Pharmacological profiles of 5‐HT _2A Receptor (5‐HT _2A R):5‐HT _2C R Interactions In Vitro

Aims The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR have garnered intense interest from drug discovery programs due to their role in metabolic (e.g., obesity) and neuropsychiatric disorders (e.g., addiction, depression, psychosis). Functional and physical interactions between the 5-HT2AR and 5-HT2CR occur in living cells (Felsing et al., PLoS One 13(8):e0203137, 2018) and rat brain (Price et al., ACS Chem Neurosci 10:3241, 2019). Intriguingly, when the two receptors are in complex, the 5-HT2CR is functionally dominant, i.e., silences signaling from the 5-HT2AR in vitro (Moutkine et al., J Biol Chem 292:6352, 2017). In the present study, we have extended these observations to uncover additional cellular signaling output associated with the 5-HT2AR:5-HT2CR complex and explore disruption of the receptor interfaces important for their interaction and signaling. Methods . A split luciferase complementation assay (LCA) was employed to assess the direct coupling of 5-HT2AR and 5-HT2CR in live HEK293 cells. The cDNA for the human 5-HT2AR and 5-HT2CR fused with complementary N-terminal (NLuc) and C-terminal (CLuc) inactive fragments, respectively, were transfected into HEK293 cells using template mammalian expression vectors and standard cloning techniques. Association of the receptor constructs in vitro within ~50 nm reconstitutes luciferase activity and light is released in the presence of D-luciferin. The expression of the activity-regulated immediate early gene early growth response 1 (EGR1) was used to trace cellular responsivity to 5-HT in cells. A truncated version of the 5-HT2CR transmembrane helix IV (TM4) domain conjugated to a polyethylene glycol chain (PEG-TM4 5-HT2CR) was synthesized as a tool to disrupt formation of the 5-HT2AR:5-HT2CR complex. Results . Serotonin (5-HT) readily induced robust expression of EGR1 mRNA in cells solely expressing 5-HT2AR, but not 5-HT2CR; 5-HT-evoked EGR1 expression was blunted in cells co-expressing the 5-HT2CR plus 5-HT2AR. In the LCA, vehicle (0.1% DMSO) treated cells exhibit robust complementation (i.e., increased luminescence) between the 5-HT2AR and 5-HT2CR. The PEG-TM4 5-HT2CR peptide significantly decreased complementation (p<0.05) in a concentration-dependent manner indicating an inhibition of association between the 5-HT2AR and 5-HT2CR. Conclusions Blunted 5-HT-evoked EGR1 expression in cells expressing both receptors supports the “master regulator” role for the 5-HT2CR when in complex with the 5-HT2AR. Secondly, the formation of the 5-HT2AR:5-HT2CR complex in vitro is dependent on an interaction interface located at the 5-HT2CR TM4. Ongoing studies are testing the hypothesis that the PEG-TM4 5-HT2CR peptide restores a critical signaling output of 5-HT2AR function, which will indicate the importance of allosteric regulation by the heteromer complex. Future studies with our novel TM domain peptides will help elucidate determinants of receptor complex formation and signaling and the physiological relevance of the 5-HT2AR:5-HT2CR complex in vivo.