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Biophysical identification of the 5‐HT2A receptor:5‐HT2C receptor interaction in vitro

Disruption of serotonin 5‐HT2A receptor (5‐HT2AR) and 5‐HT2CR function is implicated in a range of neuropsychiatric disorders with these receptors being essential sites of action for anti‐psychotic and anti‐obesity medications. Based on evidence that the 5‐HT2AR and 5‐HT2CR co‐localize and co‐immunoprecipitate from neurons in the medial prefrontal cortex of the rat (Anastasio et al, ACS Chem. Neurosci. 15:1248, 2015), we hypothesize that these two receptors form a heteromeric complex in living cells. Here, we utilize three distinct biophysical and immunocytochemistry‐based approaches to identify and study this receptor complex in living cells. First, we generated a split luciferase complementation assay to test the hypothesis that the 5‐HT2AR and 5‐HT2CR are in close proximity (< 50 nm). We predicted that inactive luciferase fragments fused to each receptor would be brought into close proximity when receptors form a complex, thereby reconstituting the enzymatic activity of luciferase. A robust complementation (i.e., increased luminescence) was observed upon addition of the substrate D‐luciferin, an outcome that was dependent on protein levels assessed in radioligand binding studies (n=3, p<0.001). Co‐transfection with either the wildtype 5‐HT2AR or 5‐HT2CR inhibited the complementation (n=3, p<0.01). Using a proximity ligation assay, we demonstrate expression of the existence of 5‐HT2AR:5‐HT2CR heteroreceptor complexes in vitro, indicating the protomers are within 40 nm of each other (n=5, p<0.05). To further delineate the receptors are within very close proximity (< 10 nm) in living cells, we generated 5‐HT2AR and 5‐HT2CR constructs for use in a bioluminescence resonance energy transfer (BRET) assay. Preliminary results indicate that the 5‐HT2AR and 5‐HT2CR form a robust BRET interaction creating a maximal signal of 230 mBRET units and a BRET50 of 2.1, which was more than double the signal generated in control studies with the β2‐adrenergic receptor. Taken together, we have identified a very close 5‐HT2AR:5‐HT2CR interaction in living cells. Ongoing research will employ these technologies to further investigate the formation and function of the receptor complex.