Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0017638 (glioma)
 30,880 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor responses. NG108-15 mouse neuroblastoma X rat glioma cells express a wide range of G protein-coupled receptors and significant levels of GRK2. Therefore, to determine the role of GRK2 in agonist-induced desensitization of various G(s)-coupled receptors in NG108-15 cells, we stably transfected cells with a dominant negative mutant GRK2 construct (Lys220Arg). In homogenates prepared from cells overexpressing the dominant negative mutant GRK2, the acute stimulation of adenylyl cyclase by various receptor and nonreceptor agonists was the same as in control cells stably transfected with plasmid only. NG108-15 cells express both A2a and A2b adenosine receptors, which mediate activation of adenylyl cyclase, with both of these responses being subject to agonist-induced desensitization with a t1/2 of 15-20 min. In dominant negative mutant GRK2 cells, the rates of desensitization of A2a and A2b receptor-stimulated adenylyl cyclase were markedly slower than in plasmid transfected controls, with the latter being similar to wild-type cells. After a 20-min treatment with an adenosine agonist, the desensitization of A2a and A2b receptor-stimulated adenylyl cyclase in dominant negative mutant GRK2 cells was less than half that seen in plasmid transfected control cells. On the other hand, the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was the same in dominant negative mutant GRK2 cells as in plasmid transfected control cells. These results indicate that in intact cells, GRK2 may mediate the desensitization of adenosine A2 receptors. Furthermore, there seems to be selectivity of GRK2 action between G(s)-coupled receptors because the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was not affected by dominant negative mutant GRK2 overexpression.
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PMID:A dominant negative mutant of the G protein-coupled receptor kinase 2 selectively attenuates adenosine A2 receptor desensitization. 918 65

The wild-type delta opioid receptor (DOR) and a carboxyl terminus-truncated mutant DOR lacking the last 31 amino acids (DOR-T) were expressed in neuroblastoma x glioma hybrid NG108-15 cells to investigate the role of the carboxyl terminus of DOR in agonist-dependent receptor phosphorylation. Stimulation of the cells with delta specific agonists significantly induced DOR phosphorylation whereas no phosphorylation of DOR-T was detected under the same conditions. Neither overexpression of G protein-coupled receptor kinases (GRK2 or GRK5) nor activation of protein kinase C promoted agonist-induced phosphorylation of DOR-T, in contrast to their strong stimulatory effect on the agonist-dependent phosphorylation of DOR. Furthermore, DOR-T failed to be internalized after agonist stimulation, probably due to its inability to be phosphorylated. Our results indicate that the carboxyl terminus of DOR is required for agonist-dependent receptor phosphorylation and the phosphorylation site(s) of DOR is likely located at its carboxyl terminus.
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PMID:Carboxyl terminus of delta opioid receptor is required for agonist-dependent receptor phosphorylation. 929 54

Beta-adrenergic receptor kinase (betaARK, EC 2.7.1.-) has been implicated in the phosphorylation of G protein-coupled receptors, including opioid receptors. Since delta-opioid receptors of mouse neuroblastoma x rat glioma hybrid cells (NG 108-15) desensitize upon activation, this investigation was designed to find out whether NG 108-15 cells contain betaARK activity. Using the reverse transcription polymerase chain reaction technique, we identified two mRNAs, one coding for rat betaARK1 and the other for rat betaARK2. No hint was found for the presence of mouse betaARK. Examining the cytosolic betaARK activity in these hybrid cells using rhodopsin as substrate, we found a strict functional dependence on the presence of exogenous G protein subunit Gbetagamma. This relationship reflects a characteristic for betaARK1 and 2 out of the known G protein-coupled receptor kinases. Finally, highly purified recombinant betaARK1 proved active to phosphorylate enriched delta-opioid receptor preparations in an opioid agonist-dependent manner. The results reported here provide the basis to study more closely the molecular function of G protein-coupled receptor kinases in a cell line (NG 108-15) most frequently used to investigate acute and chronic opioid actions.
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PMID:Rat beta-adrenergic receptor kinases 1 and 2 in mouse neuroblastoma X rat glioma NG 108-15 hybrid cells. 941 31

1. To approach the mechanisms underlying desensitization of the opioid receptor-mediated Ca2+ channel inhibition, the effects of prolonged application of [D-Ala2, D-Leu5]enkephalin (DADLE) on Ba2+ currents (I(Ba)) through Ca2+ channels were analysed in NG108-15 neuroblastoma x glioma hybrid cells. 2. Inhibition of I(Ba) by 100 nM DADLE desensitized by 57% with a time constant of 4.4 min. 3. Maximal desensitization of the delta-opioid receptor-Ca2+ channel coupling was attained by 1 microM DADLE. The EC50 value for desensitization was estimated to be 78 nM. 4. RNA blot hybridization analysis and immunoblot analysis revealed the expression of beta-adrenoceptor kinase-1 (betaARK1) in NG108-15 cells. 5. Heparin, an inhibitor of betaARK, significantly reduced the magnitude and rate of desensitization, whereas Rp-cyclic AMPS and PKI (14-24)amide, inhibitors of cyclic AMP-dependent protein kinase (PKA), or long-term treatment with phorbol 12-myristate 13-acetate to induce down-regulation of protein kinase C (PKC) had no significant effect. 6. Recovery from desensitization (resensitization) proceeded with a time constant of 6.7 min. Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, significantly attenuated the degree of resensitization. 7. In summary, we have characterized the time course and concentration-dependence of the desensitization of DADLE-induced I(Ba) inhibition in NG108-15 cells. This desensitization was reversible after removal of DADLE. It is suggested that betaARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.
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PMID:Desensitization and resensitization of delta-opioid receptor-mediated Ca2+ channel inhibition in NG108-15 cells. 955 94

The effect of endocytosis inhibitors on 5-hydroxytryptamine(2A) (5-HT(2A)) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT(2A) receptors. In HEK-293 cells, 5-HT(2A) receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319-418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, beta 2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319-418), conA, and PAO each resulted in the potentiation of 5-HT(2A) and beta-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319-418) on 5-HT(2A) receptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although beta 2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT(2A) receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT(2A) receptor resensitization in HEK-293 cells. In addition, Arr2(319-418) had no effect on 5-HT(2A) receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the beta 2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319-418) significantly reduced 5-HT(2A) receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT(2A) receptor desensitization and resensitization exist in HEK-293 cells.
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PMID:Cell-type specific effects of endocytosis inhibitors on 5-hydroxytryptamine(2A) receptor desensitization and resensitization reveal an arrestin-, GRK2-, and GRK5-independent mode of regulation in human embryonic kidney 293 cells. 1164 30

Activation of G protein-coupled receptors (GPCRs) may bring about their disappearance from the cell membrane, and it is commonly accepted that G protein-coupled receptor kinases (GRKs) play a key function in this mechanism. Opioid receptors belong to the family of GPCRs and are substrates of GRKs. We examined the fate of delta- and mu-opioid receptors and GRK2 and 3 in living cells during the process of receptor sequestration, using laser scanning microscopy. For visualization purposes, receptors and kinases were tagged at their respective C terminus with a fluorophore. The opioid receptors were fused to enhanced green fluorescence protein (EGFP), and the GRKs were linked to red fluorescence protein (DsRed). The cDNAs of these constructs served for transfection of human embryonic kidney 293 cells and neuroblastoma-glioma hybrid cells (NG 108-15), respectively. We report that activation of delta-opioid-EGFP receptors triggers a rapid translocation of cytosolic GRK-DsRed toward the cell membrane, which in turn releases vesicles carrying both green fluorescent delta-receptors and red fluorescent GRKs. Phosducin, a Gbetagamma scavenger, completely prevents translocation of GRKs and the formation of vesicles. In analogous experiments with mu-opioid receptors fused to EGFP, we observed that receptor activation also discharges green fluorescent vesicles. In contrast to delta-receptors, mu-receptors failed to trigger accumulation of GRK2 or 3 at the membrane, and no cointernalization of mu-opioid receptors with GRK2 or 3 was observed. The results suggest that delta-opioid receptors, but not mu-receptors, cointernalize with GRK2 or 3.
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PMID:Opioid receptor types selectively cointernalize with G protein-coupled receptor kinases 2 and 3. 1180 94

We have examined the cellular processes underlying the desensitization of the 5-hydroxytryptamine (5-HT)(2A) receptor induced by agonist or antagonist exposure. Treatment of C6 glioma cells with either 5-HT or the 5-HT(2A) receptor antagonist ketanserin resulted in an attenuation in 5-HT(2A) receptor function, specifically the accumulation of inositol phosphates stimulated by the partial agonist quipazine. 5-HT-induced desensitization of the 5-HT(2A) receptor involved receptor internalization through a clathrin- and dynamin-dependent process because it was prevented by concanavalin A, monodansylcadaverine, and by expression of the dominant negative mutants beta-arrestin (319-418) and dynamin K44A. Although short-term (i.e., 10 min) 5-HT and ketanserin exposure resulted in the same degree of desensitization, ketanserin-induced desensitization was not prevented by these agents and did not involve receptor internalization. In contrast, prolonged ketanserin exposure (i.e., 2 h) resulted in 5-HT(2A) receptor internalization through a clathrin- and dynamin-dependent process, as was observed after agonist treatment. Inhibitors of protein kinase C or calcium-calmodulin kinase II did not attenuate or prevent 5-HT-induced desensitization of the receptor. 5-HT(2A) receptor desensitization induced by 5-HT and prolonged ketanserin treatment, but not by short-term ketanserin treatment, was prevented by the expression of the dominant negative mutant of G protein-coupled receptor kinase (GRK)2, GRK2-K220R, and by an anti-GRK2/3 antibody. Our data indicate a dual mechanism of early and late desensitization by the antagonist ketanserin. Short-term ketanserin treatment reduced the specific binding of the agonist radioligand [(125)I](+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([(125)I]DOI) and the ability of 5'-guanylylimidodiphosphate to attenuate this binding, suggesting that at the early stage of antagonist-induced desensitization the capacity of the 5-HT(2A) receptor to couple to G protein is impaired.
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PMID:Mechanisms of ligand-induced desensitization of the 5-hydroxytryptamine(2A) receptor. 1180 6

1. To determine the role of G protein-coupled receptor kinases (GRKs) in the regulation of endogenous secretin receptor responsiveness, we have transiently overexpressed both wild-type (WT) and dominant negative mutant (DNM) GRKs in NG108-15 mouse neuroblastoma x rat glioma hybrid cells and investigated the effects of this on agonist-stimulated adenylyl cyclase activity. 2. Overexpression of WT GRK6 selectively inhibited secretin-stimulated cyclic AMP accumulation (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.1+/-2.0 and 6.2+/- 0.8 in control and WT GRK overexpressing cells, respectively) without affecting cyclic AMP responses mediated by the adenosine A(2) receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) or the prostanoid-IP receptor agonist iloprost, or the direct activator of adenylyl cyclase, forskolin. On the other hand DNM GRK6 (Lys(215)Arg) overexpression produced the opposite effect--a selective increase in the secretin-stimulated cyclic AMP response was observed in cells overexpressing DNM GRK6 compared to plasmid-transfected cells (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.6+/-2.7 and 29.6+/-5.6 for control and DNM GRK6-overexpressing cells, respectively). 3. Overexpression of WT GRK5 likewise inhibited the secretin-stimulated cyclic AMP response, however, this effect was not as selective as with GRK6, since adenosine A(2) receptor responsiveness was also suppressed by GRK5 overexpression. Unlike DNM GRK6, overexpression of DNM GRK5 failed to modulate secretin or A(2) adenosine receptor signalling suggesting that endogenous GRK5 is unlikely to regulate desensitization of these receptors in NG108-15 cells. 4. Overexpression of WT GRK2 did not affect secretin-stimulated cyclic AMP accumulation. Instead, GRK2 overexpression selectively inhibited A(2) adenosine receptor responsiveness, confirming our previous findings. 5. Together these results suggest a selective role of endogenous GRK6 in regulating secretin receptor responsiveness in NG108-15 cells. In addition, these data indicate that GRKs exert a surprising degree of selectivity in the regulation of natively expressed GPCR responses.
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PMID:G protein-coupled receptor kinase 6 (GRK6) selectively regulates endogenous secretin receptor responsiveness in NG108-15 cells. 1259 20

Searching for amplifications in low grade and high grade gliomas we observed an interesting correlation between the recurrence and progression of astrocytic low grade gliomas and the amplification of the STK15 gene located in the chromosomal region 20q13. Chromosome copy gains in this region have been reported previously in astrocytic gliomas and glioma cell lines and in many cancer types including breast, colorectal and ovarian cancers. The putative serine/threonine kinase STK15 has been reported to be amplified and overexpressed in breast cancer cell lines and colorectal cancer. Another candidate gene located in this region is PTPN1, a protein tyrosine phosphatase non-receptor type 1 that might play a role in cell cycle control. We used comparative PCR for quantitative DNA analysis to search for STK15 and PTPN1 amplification in gliomas previously characterized by CGH. Five out of 16 tumors (31%) of different WHO grade (1x grade II, 1x grade III, 3x grade IV) showed DNA amplification of STK15 whereas we did not detect amplification of PTPN1. We hypothesize that amplification of the STK15 gene may be a non-random genetic alteration in human gliomas playing a role in the genetic pathways of tumorigenesis.
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PMID:The putative serine/threonine kinase gene STK15 on chromosome 20q13.2 is amplified in human gliomas. 1288 93

Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J. Neurochem. 89, 442.]. In the present study, the mechanisms involved in this response were further elucidated. In cells transfected with the Gbetagamma-scavenger beta-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gbetagamma-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCzeta decreased basal and 2MeSADP-stimulated phosphorylation of PKB indicating a role for this PKC isoform in PKB signalling. Although the increased PKB phosphorylation was abolished in the presence of the IGF-I receptor tyrosine kinase inhibitor AG 1024, 2MeSADP did not significantly increase receptor phosphorylation. Nevertheless, phosphorylation of a 120 kDa IGF-I receptor-associated protein was observed. The latter protein was identified by MALDI-TOF/TOF-MS as the proline-rich tyrosine kinase 2 (Pyk2) that co-operates with Src in a PLD2-dependent manner. Consistent with the signalling towards Rap1 and PKB, activation of Pyk2 was abrogated by Ca2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y12 and IGF-I receptors that proceeds through Gbetagamma-, Ca2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.
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PMID:P2Y12 receptor signalling towards PKB proceeds through IGF-I receptor cross-talk and requires activation of Src, Pyk2 and Rap1. 1623 84


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