Measurement of Inositol (Poly)phosphate Formation Using [3H]Inositol Labeling Protocols in Permeabilized Cells

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Abstract

Hormones, neurotransmitters, chemoattractants, and growth factors all elicit intracellular responses, on binding to cell surface receptors, by activating inositol phospholipid-specific phospholipase C (PLC). Activated PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2), a minor membrane phospholipid, to form two second messengers, diacylglycerol (DAG) and inositol (1,4,5)trisphosphate [Ins(1,4,5,)P3]. DAG is a direct activator of protein kinase C isozymes, Ins(1,4,5)P3 mobilizes intracellular Ca2+. G protein-coupled receptors couple to the PLC-β family via G proteins, and tyrosine kinase receptors activate PLCγ isozymes (1). Regardless of the PLC isozyme activated, the product is invariantly Ins(1,4,5)P3.