-2, were identified in human heart. Quantitation by reverse-transcription polymerase chain reactions showed that in heart failure there were no changes of the mRNA levels for beta-arrestin-1 and beta-arrestin-2, a slight (< 50%) increase of the mRNA for beta ARK-2, and a threefold increase for beta ARK-1 mRNA. At the protein level, beta-arrestin-1 was readily detected by Western blotting in human heart. Its absolute values were approximately 350 fmol/mg cytosolic protein, and its expression was not changed in heart failure. beta-Arrestin-2 levels were too low to be detectable using the same methods. beta ARK levels as determined by enzymatic activity were approximately 20 fmol/mg cytosolic protein (beta ARK-1 plus beta ARK-2) and thus almost 20-fold lower than those of beta-arrestin. beta ARK levels were increased approximately twofold in heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗