ANNEXINS IN THE HUMAN NEUROBLASTOMA SH-SY5Y - DEMONSTRATION OF RELOCATION OF ANNEXIN-II AND ANNEXIN-V TO MEMBRANES IN RESPONSE TO ELEVATIONOF INTRACELLULAR CALCIUM BY MEMBRANE DEPOLARIZATION AND BY THE CALCIUM IONOPHORE A23187

The human neuroblastoma SH-SY5Y was found to express annexins I, II, I V, V, and VI by western blot analysis, Calcium-dependent membrane-bind ing proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with M(r) and pi values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting, The membrane asso ciation of annexins II and V was studied in cells that had been stimul ated to release noradrenaline by K+ depolarisation or by treatment wit h the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA an d required Triton X-100 for their solubilisation, Homogenisation of ce lls in calcium-containing buffers also resulted in the formation of EG TA-resistant membrane-associated annexins II and V, The results demons trate calcium-dependent relocation of annexins II and V to membranes i n intact cells and suggest that these annexins bind in a calcium-depen dent manner to non-phospholipid components of SH-SY5Y membranes. Exami nation of cells by immunofluorescence microscopy demonstrated that ann exin II was homogeneously associated with the plasma membrane before t reatment with ionophore and relocated to discrete patches of staining after treatment, Annexin V was found by immunofluorescence to be prese nt in the cytoplasm and in the nucleus, Stimulation of the cells produ ced no change in the cytoplasmic staining pattern but resulted in a pa rtial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium sig nalling at discrete intracellular locations. The results are not consi stent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.