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We previously reported that co-expression of the Gi-coupled metabotropic glutamate receptor 2 (mGluR) and the Gq-coupled serotonin (5-HT) 2A receptor (2AR) in Xenopus oocytes (Fribourg et al. Cell 147:1011-1023, ) 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 mGluR/2AR heteromer integrates the actions of psychedelic and antipsychotic drugs. To investigate whether mGluR 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 responses by the inverse mGluR 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 mGluR binding sites and higher density of 2AR binding sites in total membrane preparations, and (c) higher ratios of mGluR/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. [ABSTRACT FROM AUTHOR]