Membrane-specific association of annexin I and annexin II in anterior pituitary cells.
Calpactins are members of the annexin family of structurally related Ca2(+)-dependent membrane binding proteins. Recent studies suggest a role for calpactins in the membrane fusion event of exocytosis. We show in this work that two members of the annexin family which are immunologically related to calpactin I (p36, annexin II) and calpactin II (p35, annexin I) are present in anterior pituitary cells. When sheep adenohypophyseal cells are disrupted in the absence of a Ca2+ chelator, immunoreactive calpactins associate with the crude vesicle fraction. Further purification of this subcellular fraction by sucrose density gradient centrifugation revealed a differential distribution: calpactin I was associated with secretory granule membranes and with plasma membranes, whereas calpactin II was found primarily with the plasma membrane fraction. Consistent with the Ca2+ and phospholipid binding properties of the calpactins, extraction of these proteins from the pituitary membranous fractions required sequential treatment with a detergent, octylglucoside, in the presence of 1 mM Ca2+ followed by solubilization with EGTA. Calpactins contain sites for phosphorylation by protein kinase C, and in this study we found phosphoprotein substrates for protein kinase C associated with secretory granule and plasma membranes which could be immunoprecipitated with calpactin antisera. In summary, the characteristics in anterior pituitary secretory cells of these two members of the annexin family lend support to the hypothesis that calpactins, potentially regulated by Ca2+ and by phosphorylation, may have a role in exocytosis.
Turgeon
J L
JL
Department of Human Physiology, School of Medicine, University of California, Davis 95616.
Cooper
R H
RH
Waring
D W
DW
D000818:Animals; D017302:Annexins; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient / Q000379:methods; D003594:Cytoplasmic Granules / Q000737:chemistry; D005260:Female; D007425:Intracellular Membranes / Q000737:chemistry; D008970:Molecular Weight; D010903:Pituitary Gland, Anterior / Q000737:chemistry*; D012756:Sheep
D017302:Annexins; D002135:Calcium-Binding Proteins