A FRET-Based approach to study the SUMOylation of Serotonin 1A Receptors
University of Kansas
Pharmacology & Toxicology
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Serotonin 1A receptors are an inhibitory G-protein coupled receptor that are known to play a key role in the regulation of mood and cognition. Dysregulation of serotonin 1A receptors has been implicated in mood related disorders such as depression and anxiety. Post translational modifications including palmitoylation and phosphorylation are found to regulate the function of serotonin 1A receptors. Previous studies in our lab demonstrated that serotonin 1A receptors are SUMOylated, however the impact of SUMOylation on serotonin 1A receptor function is yet to be elucidated. Acute agonist stimulation of serotonin 1A receptors was found to increase the levels of the SUMOylated receptors in rat cortex. This study employed acceptor photobleach FRET to further investigate the interaction between SUMO-1 and serotonin 1A receptor and identify the sites of SUMOylation on the serotonin 1A receptor. We used cell lines expressing both endogenous (N2A) and transfected serotonin 1A receptors (HEK293 and SHSY5Y) and observed FRET between serotonin 1A receptor and SUMO-1 in all the cell lines. Using acceptor photobleach FRET, we found three lysine residues on the serotonin 1A receptor (232, 235, 324) that are possibly involved in SUMOylation. We also conducted an immunocytochemistry-based approach, to study the effect of agonist stimulation of serotonin 1A receptors on SUMOylation of the receptors in the cell membrane. We observed similar extent of colocalization of serotonin 1A receptor and SUMO-1 antibody in both the 8-OH-DPAT and vehicle treated groups. This was observed due to the limitations of light microscopy to distinguish between objects closer than 100nm as two different entities. Further studies need to be performed using techniques with higher resolution such as electron microscopy to study the effect of agonist stimulation on the SUMOylated serotonin 1A receptors. Our data provides some important insights about the putative sites of SUMOylation on the serotonin 1A receptor. The identification of the primary site of SUMOylation along with the knowledge about the effect of agonist stimulation on the SUMOylation of serotonin 1A receptors would help us decipher the role of SUMOylation in serotonin 1A receptor desensitization. Further understanding the regulation of serotonin 1A receptors by SUMOylation will aid in elucidating the role of serotonin 1A receptors in various mood disorders such as depression.
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