Translocation of cytosolic annexin 2 to a Triton-insoluble membrane subdomain upon nicotine stimulation of chromaffin cultured cells.

To gain a better understanding of the function of annexin 2, we have investigated the subcellular distribution of the monomeric and heterotetrameric forms of annexin 2 and their relationship to the cytoskeleton upon stimulation of chromaffin cells. Quantitative immunoblotting has revealed that in resting cells a large amount of annexin 2 is monomeric and cytosolic. Upon nicotine stimulation 80% of total annexin 2 becomes associated with a Triton-X100-insoluble fraction where the monomeric and the heterotetrameric forms are found. The translocation of monomeric annexin 2 is Ca2+-dependent and complete at 1 microM free Ca2+. We have shown that about 66% of the annexin 2 associated with the Triton-X100-insoluble fraction is soluble in octylglucoside while the remnants are insoluble in the detergent and remain likely associated with actin filaments and associated cytoskeleton proteins. The octylglucoside-soluble fraction contains integral proteins from the plasma membrane and from granule membrane, but does not contain caveolin. Moreover, upon nicotine stimulation, a redistribution of proteins was detected in this fraction. These dynamic processes appear concomitantly with the phosphorylation of annexin 2 in this compartment and with catecholamine release. It is suggested that the soluble octylglucoside fraction may represent a special lipidic membrane compartment where the NSF attachment proteins and the cytosolic proteins like annexin 2 and rab3a may become concentrated upon stimulation of the cell. The presence of annexin 2 is consistent with its proposed function on granule and target membrane proteins required for the close apposition of two distinct membranes and supports its functional role in the regulated exocytosis/endocytosis process.