Localization of phospholipase C beta 1 on the detergent-resistant membrane microdomain prepared from the synaptic plasma membrane fraction of rat brain.

The membrane microdomain (MD), such as detergent-resistant low-density membrane microdomain fraction (DRM), has been paid much attention because many signal-transducing molecules are recovered in this fraction, although precise localization and interactions of these molecules are largely unclear. To identify neuronal MD-localized proteins, monoclonal antibodies (mAbs) against the DRM-components of synaptic plasma membrane fraction (SPM) were produced and the antigens were characterized. One of the antigens reacted with two closely positioned bands of about 140 kDa in SDS-PAGE and the antigen showed age-dependent localization on DRM. The antigen was immunoprecipitated with the mAb after partial solubilization with 0.6 M NaCl from SPM-derived DRM and identified as phospholipase C beta 1 through mass analysis. The identity was further confirmed with Western blotting using a specific polyclonal antibody. The enzyme purified from the DRM was activated by the alpha subunit of trimeric G protein, Gq, expressed in HEK293 cells. The lipid composition of the liposomes affected the enzymatic activity and the addition of NAP-22, a neuronal DRM-localized protein, inhibited the activity. These results suggest that there exists a signal-transducing MD that performs important roles in neuronal functions through PIP(2) signaling and Ca(2+) mobilization. (c) 2007 Wiley-Liss, Inc.