Measurement of intracellular Ca²⁺ mobilization to study GPCR signal transduction

Understanding G protein-coupled receptor (GPCR) structure-function relationship and its activation mechanism has been broadly explored using mutational strategy due to problems in GPCR crystallization. Probing into GPCR: effector (G protein/β-arrestin) interactions and downstream signaling are important aspects of GPCR research. Among the G proteins, though there are some approaches to investigate G_q-mediated signaling, they involve the use of radioactivity and are qualitative in nature. Our method described here makes use of the cell permeable nature of fluorescent Ca²⁺ indicator dye, fura2AM, that binds with the Ca²⁺ released in response to GPCR: G_q interaction on ligand treatment. Using this spectrophotometric method, EC₅₀ values of the GPCR: ligand binding can be calculated and the binding affinity can be analyzed.