Functional analysis of olfactory receptors expressed in a HEK-293 cell system by using cameleons.
Cameleon is a genetically engineered Ca2+ sensing molecule consisting of two variants of the green fluorescent protein (GFP), calmodulin and calmodulin-binding protein, M13. HEK-293 cells stably expressing three types of cameleons, yellow cameleon-2, cameleon-3er, and cameleon-2nu, were constructed, and the expression and localization of these cameleons were confirmed by fluorescent imaging. Among the cameleons, the yellow cameleon-2 was selected for analyzing the change in Ca2+ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic Ca2+ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-l0 and 17, and the expression of the olfactory receptor genes were examined using immunocytochemical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular Ca2+ level, which was measured using yellow cameleon-2. These results demonstrate that yellow cameleon-2 can be conveniently used to examine the function of the olfactory receptors expressed in heterologous cells.
Ko
Hwi Jin
HJ
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Korea.
Park
Tai Hyun
TH
D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D018035:Receptors, Odorant / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D002135:Calcium-Binding Proteins; D018035:Receptors, Odorant; C495639:yellow cameleon; D002118:Calcium