Dopamine D1 receptor distribution in Sf9 cells imaged by confocal microscopy: a quantitative evaluation.
A c-myc epitope-tagged human dopamine D1 receptor (c-myc D1 receptor) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. In the basal state, immunofluorescently labeled c-myc D1 receptors imaged by confocal microscopy appeared as a bright ring of label predominantly on the cell surface, and to a lesser extent as intracellular clusters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fractions using the D1 receptor-antagonist [3H]-SCH-23390. After exposure to dopamine, c-myc D1 receptors were redistributed on the cell surface, changing from a continuous ring to a discontinuous pattern of label. Analysis of fluorescence intensity and three-dimensional computer reconstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by radioligand-binding analysis. These findings constituted the first direct evidence of agonist-induced D1 receptor internalization. The results showed that the combination of confocal microscopy and three-dimensional reconstruction can be used to visualize and assess receptor distribution in Sf9 cells.
Trogadis
J E
JE
Eye Research Institute of Canada, Toronto Hospital, Ontario.
Ng
G Y
GY
O'Dowd
B F
BF
George
S R
SR
Stevens
J K
JK
D000818:Animals; D002478:Cells, Cultured; D004298:Dopamine / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007313:Insecta; D018613:Microscopy, Confocal; D011869:Radioligand Assay; D017447:Receptors, Dopamine D1 / Q000032:analysis* / Q000037:antagonists & inhibitors
D017447:Receptors, Dopamine D1; D004298:Dopamine