Molecular Interactions Between the Glutaminergic and Dopaminergic Systems

C Pou1, C Mannoury La Cour2, MJ Millan2, G Milligan1. 1University of Glasgow, Glasgow, Scotland, UK, 2Institut de Recherches Servier, Unité de recherche et découverte en neurosciences, Croissy sur Seine, France

Along with serotonin and norepinephrine, the neurotransmitters dopamine and glutamate have been described as major players in the pathophysiology and potential treatment of psychological diseases such as schizophrenia and depression disorders (1-3). To date, most therapeutic drugs have targeted the monoaminergic systems and comprise tricyclic antidepressants, selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors and compounds targeting the dopaminergic system to re-establish equilibrium in these pathways. Hence, there is a need to find new approaches to tackle these disorders. While implication of the dopaminergic pathway is well documented, there is growing evidence that the glutaminergic systems potentially play an important role in the neurobiology and pathophysiology of depressive disorders (1-3, 5). Indeed, Group II metabotropic glutamate receptors mGlu2 and mGlu3 are widely distributed throughout the CNS where they display moderate to high expression in brain regions associated with major depressive disorders (MDD) (e.g. hippocampus, prefrontal cortex and amygdala) (3), and some mGlu2/3 receptor antagonists such as LY341495 have shown antidepressant-like effects in animal models of depression (6).

The aim of this study is to examine the potential molecular interaction between two receptors potentially involved in the aetiology of these disorders: the dopaminergic receptor D2 and the metabotropic glutaminergic receptor mGlu2. To this effect, the mGlu2 and D2L receptors were modified by N-terminal addition of respectively the SNAP form of O6-alkylguanine-DNA-alkyltransferase or HaloTag, a modified haloalkane dehalogenase, plus a peptide epitope tag. In addition to co-immunoprecipitation of co-expressed pairs of HA-HALO-D2L and VSV-G-SNAP-mGlu2 receptors, cells able to express each or both of these constructs were established and used to confirm cell surface expression and the specificity of SNAP- and HALO-tag covalent labelling reagents using a homogenous time-resolved fluorescence resonance energy transfer assay (increased specific fluorescence at 620 nm p<0.001 compared to untreated cells). The presence of cell surface HA-HALO-D2L and VSV-G-SNAP-mGlu2 receptor homomers and heteromers were detected by a significant (p<0.001 compared to untreated cells) and saturable increase of specific fluorescence at 665 nm. Following expression of reciprocally tagged receptor constructs and labelling with compatible fluorophores, the presence of cell surface D2L-mGlu2 heteromers was also observed. This did not prevent the observation of homomers of each receptor, suggesting that such homomers and heteromers co-exist at the surface of these cells. A second approach involved the pharmacological characterisation of these interactions using selective ligands for each receptor. These studies demonstrate the ability of dopamine D2L and mGlu2 receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady-state. These data are of potential importance both to disorders in which D2L and mGlu2 receptors are implicated such as schizophrenia and depression but may also broaden our understanding of current therapies.

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