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.
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Korea.
Hwi Jin
HJ
Ko
Tai Hyun
TH
Park
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