Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The existence of lipolytic beta-adrenoceptor (BAR) resistance was investigated in vivo and in isolated abdominal subcutaneous adipocytes in 65 healthy and drug-free subjects. The concentration of isoprenaline (nonselective BAR agonist) causing half-maximum lipolysis effect (ED50) varied bimodally and 10(6)-fold between individuals but was almost constant in the same subject when measured two times at rest or before and 30 min after exercise. The subjects were categorized as having either high or low isoprenaline sensitivity. The former group had a 50% reduced in vivo lipolytic response to exercise and mental stress, despite a 50% increased plasma noradrenaline response (P < 0.01) and a 350% increased plasma adrenaline response (P < 0.02). In fat cells the lipolytic ED50 values for noradrenaline and terbutaline (BAR2 agonist) were 10 times lower (P < 0.001) in low-sensitive subjects, but the maximum lipolytic actions of these agents (and of isoprenaline) were similar in both groups. The action on lipolysis of dobutamine (BAR1 agonist), forskolin (stimulating adenylate cyclase), dibutyryl cyclic AMP (activating protein kinase), clonidine (alpha 2-adrenergic agonist), or phenyl isopropyladenosine (adenosine receptor agonist) were almost identical in high- and low-sensitivity subjects. ED50 for isoprenaline correlated with ED50 for terbutaline (r = 0.75), but not with ED50 for dobutamine. In high-sensitivity subjects the number of BAR2 was almost three-fold increased (P < 0.002) and the steady-state adipocyte mRNA level for BAR2 was sixfold increased (P < 0.005). BAR2 affinity as well as BAR1 number, affinity and mRNA expression were similar in both groups. In 11 cholecystectomy patients (otherwise healthy) lipolytic ED50 for beta agonists correlated in omental and subcutaneous fat cells (r = 0.85 for isoprenaline; r = 0.95 for terbutaline). In conclusion, lipolytic resistance to catecholamines is present in vivo in apparently healthy subjects due to reduced expression of BAR2 in adipocytes.
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PMID:Lipolytic catecholamine resistance due to decreased beta 2-adrenoceptor expression in fat cells. 133 70

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
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PMID:Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants. 862 39

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores--melanocyte stimulating hormone, light, (-) norepinephrine, 5-hydroxytrptamine, and the beta2-adrenergic receptor--were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP3 in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide beta responses were blocked only by H89, predicting coupling to adenylyl cyclase.
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PMID:Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C. 869 26

We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.
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PMID:Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase. 870 33

Phosducin is a member of the large group of proteins that bind to G-protein betagamma-subunits (Gbetagamma) and whose biological functions are often unknown. Human A431 cells do not contain detectable amounts of phosducin. We generated A431 cells expressing phosducin at a level of approximately 1 pmol/mg of cytosolic protein, which is approximately 10% of the phosducin level in brain. cAMP accumulation in response to beta2-adrenergic receptor agonists was enhanced at early times in phosducin-expressing cells, but reached a lower plateau than in control cells. Permeabilization of the cells with digitonin did not change this pattern, but allowed the introduction of specific inhibitors: antibodies to phosducin abolished all differences between the two cell lines. Inhibitors of the beta-adrenergic receptor kinase abolished the differences at early time points. An almost complete loss of beta2-adrenergic receptor desensitization in the phosducin-expressing cells was also observed when intact cells were desensitized and receptor function was then determined in membrane preparations. Inhibition of protein kinase A accentuated the effects of phosducin, suggesting that also in vivo phosducin is regulated by this kinase. These data indicate that phosducin affects G-protein-mediated signaling in at least two ways: it dampens the overall responsiveness, and it impairs the rapid desensitization mediated by the beta-adrenergic receptor kinase.
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PMID:Expression of phosducin in a phosducin-negative cell line reveals functions of a Gbetagamma-binding protein. 879 22

The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. In an effort to gain a better understanding of the regulation of M2 receptors, we have investigated homologous and heterologous regulation of M2 muscarinic receptor protein and gene expression in human embryonic lung fibroblasts (HEL 299 cells). HEL 299 cells constitutively express m2 receptors, with no evidence of other muscarinic receptor subtypes. We have shown that M2 receptors in these cells can be down-regulated by muscarinic and beta2-adrenergic receptor agonists. Unlike the down-regulation mediated by muscarinic and beta-adrenergic stimulation, activation of PKC with PDBu was mediated through changes in m2 muscarinic receptor mRNA through reduced gene transcription. Because of the inflammatory nature of asthma, we have focused on delineating the interactions between cytokines and M2 receptors in an attempt to define potential endogenous modulators of M2 receptor expression. We have shown that the multi-functional cytokine, transforming growth factor beta1 (TGF-beta1), which is involved in several inflammatory conditions induces desensitization and down-regulation of M2 muscarinic receptor protein and gene expression that was mediated through a reduction in the rate of m2 receptor gene transcription. Other cytokines of interest are tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) which are elevated in asthma. We have demonstrated that TNF-alpha and IL-1beta synergise to induce down-regulation of M2 muscarinic receptor protein and mRNA which was associated with functional desensitization of the receptor protein. The M2 receptor mRNA down-regulation appeared to be mediated through a reduction in the rate of m2 receptor gene transcription which may be dependent on the transcription and translation of unknown protein factor(s). Moreover, a role of PKA and ceramide pathways in M2 receptor regulation is suggested. Collectively, our work provides a mechanistic explanation of previous reports indicating altered function of M2 receptors in asthma. Ours results also suggest that the expression of this receptor subtype may be under the control of a cytokine network at the airways.
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PMID:Regulation of muscarinic M2 receptors. 912 42

We report a strategy of tumor growth inhibition based on the expression of a foreign protein with both potential anti-proliferative and immunogenic properties. To validate our approach, we used 2 ras-mutated murine carcinoma cell lines (carB and C57/PDV) transfected with the gene encoding a fusion protein containing the human beta2-adrenergic receptor and the alpha subunit of the Gs protein (beta2Gs). We previously showed that the sustained activation of the beta2Gs fusion protein expressed in carB cells (carB beta2Gs cells) induced a cAMP-dependent inhibition of cell growth in vitro. Here, we observed inhibition of tumor growth after s.c. inoculation of 2 carB beta2Gs clones (10C2 and 20F4) in syngeneic ICFW mice. We thus selected 3 C57/PDV beta2Gs clones (2D3, 5F3 and 1G1) in which activation of the fusion protein was not efficiently coupled to the cAMP-PKA signaling pathway. Contrasting with carB beta2Gs clones, activation of the fusion protein in these C57/PDV beta2Gs clones did not have any anti-proliferative effect in vitro. Therefore, they were good candidates to assess the immunogenic property of the fusion protein. Accordingly, none of the C57/PDV beta2Gs clones formed tumors in immunocompetent syngeneic C57BL/6 mice, while they were still tumorigenic in nude mice. Most interestingly, all of the beta2Gs clones that did not form tumors, from both cell lines, provided protection against respective wild-type tumor development. Our results show that expression of the beta2Gs fusion protein in cancer cells elicits inhibition of cell proliferation and/or immune rejection of both beta2Gs-modified and wild-type tumor cells.
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PMID:Human beta2-adrenergic receptor/GS alpha fusion protein, expressed in 2 ras-dependent murine carcinoma cell lines, prevents tumor growth in syngeneic mice. 918 7

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.
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PMID:Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes. 919 91

Previous studies indicated that partial agonists cause less desensitization of the beta2-adrenergic receptor (betaAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of betaAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the betaAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified betaAR behaved identically to the wild type receptor with regard to agonist Kd, coupling efficiency, and desensitization. The coupling efficiencies for betaAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of betaAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine >/= fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the betaAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, betaAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of betaAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of betaAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of betaARK phosphorylation of the betaAR.
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PMID:beta2-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists. 929 36

Many of the G-protein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through several different classes of G protein. For example, although most of the actions of the prototype beta2-adrenergic receptor are mediated through Gs proteins and the cyclic-AMP-dependent protein kinase (PKA) system, beta-adrenergic receptors can also couple to Gi proteins. Here we investigate the mechanism that controls the specificity of this coupling. We show that in HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta2-adrenergic receptor is mediated by the betagamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras. Activation of this pathway by the beta2-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase, the enzyme that generates cAMP, but not MAP kinase. Our results demonstrate that a mechanism previously shown to mediate uncoupling of the beta2-adrenergic receptor from Gs and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to 'switch' coupling of this receptor from Gs to Gi and initiate a new set of signalling events.
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PMID:Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. 936 96


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