ABSTRACT
Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB(1) and CB(2) receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [(35)S]GTPgammaS binding. Western blot analysis showed that CB(1) receptor immunoreactivity was significantly lower in glioblastoma multiforme (-43%, n=10; p<0.05) than in normal post-mortem brain tissue (n=16). No significant differences were found for astrocytoma (n=6) and meningioma (n=8) samples. Conversely, CB(2) receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n=9; p<0.05) and astrocytoma (471%, n=4; p<0.05) than in control brain tissue (n=10). Finally, the maximal stimulation of [(35)S]GTPgammaS binding by WIN 55,212-2 was significantly lower in glioblastomas (134+/-4%) than in control membranes (183+/-2%; p<0.05). The basal [(35)S]GTPgammaS binding and the EC(50) values were not significantly different between both groups. The present results demonstrate opposite changes in CB(1) and CB(2) receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.
Subject(s)
Brain Neoplasms/chemistry , Glioma/chemistry , Receptor, Cannabinoid, CB1/analysis , Receptor, Cannabinoid, CB2/analysis , Adult , Aged , Astrocytoma/chemistry , Benzoxazines/pharmacology , Blotting, Western , Brain Chemistry , Cannabinoids/pharmacology , Cell Membrane/chemistry , Female , Glioblastoma/chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Male , Meningioma/chemistry , Middle Aged , Morpholines/pharmacology , Naphthalenes/pharmacology , Receptor, Cannabinoid, CB1/drug effects , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/drug effects , Receptor, Cannabinoid, CB2/metabolism , Sulfur RadioisotopesABSTRACT
The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3zeta, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585-712). Expression of this fragment suppresses receptor-induced cofilin-PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions.
Subject(s)
Nuclear Proteins/metabolism , Phospholipase D/metabolism , Protein Processing, Post-Translational/physiology , Receptors, Muscarinic/metabolism , 14-3-3 Proteins , Actins/genetics , Actins/metabolism , Cell Line , Gene Expression , Humans , Lim Kinases , Mutation , Nuclear Proteins/genetics , Phospholipase D/genetics , Phosphorylation , Protein Binding/physiology , Protein Kinases/genetics , Protein Kinases/metabolism , Receptors, Muscarinic/geneticsABSTRACT
The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical G(s)-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation.
