Optimization of a Protein Binding Assay for cyclic AMP to Study the Function of Melatonin Receptors
Stroh, Benjamin A.
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Melatonin, a hormone produced by the pineal gland and retina, has become a popular subject of interest in the field of neurobiology. It plays a major role in the lives of all vertebrates by regulating circadian rhythms, which govern sleep/wake cycles as well as seasonal behavior patterns. Melatonin is currently used in several therapeutic applications, but the extent of the physiological effects of the hormone is not fully understood. It has been determined that the hormone initiates a signaling cascade within cells via at least two specific inhibitory G-protein linked receptors (Reppert, 1997). These receptors activate a G-protein that inhibits the enzyme adenylyl cyclase, which is responsible for the production of cyclic-AMP. The measurement of this inhibition is the basis for an assay to study the function of these melatonin receptors under different conditions. The aim of this project was to determine the optimal parameters and procedures for this measurement. When put to use, the newly modified assay produced promising results. They suggest that the assay is more sensitive in measuring the inhibition of forskolin stimulated cAMP production in cells expressing a high number of melatonin receptors than previous methods. However, the assay yields less reliable results in situations where less inhibition is taking place. The modified assay has the potential to allow researchers to gain a better understanding of the functions of melatonin receptors.