Abstract
Abstract: :
Purpose: Most living organisms synchronize biochemical and behavioral events to the dark /light cycles of their environment. Organisms must be able to adapt to daily and seasonal changes in environmental light conditions for survival. Timing of these events in mammals is under the regulation of the circadian clock located in the suprachiasmatic nucleus (SCN) region of the hypothalamus. In mammals the circadian oscillators for this biological clock get light signaling information from ocular photoreceptors. The light information was thought to come exclusively from rod and cone visual photoreceptors, but recent evidence has suggested that melanopsin, a novel opsin–like protein, is necessary for normal photoentrainment in mammals. Melanopsin expression has been identified in a subset of retinal ganglion cells (RGCs) in the inner retina of mammals that are photosensitive. This subset of RGCs have axons that project directly to the SCN. Melanopsin has been suggested to function as a light–activated sensor in a non–image forming signaling system that helps to regulate photoentrainment of circadian responses in mammals. The protein structure and location of expression of melanopsin suggests its function as a photopigment. We hypothesize that melanopsin is the retinal photopigment receiving light input and transducing a biochemical signal for the regulation of photoentrainment in mammals. Methods: We are using a molecular and biochemical approach to identify the spectral properties and the signaling pathway for melanopsin. We express mouse melanopsin using a heterologous mammalian cell lines expression system. The expressed melanopsin has a 1D4 epitope tag (the last 18 amino acids of Bovine Rhodopsin) appended to its C–terminus. Expressed melanopsin is reconstituted with chromophore and then purified using immuno–affinity column chromatography. Purified melanopsin is analyzed using a spectrophotometer to determine the wavelength absorbance spectrum for the photopigment. Purified membranes containing melanopsin are used in a filter–binding assay to determine the photopigments rate of G–protein activation Results: We have recently shown that melanopsin reconstituted with 11–cis–retinal has a maximum wavelength absorbance of 424nm, and that this photopigment can activate the G–protein transducin (Gt) in a light dependent manner. Conclusion: These results show that melanopsin forms a functional photopigment in vitro. These results also support the hypothesis that melanopsin is involved in the regulation of photoentrainment of the circadian clock in mammals.
Keywords: opsins • ganglion cells • signal transduction