Cross-signaling in metabotropic glutamate 2 and serotonin 2A receptor heteromers in mammalian cells
- 786 Downloads
We previously reported that co-expression of the Gi-coupled metabotropic glutamate receptor 2 (mGlu2R) and the Gq-coupled serotonin (5-HT) 2A receptor (2AR) in Xenopus oocytes (Fribourg et al. Cell 147:1011–1023, 2011) results in inverse cross-signaling, where for either receptor, strong agonists suppress and inverse agonists potentiate the signaling of the partner receptor. Importantly, through this cross-signaling, the mGlu2R/2AR heteromer integrates the actions of psychedelic and antipsychotic drugs. To investigate whether mGlu2R and 2AR can cross-signal in mammalian cells, we stably co-expressed them in HEK293 cells along with the GIRK1/GIRK4 channel, a reporter of Gi and Gq signaling activity. Crosstalk-positive clones were identified by Fura-2 calcium imaging, based on potentiation of 5-HT-induced Ca2+ responses by the inverse mGlu2/3R agonist LY341495. Cross-signaling from both sides of the complex was confirmed in representative clones by using the GIRK channel reporter, both in whole-cell patch-clamp and in fluorescence assays using potentiometric dyes, and further established by competition binding assays. Notably, only 25–30 % of the clones were crosstalk-positive. The crosstalk-positive phenotype correlated with (a) increased colocalization of the two receptors at the cell surface, (b) lower density of mGlu2R binding sites and higher density of 2AR binding sites in total membrane preparations, and (c) higher ratios of mGlu2R/2AR normalized surface protein expression. Consistent with our results in Xenopus oocytes, a combination of ligands targeting both receptors could elicit functional crosstalk in a crosstalk-negative clone. Crosstalk-positive clones can be used in high-throughput assays for identification of antipsychotic drugs targeting this receptor heterocomplex.
KeywordsG protein-coupled receptor (GPCR) Metabotropic glutamate 2 (mGlu2) receptor 5-HT2A receptor Cross-signaling Calcium intracellular release Membrane potential probes Mammalian cells
The authors wish to thank Dr. Taihao Jin (University of California, San Francisco) for developing an automated program to read fluorescent data from 96-well microplates in a Flex Station 3 reader, Dr Clive M. Baumgarten (Virginia Commonwealth University) for kindly offering access to his patch-clamp rigs, Dr George Liapakis (University of Crete, Greece) for preliminary binding experiments in the clonal cell lines, Drs Carlos A. Villalba-Galea (Virginia Commonwealth University) and Qiong Yao Tang (Xuzhou Medical College, Xuzhou, Jiangsu Province, China) for help with electrophysiology experiments, and Junghoon Ha for analysis and with the presentation of Fig. S3. We also thank all members of the Logothetis lab for critical feedback on the work and the manuscript. We acknowledge Heikki Vaananen and Nada Marjanovic for technical support and the Icahn School of Medicine at Mount Sinai Quantitative PCR Core Facility.
This work was supported by the National Institutes of Health grants R01HL59949 and R01 HL090882 to D.E.L, R01MH084894 to J.G-M, and T32 MH096678 training grant to M.F.
- 6.Delille HK, Becker JM, Burkhardt S, Bleher B, Terstappen GC, Schmidt M, Meyer AH, Unger L, Marek GJ, Mezler M (2012) Heterocomplex formation of 5-HT2A-mGlu2 and its relevance for cellular signaling cascades. Neuropharmacology 62:2184–2191. doi: 10.1016/j.neuropharm.2012.01.010 CrossRefPubMedGoogle Scholar
- 8.Fell MJ, Svensson KA, Johnson BG, Schoepp DD (2008) Evidence for the role of metabotropic glutamate (mGlu)2 not mGlu3 receptors in the preclinical antipsychotic pharmacology of the mGlu2/3 receptor agonist (−)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039). J Pharmacol Exp Therapeut 326:209–217. doi: 10.1124/jpet.108.136861 CrossRefGoogle Scholar
- 11.Fribourg M, Moreno JL, Holloway T, Provasi D, Baki L, Mahajan R, Park G, Adney SK, Hatcher C, Eltit JM, Ruta JD, Albizu L, Li Z, Umali A, Shim J, Fabiato A, MacKerell AD Jr, Brezina V, Sealfon SC, Filizola M, Gonzalez-Maeso J, Logothetis DE (2011) Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs. Cell 147:1011–1023. doi: 10.1016/j.cell.2011.09.055 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Gonzalez-Maeso J, Ang RL, Yuen T, Chan P, Weisstaub NV, Lopez-Gimenez JF, Zhou M, Okawa Y, Callado LF, Milligan G, Gingrich JA, Filizola M, Meana JJ, Sealfon SC (2008) Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature 452:93–97. doi: 10.1038/nature06612 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C, Huang LY, Tang C, Shen Q, Salon JA, Morse K, Laz T, Smith KE, Nagarathnam D, Noble SA, Branchek TA, Gerald C (1998) GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 396:674–679. doi: 10.1038/25348 CrossRefPubMedGoogle Scholar
- 27.Markey KA, Koyama M, Gartlan KH, Leveque L, Kuns RD, Lineburg KE, Teal BE, MacDonald KP, Hill GR (2014) Cross-dressing by donor dendritic cells after allogeneic bone marrow transplantation contributes to formation of the immunological synapse and maximizes responses to indirectly presented antigen. J Immunol 192:5426–5433. doi: 10.4049/jimmunol.1302490 CrossRefPubMedGoogle Scholar
- 28.Moreno JL, Miranda-Azpiazu P, Garcia-Bea A, Younkin J, Cui M, Kozlenkov A, Ben-Ezra A, Voloudakis G, Fakira AK, Baki L, Ge Y, Georgakopoulos A, Moron JA, Milligan G, Lopez-Gimenez JF, Robakis NK, Logothetis DE, Meana JJ, Gonzalez-Maeso J (2016) Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia. Sci Signal (in press)Google Scholar
- 29.Moreno JL, Muguruza C, Umali A, Mortillo S, Holloway T, Pilar-Cuellar F, Mocci G, Seto J, Callado LF, Neve RL, Milligan G, Sealfon SC, Lopez-Gimenez JF, Meana JJ, Benson DL, Gonzalez-Maeso J (2012) Identification of three residues essential for 5-hydroxytryptamine 2A-metabotropic glutamate 2 (5-HT2A.mGlu2) receptor heteromerization and its psychoactive behavioral function. J Biol Chem 287:44301–44319. doi: 10.1074/jbc.M112.413161 CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Patil ST, Zhang L, Martenyi F, Lowe SL, Jackson KA, Andreev BV, Avedisova AS, Bardenstein LM, Gurovich IY, Morozova MA, Mosolov SN, Neznanov NG, Reznik AM, Smulevich AB, Tochilov VA, Johnson BG, Monn JA, Schoepp DD (2007) Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized phase 2 clinical trial. Nat Med 13:1102–1107. doi: 10.1038/nm1632 CrossRefPubMedGoogle Scholar
- 34.Saugstad JA, Segerson TP, Westbrook GL (1996) Metabotropic glutamate receptors activate G-protein-coupled inwardly rectifying potassium channels in Xenopus oocytes. J Neurosci: Off J Soc Neurosci 16:5979–5985Google Scholar
- 36.Spooren WP, Gasparini F, van der Putten H, Koller M, Nakanishi S, Kuhn R (2000) Lack of effect of LY314582 (a group 2 metabotropic glutamate receptor agonist) on phencyclidine-induced locomotor activity in metabotropic glutamate receptor 2 knockout mice. Eur J Pharmacol 397:R1–R2CrossRefPubMedGoogle Scholar