Received for publication May 9, 2007.
Revised June 15, 2007.
Accepted for publication June 21, 2007.

A key role for diacylglycerol lipase- in metabotropic glutamate receptor-dependent endocannabinoid mobilization

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- (DGL-), in mGlu receptor-dependent 2-AG mobilization. DGL- 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- by RNA interference elicited opposite lipidomic changes to those of DGL- overexpression, and abolished group-I mGlu receptor-dependent 2-AG mobilization. Coimmunoprecipitation and site-directed mutagenesis experiments revealed that DGL- 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- 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- mediates group-I mGlu receptor-induced 2-AG mobilization. They further suggest that the interaction of CC-Homer with DGL- is necessary for appropriate function of this lipase.

Key words: Cannabinoid, Metabotropic glutamate, IP3/DAG, Fluorescence techniques, Immunocytochemistry, Mass Spectroscopy, Mutagenesis/Chimeric approaches, RNA/siRNA

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