Pharmacological profiles of 5‐HT 2A Receptor (5‐HT 2A R):5‐HT 2C R Interactions In Vitro

Aims The serotonin 5‐HT 2A receptor (5‐HT 2A R) and 5‐HT 2C R 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‐HT 2A R and 5‐HT 2C R 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‐HT 2C R is functionally dominant, i.e., silences signaling from the 5‐HT 2A R 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‐HT 2A R:5‐HT 2C R 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‐HT 2A R and 5‐HT 2C R in live HEK293 cells. The cDNA for the human 5‐HT 2A R and 5‐HT 2C R 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‐HT 2C R transmembrane helix IV (TM4) domain conjugated to a polyethylene glycol chain (PEG‐TM4 5‐HT 2C R) was synthesized as a tool to disrupt formation of the 5‐HT 2A R:5‐HT 2C R complex. Results . Serotonin (5‐HT) readily induced robust expression of EGR1 mRNA in cells solely expressing 5‐HT 2A R, but not 5‐HT 2C R; 5‐HT‐evoked EGR1 expression was blunted in cells co‐expressing the 5‐HT 2C R plus 5‐HT 2A R. In the LCA, vehicle (0.1% DMSO) treated cells exhibit robust complementation (i.e., increased luminescence) between the 5‐HT 2A R and 5‐HT 2C R. The PEG‐TM4 5‐HT 2C R peptide significantly decreased complementation (p<0.05) in a concentration‐dependent manner indicating an inhibition of association between the 5‐HT 2A R and 5‐HT 2C R. Conclusions Blunted 5‐HT‐evoked EGR1 expression in cells expressing both receptors supports the “master regulator” role for the 5‐HT 2C R when in complex with the 5‐HT 2A R. Secondly, the formation of the 5‐HT 2A R:5‐HT 2C R complex in vitro is dependent on an interaction interface located at the 5‐HT 2C R TM4. Ongoing studies are testing the hypothesis that the PEG‐TM4 5‐HT 2C R peptide restores a critical signaling output of 5‐HT 2A R 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‐HT 2A R:5‐HT 2C R complex in vivo .