Functional Crosstalk between mGluR2 and 5-HT2A Depends on their Expression Ratios

Abstract

We recently reported that a heteromeric complex formed between the Gi-coupled metabotropic glutamate 2 receptor (mGluR2) and the Gq-coupled serotonin 5-HT2A receptor (2AR) integrates the actions of serotonergic and glutamatergic drugs, modulating the balance between Gi and Gq signaling in a way that can be used to predict the psychoactive properties of these drugs (Fribourg et al. 2011, Cell. 147(5):1011-23). The biological relevance of the mGluR2/2AR crosstalk was challenged by a recent study in which co-expression of the two receptors in HEK-293 cells had no significant effect on either Gi or Gq signaling in response to several serotonergic and glutamatergic drugs (Delille et al., 2012, Neuropharmacology 62(7):2184-91). This controversy motivated us to address the significance of the ratio of the expression levels of two receptors on the degree of their functional crosstalk. To this end we generated several clones of HEK-293 cells expressing different ratios of the two receptors in the background of the G protein inwardly rectifying GIRK1/GIRK4 channel, which can serve as a reporter for both Gi and Gq signaling. The fluorescent potensiometric dye DiBAC4(3) was used as a probe for membrane potential changes in response to GIRK1/GIRK4 channel activity, whereas the ratiometric calcium indicator Fura-2 was used as an alternative reporter of Gq signaling. Here we report that drugs targeting either of the two receptors were able to modify both Gi and Gq signaling in some, but not all of our clones. Real time RT-PCR, radio-ligand binding assays and flow-cytometry were employed to correlate receptor expression levels to the degree of their functional crosstalk. Clones showing the most effective crosstalk will be used in DiBAC4(3) assays for high-throughput screening of ligands targeting the heteromeric 2AR/mGluR2 complex.