5-HT receptors are members of the G-protein coupled receptor (GPCR) superfamily. GPCR are present in most if not all cells in the human body and play vital roles in cell communication and survival. They are targets for more than 50% of currently marketed pharmaceuticals. Therefore, significant emphasis has been placed on understanding molecular mechanisms governing GPCR function. GPCR are believed to form dimeric or oligomeric complexes. However, little is known about how dimer/oligomer biogenesis occurs and how it regulates GPCR function. Currently, we are focusing on investigating receptor dimerization and how it affects receptor function using the 5-HT2C receptor as a model system. Fluorescent tags (CFP, YFP) are attached to the intracellular region of the 5-HT2C receptor and confocal microscopy is used to visualize receptor synthesis in the endoplasmic reticulum and subsequent trafficking of receptors to the plasma membrane. The effect of drug treatments on this process can be monitored. Confocal microscopy combined with fluorescence resonance energy transfer (FRET) allows us to identify potential interacting proteins, such as receptor homodimers/oliogomers or receptor interactions with other signaling proteins such as beta-arrestin.
GFP-tagged 5-HT2C antibodies have been created and are being used to label native 5-HT2C receptors endogenously expressed in primary hippocampal neurons. State-of-the-art confocal microscopy combined with fluorescence correlation spectroscopy (FCS) and bimolecular fluorescence complementation allow direct visualization of 5-HT2C receptors and will be used to provide evidence for homodimerization of native receptors expressed in living primary cell cultures.
5-HT pharmacology, GPCR structure and function.
The long-term objective of our research is to gain a better understanding of the physiological roles of serotonin (5-HT) in the central nervous system. 5-HT produces its actions by binding to specific receptors located in cell membranes. To date, 14 different 5-HT receptors have been cloned. Drugs that interact with 5-HT receptors are prescribed for anxiety, depression, schizophrenia, appetite regulation, migraine, and emesis. Drugs of abuse, such as LSD, stimulate 5-HT receptors in the brain. Molecular biology techniques are used to create genetic mutations in 5-HT receptor genes to gain a better understanding of how receptor structure is related to function. Genes for normal and mutant receptors are expressed in recombinant cell systems and evaluated for function using standard radiodligand binding and second messenger assays. Confocal microscopy is used to visualize trafficking of native and mutant receptors tagged with fluorescent proteins. Biophysical techniques (FRET, BRET) are used to explore the formation of receptor complexes (homodimers).