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Selective Actions of Novel Allosteric Modulators Reveal Functional Heteromers of Metabotropic Glutamate Receptors in the CNS

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Selective Actions of Novel Allosteric Modulators Reveal Functional Heteromers of Metabotropic Glutamate Receptors in the CNS The Journal of Neuroscience, January 1, 2014 • 34(1):79–94 • 79 Cellular/Molecular Selective Actions of Novel Allosteric Modulators Reveal Functional Heteromers of Metabotropic Glutamate Receptors in the CNS Shen Yin,1,2 Meredith J. Noetzel,1,2 Kari A. Johnson,1,2 Rocio Zamorano,1,2 Nidhi Jalan-Sakrikar,1,2 Karen J. Gregory,1,2,3 P. Jeffrey Conn,1,2 and Colleen M. Niswender1,2 1Department of Pharmacology and the 2Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, and 3Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia Metabotropic glutamate (mGlu) receptors play important roles in regulating CNS function and are known to function as obligatory dimers. Although recent studies have suggested heterodimeric assembly of mGlu receptors in vitro, the demonstration that distinct mGlu receptor proteins can form heterodimers or hetero-complexes with other mGlu subunits in native tissues, such as neurons, has not been shown.Usingbiochemicalandpharmacologicalapproaches,wedemonstrateherethatmGlu2 andmGlu4 formahetero-complexinnative rat and mouse tissues which exhibits a distinct pharmacological profile. These data greatly extend our current understanding of mGlu receptor interaction and function and provide compelling evidence that mGlu receptors can function as heteromers in intact brain circuits. Introduction subtypes, and members of Group II and III receptors coassem- The metabotropic glutamate (mGlu) receptors are members bling in vitro. of the Family C Seven Transmembrane Spanning/G-Protein- Among the Group III mGlu receptors, mGlu4 plays an impor- Coupled Receptors (7TMRs/GPCRs) and are activated by the tant role in the basal ganglia, a primary site of pathology in move- major excitatory neurotransmitter, glutamate. In their simplest ment disorders such as Parkinson’s disease (PD). Activation of context, Group I mGlus (mGlu1 and mGlu5) primarily modulate mGlu4 reduces transmission at synapses that project from the postsynaptic neuronal activity, whereas the Group II mGlus striatum to the globus pallidus (striatopallidal synapses) as well as (mGlu2 and mGlu3) are found in both presynaptic and postsyn- synapses between the subthalamic nucleus and the substantia aptic locations, and the Group III receptors (mGlu4, mGlu6, nigra pars compacta (STN-SNc synapses) (Marino et al., 2003; mGlu7, and mGlu8) are predominantly expressed presynapti- Valenti et al., 2003, 2005), two synapses that are overactive in PD. cally, where they act as autoreceptors and heteroreceptors to reg- At each of these synapses, the response to the general Group III ulate neurotransmitter release (for review, see Niswender and mGlu agonist L-AP4 is potentiated by N-phenyl-7-(hydroxyimino) Conn, 2010). The eight mGlu receptor subtypes have been his- cyclopropa[b]chromen-1a-carboxamide (PHCCC) (Marino et torically thought to function as homodimers (Romano et al., al., 2003; Valenti et al., 2005), a positive allosteric modulator 1996; Kunishima et al., 2000). However, recent in vitro stud- (PAM) that is selective for mGlu4. In contrast to findings at the ies suggest that mGlu receptors can also form heterodimers striatopallidal and STN-SNc synapses, we now report the surpris- (Doumazane et al., 2011; Kammermeier, 2012), with Group I ing observation that PHCCC is without effect in regulating mGlus interacting with each other but not associating with other mGlu4-modulated transmission at synapses between cortex and striatum (corticostriatal synapses). Received March 14, 2013; revised Oct. 14, 2013; accepted Nov. 8, 2013. Previous immunohistochemistry and in situ hybridization Author contributions: S.Y., M.J.N., K.A.J., N.J.-S., K.J.G., P.J.C., and C.M.N. designed research; S.Y., M.J.N., K.A.J., studies suggest that mGlu2 and mGlu4 are colocalized in several R.Z., and N.J.-S. performed research; S.Y., M.J.N., K.A.J., N.J.-S., K.J.G., and C.M.N. analyzed data; S.Y., M.J.N., K.A.J., K.J.G., P.J.C., and C.M.N. wrote the paper. brain regions (Ohishi et al., 1993, 1995; Neki et al., 1996; Bradley This work was supported by NIH Grants NS048334 (C.M.N. and P.J.C.), NS078262 (C.M.N.), NS031373 (P.J.C.), et al., 1999), and mGlu2 is also functionally expressed at cortico- NS067737 (K.A.J.), NS071746 (M.J.N.), an National Health and Medical Research Council (Australia) Overseas Bio- striatal synapses (Johnson et al., 2005). We tested the hypothesis medicalPostdoctoralfellowship(K.J.G.),andaNationalAllianceforResearchonSchizophreniaandDepressionMaltz that heterodimers of mGlu2/4 may display a unique profile in Young Investigator Award (K.J.G.). We thank Dr. Corey R. Hopkins, Dr. Darren Engers, and Patrick Gentry for the response to selective mGlu4 PAMs and that these mGlu subtypes synthesis of mGlu4 PAMs used in this study. M.J.N., P.J.C., and C.M.N. received research support from Bristol Myers Squibb and Astra Zeneca. The remaining form hetero-complexes in the striatum. Through evaluation of authors declare no competing financial interests. mGlu4 PAMs from different chemical scaffolds, we show here Correspondence should be addressed to Dr. Colleen M. Niswender, 12478C MRB IV, Department of Pharmacol- that hetero-interactions between mGlu2 and mGlu4 differentially ogy, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232-0697. E-mail: impact responses to individual mGlu receptor PAMs and an [email protected]. DOI:10.1523/JNEUROSCI.1129-13.2014 mGlu2-negative allosteric modulator (NAM). Furthermore, co- Copyright © 2014 the authors 0270-6474/14/340079-16$15.00/0 immunoprecipitation studies suggest that mGlu2 and mGlu4 re- • 80 • J. Neurosci., January 1, 2014 • 34(1):79–94 Yin et al. Functional mGlu2/4 Hetero-Interactions Revealed in the CNS ceptors form hetero-complexes in the striatum and the unique For coimmunoprecipitation assays in rat or mouse brain samples, pharmacological profile of effects of selected mGlu4 receptor Sprague Dawley rats of mixed gender (Charles River) and ICR (CD-1) male mice (Harlan) 22–30 d old were anesthetized under isoflurane an- PAMs, as well as an mGlu2 NAM, is recapitulated at the cortico- striatal synapse. These studies directly impact our understanding esthesia, decapitated, and brains were rapidly removed and cut into 0.5–1 of mGlu receptors and regulation by allosteric modulators in the mm coronal slices using a brain matrix or a vibratome (Leica). Slices were transferred to a chilled metal surface, and dorsal striatum and medial basal ganglia; providing critical insights into potential functions prefrontal cortex (prelimbic and infralimbic regions) were dissected us- and pharmacological properties of mGlu receptors that are coex- ing a scalpel blade and immediately frozen on dry ice. Samples were pressed in multiple regions and cell populations. homogenized in buffer containing the following (in mM): 50 Tris HCl, pH 7.4, 50 NaCl, 10 EGTA, 5 EDTA, 2 NaF, 1 Na3VO4, 1 PMSF supple- mented with 1ϫ Complete Mini protease inhibitor mixture, phos- Materials and Methods phatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Homogenized Cell line establishment and cell culture. Cell culture reagents were pur- samples were centrifuged at 16,100 ϫg for 15 min at 4°C, and pelleted chased from Invitrogen unless otherwise noted. Rat mGlu2 or rat mGlu4 membranes were stored at Ϫ80°C. Membrane pellets were resuspended was cloned into the pIRESpuro3 vector, transfected into human embry- in immunoprecipitation lysis buffer (same as immunoprecipitation lysis onic kidney (HEK)/G-protein inwardly rectifying potassium (GIRK) buffer for immunoprecipitation mGlu4 in cell lines, supplemented with cells, and selected with puromycin. Polyclonal rat mGlu2/HEK/GIRK 1mM PMSF) and rocked/rotated/incubated at 4°C for 1 h. Supernatant and rat mGlu4/HEK/GIRK cells were cultured in growth media as previ- was prepared by centrifugation at 16,100 ϫg for 15 min and precleared ously described (Niswender et al., 2008a), supplemented with nonessen- by protein A/G beads at 4°C for 2–3 h. mGlu4 antibodies or normal rabbit tial amino acids. Rat mGlu4 was also subcloned into the pIREShyg3 IgG (Millipore, catalog #12-370) were bound to protein A/G beads by vector, and the resulting plasmid was transfected into rat mGlu2/HEK/ rotating at 4°C for 2–3 h. Precleared cell lysates were then added to mGlu4 GIRK cells; cells were then selected with 200 ␮g/ml hygromycin B. Poly- antibody bound protein A/G beads or rabbit IgG-coupled beads as a clonal cells were cultured in growth media supplemented with 100 ␮g/ml negative control. After overnight rotation at 4°C, the beads were washed hygromycin B. and samples were eluted and analyzed as described above. Western blot analysis. Cells were scraped into lysis buffer (50 mM Tris- Thallium flux assays. Thallium flux assays were performed according HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40, and 0.5% deoxycholate) to methods described previously (Niswender et al., 2008b) with minor containing protease inhibitor mixture (Roche) and incubated on ice for modifications. For dye loading, media was exchanged with assay buffer 20–30 min. The supernatant was separated from cell debris by centrifu- (HBSS, containing 20 mM HEPES, pH 7.4) using an ELX405 microplate gation at 16,000 ϫg for 10 min at 4°C. Protein concentrations in cell washer (BioTek), leaving 20 ␮l/well, followed by addition of 20 ␮l/well lysates were quantified by Bio-Rad Protein Assay (Bio-Rad) or Bradford 2ϫ FluoZin-2 AM (330 nM final) indicator dye, (Invitrogen, prepared as protein assay (Bio-Rad), and aliquots of lysate were heated in SDS sample a DMSO stock and mixed in a 1:1 ratio with pluronic acid F-127) in assay buffer (containing 10% SDS and 9.3% DTT) at 65°C for 5 min. Samples buffer. Aftera1hincubation at room temperature, dye was exchanged were loaded on SDS–PAGE and transferred to nitrocellulose membranes with assay buffer, leaving 20 ␮l/well.
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