EIPA We now present a series of findings obtained in

We now present a series of findings obtained in heterologous expression systems, brain slices, and living animals that suggest that any interaction between mGlu7 and α1-adrenergic receptors exists, is specific, and is physiologically and behaviourally relevant.
Materials and methods Norepinephrine, L-2-amino-4-phosphonobutanoate (L-AP4), l-serine-O-phosphate (L-SOP), 7-hydroxy-3-(4-iodophenoxy)-4H-1-benzopyran-4-one (XAP044), 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one hydrochloride (MMPIP), (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC), 4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (UO126), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic EIPA (VU0155041) were purchased from Tocris Bioscience (Bristol, UK). Phenylephrine (PE), pargyline and ascorbic acid were purchased from Sigma-Aldrich (Steinheim, Germany). Myo-[3H]inositol (18 Ci/mmol) was purchased from PerkinElmer (Milan, Italy). Dowex resin (AG 1-X8,100-200 FORMATE) and cromatography column (ECONO-COL POLYPROP) were from Bio-Rad Laboratories (Segrate, Italy). All other drugs were purchased from Sigma-Aldrich (Milan, Italy).
Results
Discussion There are several examples of interactions between receptors coupled to Gq/11 and Gi/o proteins involving mGlu receptors. The original evidence that Gi/o-coupled mGlu2 receptors negatively regulate electrophysiological responses mediated by Gq/11-coupled 5-HT2A serotonin receptors (Aghajanian and Marek, 2000, Marek et al., 2000) was one of the milestones in the development of mGlu2/3 receptor agonists and mGlu2 receptor positive allosteric modulators (PAMs) for the treatment of schizophrenia. The elegant work of Josè Gonzales-Maeso and Associates has demonstrated that mGlu2 and 5-HT2A receptors form a multimeric complex in which the mGlu2 receptor has the important function of restraining the excessive activation of 5-HT2A receptors that may occur in response to hallucinogens or in the brain of patients affected by schizophrenia (González-Maeso et al., 2008, Baki et al., 2016, Holloway et al., 2013; reviewed by Delille et al., 2013). Another example of a cross-talk between Gq/11- and Gi/o-coupled receptors is the interaction between mGlu1 and GABAB receptors in cerebellar Purkinje cells (Tabata et al., 2004), where mGlu1 receptors play a key role in mechanisms of activity-dependent synaptic plasticity underlying motor learning (Conquet et al., 1994, Aiba et al., 1994, Ichise et al., 2000, Hansel and Linden, 2000, Nakanishi, 2005, Ohtani et al., 2014). In Purkinje cells, GABAB receptors may act as extracellular Ca2+-dependent cofactors to enhance mGlu1 receptor signalling (Tabata et al., 2004). Studies carried out in heterologous expression systems suggest that the interaction between GABAB and mGlu1 receptors is mediated by the βγ-subunits released from Gi proteins following GABAB receptor activation (Rives et al., 2009). This might represent a general mechanism of Gi-coupled receptors interacting with neighbor Gq-coupled receptors (Rives et al., 2009). Accordingly, our data obtained in HEK 293 cells indicated that the functional interaction between mGlu7 and α1-adrenergic receptors was abrogated by the C-terminal portion of GRK2, which interacts with the βγ subunits of G proteins (Premont et al., 1995). As opposed to the GABAB/mGlu1 interaction, but similarly to the mGlu2/5-HT2A interaction, we have found that activation of the Gi-coupled mGlu7 receptors negatively regulates α1-adrenergic receptor signalling. We wish to highlight that activation of mGlu7 receptors has also been shown to stimulate PI hydrolysis in cultured neurons (Perroy et al., 2002) and in cerebrocortical nerve terminals (Martín et al., 2010, Martín et al., 2011, Ferrero et al., 2011, Ferrero et al., 2013, Ferrero et al., 2016). Our data obtained in transfected HEK 293 cells suggest that the interaction between mGlu7 receptors and α1-adrenergic receptors is mediated by the βγ-subunits released from the Gi protein, which, in turn, activate the MAPK pathway (Tohgo et al., 2003, Ahn et al., 2004, Shukla et al., 2011, Eichel et al., 2016). Interestingly, the negative modulation of α1-adrenergic receptor signalling in HEK 293 cells was highly specific for mGlu7 receptors because it was not shared by other mGlu receptor subtypes coupled to Gi, such as mGlu2 and mGlu4 receptors. How a mechanism driven by βγ-subunits may be specific for an individual receptor subtype under conditions of receptor overexpression in heterologous expression systems is unclear, particularly because mGlu2, mGlu4, and mGlu7 receptors stimulate the MAPK pathway in HEK 293 cells (Iacovelli et al., 2004, Iacovelli et al., 2009, Iacovelli et al., 2014). A more in-depth analysis of signal propagation in response to the three above mentioned mGlu receptors coupled to Gi is necessary to unravel the mechanisms that confer specificity to mGlu7 receptors in inhibiting α1-adrenergic receptor signalling. This specificity was also observed in mouse cortical slices, where L-AP4 or L-SOP were found to inhibit α1-adrenergic receptor signalling only at concentrations that recruit mGlu7 receptors (the EC50 of both compounds for mGlu7 receptors is in the high micromolar range, whereas the EC50 for mGlu4, mGlu6, and mGlu8 receptors is in the low micromolar/high nanomolar range) (reviewed by Schoepp et al., 1999). In addition, L-AP4 failed to inhibit α1-adrenergic receptor signalling in cortical slices from mGlu7−/− mice, but retained the inhibitory activity in slices from mGlu4−/− mice. On the basis of these findings we are convinced that the negative modulation of α1-adrenergic receptors by mGlu7 receptors is a physiological mechanism occurring in the CNS. Thus, α1-adrenergic receptors may be added to the list of receptors that functionally interact with mGlu7 receptors. The elegant work of Prof. Sanchez-Prieto and Associates has demonstrated that the mGlu7 receptor co-localizes with A1 adenosine and GABAB receptors in a subpopulation of cerebrocortical nerve endings expressing N-type voltage-sensitive Ca2+ channels. mGlu7, A1 and GABAB receptors share the same signalling mechanism in inhibiting glutamate release (i.e., inhibition of adenylyl cyclase and Ca2+ channels), and responses to activation of the three receptors are less than additive (Martin et al., 2008). mGlu7 receptors can also enhance glutamate release after prolonged agonist exposure, an effect that appears to be mediated by the activation of phospholipase-C and translocation of the synaptic vesicle-associated protein, Munc13-1 (Martín et al., 2010). Interestingly, mGlu7 receptors and β-adrenergic receptors functionally interact in activating phospholipase C and promoting Munc13-1 translocation under conditions in which mGlu7 receptors enhance glutamate release from isolated nerve terminals (Ferrero et al., 2016). A comparison of these findings with our findings suggest that mGlu7 receptors play a complex modulatory role on central noradrenergic neurotransmission that involves a functional cross-talk with both β- and α1-adrenergic receptors.