Abstract
Activation of group I metabotropic glutamate (mGlu) receptors recruits the endocannabinoid system to produce both short- and long-term changes in synaptic strength in many regions of the brain. Although there is evidence that the endocannabinoid 2-arachidonoylglycerol (2-AG) mediates this process, the molecular mechanism underlying 2-AG mobilization remains unclear. In the present study, we used a combination of genetic and targeted lipidomic approaches to investigate the role of the postsynaptic membrane-associated lipase, diacylglycerol lipase type-α (DGL-α), in mGlu receptor-dependent 2-AG mobilization. DGL-α overexpression in mouse neuroblastoma Neuro-2a cells increased baseline 2-AG levels. This effect was accompanied by enhanced utilization of the 2-AG precursor 1-stearoyl,2-arachidonoyl-sn-glycerol and increased accumulation of the 2-AG breakdown product arachidonic acid. A similar, albeit less marked response was observed with other unsaturated and polyunsaturated monoacylglycerols, 1,2-diacylglycerols, and fatty acids. Silencing of DGL-α by RNA interference elicited lipidomic changes opposite those of DGL-α overexpression and abolished group I mGlu receptor-dependent 2-AG mobilization. Coimmunoprecipitation and site-directed mutagenesis experiments revealed that DGL-α interacts, via a PPxxF domain, with the coiled-coil (CC)-Homer proteins Homer-1b and Homer-2, two components of the molecular scaffold that enables group I mGlu signaling. DGL-α mutants that do not bind Homer maintained their ability to generate 2-AG in intact cells but failed to associate with the plasma membrane. The findings indicate that DGL-α mediates group I mGlu receptor-induced 2-AG mobilization. They further suggest that the interaction of CC-Homer with DGL-α is necessary for appropriate function of this lipase.
Footnotes
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This work was supported by National Institute on Drug Abuse grants DA12447 and DA12413 (to D.P.) and DA11322 and DA00286 (to K.M.). K.-M.J. and G.A. contributed equally to this work.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.037796.
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ABBREVIATIONS: CNS, central nervous system; DSI, depolarization-induced suppression of inhibition; 2-AG, 2-arachidonoylglycerol; PLC, phospholipase C; PIP2, phosphatidylinositol-4,5-bisphosphate; DAG, 1,2-diacylglycerol; DGL, diacylglycerol lipase; DGL-α, diacylglycerol lipase type-α; CC, coiled-coil; DHPG, (S)-3,5-dihydroxyphenylglycine; PCR, polymerase chain reaction; EGFP, enhanced green fluorescent protein; shRNA, small-hairpin RNA; RNAi, RNA interference; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; DHDG, diheptadecanoyl-sn-glycerol; HDG, 1(3)-heptadecanoyl-sn-glycerol; HDA, heptadecanoic acid; mGlu, metabotropic glutamate; LC/MS, liquid chromatography/mass spectrometry; ESI, electrospray ionization; MAG, monoacylglycerol; SIM, selected ion monitoring; SAG, 1-stearoyl,2-arachidonoyl-sn-glycerol; HEK, human embryonic kidney; IP3, inositol-1,4,5-trisphosphate; RHC80267, 1,6-bis(cyclohexyloximinocarbonylamino)hexane; U73122, 1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione.
- Received May 4, 2007.
- Accepted June 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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