Annexins in the Human Neuroblastoma SH‐SY5Y: Demonstration of Relocation of Annexins II and V to Membranes in Response to Elevation of Intracellular Calcium by Membrane Depolarisation and by the Calcium Ionophore A23187

Abstract

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and p1 values similar to those of annexins I, II, Ill, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by Kdepolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to nonphospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus. Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

Occurrence of Two Types of Secretory Vesicles in the Human Neuroblastoma SH‐SY5Y

“…A similar punctate pattern has been observed by using immunocytochemistry to determine the localization of synaptobrevin and synaptophysin in PCI 2 cells (Chilote et al, 1995); synaptotagmin, rab3a, and synaptophysin in cultured hippocampal neurons (Matteoli et al, 1991(Matteoli et al, , 1992; NPY in the neuroblastoma NG1O8-l5 (Fried et al, 1995); and secretogranin 11 in SH-SY5Y (Giudici et al, 1992). The immunocytochemical staining pattern observed in this study is very different from the diffuse, uniform staining observed in our previous work on the distribution of annexins II and V in SH-SYSY (Blanchard et al, 1996). Annexin 11 is located at the plasma membrane and annexin V is a soluble protein distributed between the cytosol and the nucleus.…”

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis

“…Most members of the annexin family, including AnxA2, are recruited to cellular membranes in response to several stimuli that induce intracellular Ca 2ϩ mobilization (20). Interaction of AnxA2 at the N terminus with p11/S100A10 or by phosphorylation has been proposed to play a regulatory role in the Ca 2ϩ -dependent association to the cell membranes (21).…”

4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-ones as N-formyl peptide receptor 1 (FPR1) antagonists

“…It should be noted that compounds having the same scaffold were also reported as small molecule blockers of the interaction between S100A10 and annexin A2 [34]. Because both annexin A2 and FPR1 are involved in pathogenesis of tumor growth and invasion of neuroblastoma, glioma, and hepatocellular carcinoma [35–37], development of FPR1 antagonists based on the 4-aroyl-3-hydroxy-5-phenyl-1 H -pyrrol-2(5 H )-one scaffold could lead to promising dual functional agents for the treatment of these diseases.…”