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. 2017:142:59-66.
doi: 10.1016/bs.mcb.2017.07.002. Epub 2017 Sep 14.

Measurement of intracellular Ca2+ mobilization to study GPCR signal transduction

Anisha Ashokan  1 , Gopala K Aradhyam  2
Affiliations

Measurement of intracellular Ca2+ mobilization to study GPCR signal transduction

Anisha Ashokan et al. Methods Cell Biol. 2017.

Abstract

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 Gq-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: Gq 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.

Keywords: EC(50); Fura2AM; G(q)-mediated signaling; GPCR: ligand-binding affinity; Intracellular Ca(2+) rise.

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