Identification of sentrin specific proteases and SUMOylation sites of 5-HT1A receptors

Abstract

The serotonin-1A receptor (5-HT1A-R) is an abundant 5-HT receptor located in both pre-synaptic and post-synaptic membranes of neurons. Activation of this receptor has been involved in the mechanism of action of antidepressant, antipsychotic and anxiolytic drugs. Thus, studies of 5-HT1A-Rs might help to manage various psychiatric diseases. SUMOylation of 5-HT1A-Rs has recently been reported and evidence suggests that SUMOylation might play an important role in the regulation of 5-HT1A-Rs. Previous studies in our lab found that 5-HT1A-Rs can be modified by SUMO-1 in rat brain and neuroblastoma2a cells. The majority of SUMO-1-5-HT1A-Rs are co-localized with endoplasmic-reticulum and trans-Golgi-network markers. Treatment with the 5-HT1A-R agonist, 8-OH-DPAT, increases SUMO-1-5-HT1A-Rs in the detergent-resistant membrane (DRM). SUMO-1-5-HT1A-Rs minimally bind to 5-HT1A-R agonist. Investigating the mechanism involved in the SUMOylation of 5-HT1A-R will help to understand the regulation of 5-HT1A-R and may lead to the development of novel therapeutic approaches for psychiatric diseases. In this study, I focused on the SUMO machinery regulating deSUMOylation of 5-HT1A-Rs and the SUMOylation sites on 5-HT1A-Rs. I found that mouse Neuroblastoma 2a (N2a) cells can express exogenous 5-HT1A-Rs and verified expression of endogenous SUMOylation of 5-HT1A-Rs. The SUMOylation of 5-HT1A-Rs was detected around 55KD in N2a cells which is consistent to our previous finding in rat brain. In addition, N2a cells can express transfected sentrin-specific proteases (SENPs) proteins in the membrane fraction. Transfection of SENP1, 2 and 6 causes a significant difference among groups but there is no significant difference between each transfected SENPs group compared to the non-transfected control group. To identify the SUMOylation sites on 5-HT1A-Rs, six possible SUMOylation sites on 5-HT1A-R: K302, K332, K324, K232 and K235 were mutated into nonSUMOylatable arginine residues. I transfected each 5-HT1A-R mutant into N2a cells and found that transfection of each 5-HT1A-R mutant does not reduce SUMOylation of 5-HT1A-Rs. However, SUMOylation of transfected rat 5-HT1A-Rs has a different molecular mass compared to SUMOylation of endogenous 5-HT1A-Rs in N2a cells.