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.
Taguchi
Katsutoshi
K
Division of Bioinformation, Department of Biosystems Science, Graduate School of Science and Technology, Kobe-University, Kobe, Japan.
Kumanogoh
Haruko
H
Nakamura
Shun
S
Maekawa
Shohei
S
D000704:Analysis of Variance; D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002461:Cell Line, Transformed; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007527:Isoenzymes / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D054799:Phospholipase C beta; D051381:Rats; D017208:Rats, Wistar; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D003902:Detergents; D007527:Isoenzymes; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; D054799:Phospholipase C beta; C518035:Plcb1 protein, rat; D043802:GTP-Binding Protein alpha Subunits, Gq-G11