Abstract
Phosphatidylinositol-4,5-bisphosphate (PIP2) is well known to be a source of the important second messengers inositol-triphosphate (IP3) and diacylglycerol (DAG). To investigate the intracellular localization of PIP2, it has been commonly utilized pleckstrin homology (PH) domain fused with green fluorescent protein (PH-GFP) as a marker. However this method is not suitable for cells difficult to be transfected. In the present study, we established the assay method of receptor-induced transient dissociation of PIP2 from the plasma membrane in rat brown adipocytes using anti-PIP2 antibody. Cells incubated with or without stimuli for 0-120 sec were fixed immediately, blocked with BSA and incubated with anti-PIP2 antibody. After washing, the cells were incubated with Alexa Fluor 546-labeled IgG and fluorescent signals were observed using confocal laser scanning microscope. In control cells, PIP2 displayed staining which outlined the cells periphery. When the cells were stimulated with 1μM noradrenaline (NA), plasma membrane PIP2 was rapidly decreased within 2.5 sec. Dissociation of PIP2 from the plasma membrane was transient and relocalized to the plasma membrane within 2 min. Stimulation of the cells with 50μM ATP showed similar response to NA. 5μM wortmannin inhibited relocalization of PIP2 to the plasma membrane in NA- or ATP-stimulated cells, indicating that phosphatidylinositol-4-kinase (PI4K) plays an important role in PIP2 reproduction. [J Physiol Sci. 2006;56 Suppl:S118]
