title: Cross-signaling in metabotropic glutamate 2 and serotonin 2A receptor heteromers in mammalian cells.
abstract: 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 Ca(2+) 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.
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
authors.forename: Lia
authors.initials: L
authors.lastname: Baki
authors.affiliation: Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
authors.forename: Miguel
authors.initials: M
authors.lastname: Fribourg
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Younkin
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
authors.forename: Jose Miguel
authors.initials: JM
authors.lastname: Eltit
authors.affiliation: Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Moreno
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
authors.forename: Gyu
authors.initials: G
authors.lastname: Park
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
authors.forename: Zhanna
authors.initials: Z
authors.lastname: Vysotskaya
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
authors.forename: Adishesh
authors.initials: A
authors.lastname: Narahari
authors.affiliation: Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.|Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.|Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Sealfon
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.|Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.|Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.|Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
authors.forename: Javier
authors.initials: J
authors.lastname: Gonzalez-Maeso
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA. delogothetis@vcu.edu.
authors.forename: Diomedes E
authors.initials: DE
authors.lastname: Logothetis
mesh_terms: D020013:Calcium Signaling; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D055503:Protein Multimerization; D044402:Receptor, Serotonin, 5-HT2A / Q000235:genetics / Q000378:metabolism*; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044402:Receptor, Serotonin, 5-HT2A; D018094:Receptors, Metabotropic Glutamate; C108822:metabotropic glutamate receptor 2
keywords: 5-HT2A receptor; Calcium intracellular release; Cross-signaling; G protein-coupled receptor (GPCR); Mammalian cells; Membrane potential probes; Metabotropic glutamate 2 (mGlu2) receptor