A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors.

Phosphorylation of extracellular signal-regulated kinase (ERK) is linked to activation of many cell surface receptors and kinases. However, phosphorylated ERK has not been used as a biochemical marker to monitor pharmacology of these biomolecules, largely because commonly used methods to detect the phosphoprotein are not quantitative and do not have sufficient throughput. In this article, a high-throughput, 384-well, cell-based functional assay to quantify dopamine agonist-induced ERK phosphorylation in D2- and D3-overexpressed cell lines is described. The assay uses infrared-labeled secondary antibodies to detect phospho-ERK, and the signals in the wells of the microtiter plate are quantified by a LI-COR infrared scanner. V(max), EC(50), and functional K(i) values of various D2 and D3 agonists and antagonists determined in this assay are similar to those in the literature. The assay is nonradioactive, is quantitative, and has a good signal-to-noise ratio. In addition, the signal is stable. This assay can be used to monitor the activities of many G protein-coupled receptors and other signaling biomolecules that are linked to phosphorylation of ERK. The methodology can potentially be used to detect the change in level of any cellular protein in which highly selective antibodies are available.