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

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Extracellular signal-regulated kinase (ERK) phosphorylation is a key indicator of cell surface receptor and kinase activation.
• Existing methods for detecting phospho-ERK lack the quantification and throughput needed for robust pharmacological studies.

Purpose of the Study:

• To develop a high-throughput, quantitative assay for measuring dopamine agonist-induced ERK phosphorylation.
• To validate the assay's utility for determining pharmacological parameters of D2 and D3 receptor ligands.

Main Methods:

• A 384-well, cell-based functional assay utilizing infrared-labeled secondary antibodies to detect phospho-ERK.
• Quantification of signals using a LI-COR infrared scanner in D2- and D3-overexpressed cell lines.

Main Results:

• The assay accurately determined V(max), EC(50), and functional K(i) values for D2 and D3 agonists and antagonists, consistent with literature values.
• The assay demonstrated to be nonradioactive, quantitative, stable, and possess a good signal-to-noise ratio.

Conclusions:

• This novel assay provides a reliable and efficient method for monitoring ERK phosphorylation.
• The methodology is applicable to studying a wide range of G protein-coupled receptors and other signaling biomolecules linked to ERK phosphorylation.
• The technique has potential for detecting changes in any cellular protein using selective antibodies.