DOPAMINE D-1 RECEPTOR DISTRIBUTION IN SF9 CELLS IMAGED BY CONFOCAL MICROSCOPY - A QUANTITATIVE-EVALUATION

A c-myc epitope-tagged human dopamine D-1 receptor (c-myc D-1 receptor ) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. I n the basal state, immunofluorescently labeled c-myc D-1 receptors ima ged by confocal microscopy appeared as a bright ring of label predomin antly on the cell surface, and to a lesser extent as intracellular dus ters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fract ions using the D-1 receptor-antagonist [H-3]-SCH-23390. After exposure to dopamine, c-myc D-1 receptors were redistributed on the cell surfa ce, changing from a continuous ring to a discontinuous pattern of labe l. Analysis of fluorescence intensity and three-dimensional computer r econstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by ra dioligand-binding analysis. These findings constituted the first direc t evidence of agonist-induced D-1 receptor internalization. The result s showed that the combination of confocal microscopy and three-dimensi onal reconstruction can be used to visualize and assess receptor distr ibution in Sf9 cells.