Measuring agonist-induced ERK MAP kinase phosphorylation for G-protein-coupled receptors.

Agonist stimulation of G-protein-coupled receptors (GPCRs) typically results in phosphorylation and activation of ERK (Extracellular-signal Regulated Kinase) which is a member of MAP kinase (Mitogen-Activated Protein kinase) family. Detection of phosphorylated ERK1/2 MAP kinase has been widely used as readout of GPCR signaling in heterologous cells, primary cells, tissues and even in animal studies. ERK1/2 phosphorylation downstream of GPCRs is now well established to arise from the activation of both, the heterotrimeric G-proteins and β-arrestins (βarrs) with distinct spatio-temporal components. Here, we present a step-by-step protocol for measuring agonist-induced ERK1/2 MAP kinase activation downstream of GPCRs using standard Western blotting assay. Note: ERK1/2 is also referred to as p44/42 MAP kinase. ERK1 and ERK2 are same as Mitogen-Activated Protein Kinase 3 (MAP3) and Mitogen-Activated Protein Kinase 1 (MAP1), respectively.