Biophysical validation of serotonin 5-HT_2A and 5-HT_2C receptor interaction

The serotonin (5-HT) 5-HT_2A receptor (5-HT_2AR) and 5-HT_2C receptor (5-HT_2CR) in the central nervous system are implicated in a range of normal behaviors (e.g., appetite, sleep) and physiological functions (e.g., endocrine secretion) while dysfunctional 5-HT_2AR and/or 5-HT_2CR are implicated in neuropsychiatric disorders (e.g., addiction, obesity, schizophrenia). Preclinical studies suggest that the 5-HT_2AR and 5-HT_2CR may act in concert to regulate the neural bases for behavior. Here, we utilize three distinct biophysical and immunocytochem-istry-based approaches to identify and study this receptor complex in cultured cells. Employing a split luciferase complementation assay (LCA), we demonstrated that formation of the 5-HT_2AR:5-HT_2CR complex exists within 50 nm, increases proportionally to the 5-HT_2CR:5-HT_2AR protein expression ratio, and is specific to the receptor interaction and not due to random complementation of the luciferase fragments. Using a proximity ligation assay (PLA), we found that cells stably expressing both the 5-HT_2AR and 5-HT_2CR exhibit 5-HT_2AR:5-HT_2CR heteroreceptor complexes within 40 nm of each other. Lastly, bioluminescence resonance energy transfer (BRET) analyses indicates the formation of a specific and saturable 5-HT_2AR:5-HT_2CR interaction, suggesting that the 5-HT_2AR and 5-HT_2CR form a close interaction within 10 nm of each other in intact live cells. The bioengineered receptors generated for the LCA and the BRET exhibit 5-HT-mediated intracellular calcium signaling as seen for the native receptors. Taken together, this study validates a very close 5-HT_2AR:5-HT_2CR interaction in cultured cells.