Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta 2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta ARK phosphorylation of beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.
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PMID:Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms. 134 86

1. The function of trout RBC Na+/H+ antiport is unrelated to cell volume or cell pH regulation. Its role is to improve oxygen transport capacity when the supply of oxygen becomes limited. 2. Antiport activation, mediated by cAMP, promotes complex changes in blood pH which have been analyzed in vivo and in vitro. 3. The regulation of antiport (activation, desensitization, control by molecular oxygen and by a newly discovered cytosolic protein, arrestin) is presented. 4. Molecular cloning of the antiport shows that two typical site motifs of phosphorylation by cAMP-dependent protein kinase are localized on the cytoplasmic region.
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PMID:Regulation of Na+/H+ exchange and pH in erythrocytes of fish. 135 21

Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR was enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5.
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PMID:Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. 762 2

Calcium (Ca2+) plays an integral role in the light response of the photoreceptors in both vertebrate and invertebrate organisms. In the ventral eye of the horseshoe crab, Limulus polyphemus, a flash of light delivered to a dark-adapted photoreceptor stimulates a rapid rise in intracellular free calcium concentration ([Ca2+]i), which in turn mediates light adaptation. It has previously been demonstrated that in Limulus photoreceptors light, via Ca2+, activates a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase which increases the phosphorylation of arrestin. We now have identified biochemically, a calcium/calmodulin-dependent protein phosphatase (Ca2+/CaM PP) in homogenates of the Limulus lateral and ventral eye, brain, and lateral optic nerve using as a substrate, a 32P-labeled peptide fragment of the regulatory subunit of cAMP-dependent protein kinase (RII). This protein phosphatase shares biochemical properties with calcineurin, a Ca2+/CaM-dependent protein phosphatase (type-2B). Its activity is enhanced by Ca2+, calmodulin and Mn2+; and is inhibited by mastoparan, a calmodulin antagonist, and a synthetic peptide corresponding to the autoinhibitory domain of mammalian calcineurin. Most importantly, light regulates the Ca2+/CaM PP activity in the lateral eye. While there is no difference in basal activity in long-term dark- or light-adapted preparations, Ca2+ enhances Ca2+/CaM PP activity only in long-term light-adapted eyes.
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PMID:Characterization of a calcium/calmodulin-dependent protein phosphatase in the Limulus nervous tissue and its light regulation in the lateral eye. 794 99

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.
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PMID:Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor. 1053 54

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.
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PMID:Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability. 1470 42

Agonist-stimulated desensitization of the beta2-adrenergic receptor (beta2AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to beta-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human beta2AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E1, and forskolin at maximum concentrations stimulated phosphorylation of the beta2AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still approximately 200-fold greater than for the PKA site. The EC50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the beta2AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agonist-stimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine > or = formoterol = fenoterol > terbutaline = zinterol = albuterol > salmeterol > dobutamine > or = ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous beta2AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.
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PMID:Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies. 1472 51

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the beta2AR (beta2-adrenergic receptor). In the present paper, we show that challenge of HEK-293beta2 [human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP (green fluorescent protein)] cells with the beta-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the beta2AR by GRK2, membrane translocation of beta-arrestin and internalization of beta2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293beta2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the beta2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the beta2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.
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PMID:Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293beta2 cells and cardiac myocytes. 1635 65

Beta2-ARs (beta2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta2-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the beta-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta2-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu215-His220, which showed little or no PDE4D5 binding, but was still recruited to the beta2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta2-AR regulation.
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PMID:Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of beta-arrestin using spot-immobilized peptide arrays. 1728 40

The PDE4 (phosphodiesterase-4) enzyme family consists of a distinct array of N-terminal splice variant isoforms arising from four subfamily genes (4A, 4B, 4C and 4D). These all hydrolyse specifically the intracellular second messenger cAMP. Although identical in catalytic function, each isoform appears to serve a non-superfluous regulatory role. For example, a beta-arrestin-sequestered subpopulation of the PDE4D5 isoform specifically regulates the phosphorylation of the beta(2)-AR (beta(2)-adrenergic receptor) by PKA (protein kinase A; also called cAMP-dependent protein kinase). This was elucidated by the use of novel technologies, including dominant-negative approaches, siRNA (small interfering RNA) knockdown and spot-immobilized peptide array analyses. Functional phenotypes uncovered using these methodologies have shown that beta-arrestin-sequestered PDE4D5 shapes the spatial cAMP gradient around the membrane-bound beta(2)-AR, regulating its phosphorylation by PKA and its ability to activate ERK (extracellular-signal-regulated kinase) through G(i) in cardiomyocytes and HEK-293 (human embryonic kidney)-B2 cells. This approach has provided the very first identification of a non-redundant and specific role for a PDE isoform. The fact that phenotypes can be uncovered by displacing PDE4 isoforms from specific anchor sites using dominant-negative constructs and cell-permeable peptides points to novel means for developing therapeutics aimed at disrupting specifically sequestered PDE isoforms and even specifically sequestered subpopulations of individual isoforms.
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PMID:cAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling. 1795 50


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