Comparing Chronic Treatments of the INI and VSV Isoforms of the Human Serotonin 2C Receptor Impacting Phosphorylation of STAT3
Serotonin (5-HT), an indolamine neurotransmitter derivative of tryptophan, is located solely in the central nervous system, as well as the peripheral nervous system. 5-HT has a significant role in the human anatomy due to its involvement in an abundance of physiological functions such as modulating cognitive ability, appetite, mood, sleeping patterns, neuronal excitability, and homeostasis of glucose and energy. 5-HT elicits its effects by binding to at least 14 different receptor subtypes, one in particular, 5-HT2C. Previously discovered as a 7-transmembrane spanning G protein-coupled receptor (GPCR), the 5-HT2C receptor subtype is only GPCR currently to perform RNA editing, resulting in at least 14 different isoforms in the human brain. In addition, this receptor has served as a target for atypical antipsychotic medication in order to treat patients compromised with psychological deficiencies. The 5-HT2C receptor has also been considered a prime research candidate in intercellular activity due to its ability to influence signaling pathways of several proteins. The purpose of this study was to determine to what extent the human 5-HT2C receptor manipulates phosphorylated activity of the STAT3 protein in the JAK/STAT pathway by comparing two different receptor isoforms exposed to chronic drug treatments. Human Embryonic Kidney (HEK) 293 cells were transfected with human 5-HT2C receptors in the INI or VSV isoform. 5-HT2C-INI/VSV receptor transfected cells were treated with control, 5-HT, water, olanzapine, SB206553, or MK-212 for 1 hour and were also tested against untransfected HEK cells. All of the previous conditioned cells were made into lysates, proteins were separated by SDS-PAGE, and levels of phosphorylated STAT3 and unphosphorylated STAT3 were analyzed using western blots. NBT/BCIP developing solution detected untransfected cell (control) samples to contain and express phosphorylated STAT3 bands. The control was not supposed to have any protein bands present after antibody detection. This problem indicated that the particular experimental cells might have already contained a particular 5-HT2C receptor without our knowledge beforehand. Experiments will need to be restarted under the same transfections and 1 hour stimulations, but with completely new source of HEK293 cells.
"Comparing Chronic Treatments of the INI and VSV Isoforms of the Human Serotonin 2C Receptor Impacting Phosphorylation of STAT3"
ETD Collection for Tennessee State University.