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Glycosyl-phosphatidylinositol (GPI) anchored proteins are surveyed in two insulin sensitive cell types by surface labeling and phospholipase C-induced release into the medium. Serum starvation selectively increases both the number and intensity of a subset of GPI-anchored proteins. After serum starvation, loss of cell-surface GPI-anchored proteins is induced acutely by either serum re-exposure or insulin, suggesting that hormonal treatment may promote the release of these proteins from the cell surface.

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Treatment of PC-12 pheochromocytoma cells with nerve growth factor (NGF) results in the differentiation of these cells into a sympathetic neuron-like phenotype. Although the initial intracellular signals elicited by NGF remain unknown, some of the cellular effects of NGF are similar to those of other growth factors, such as insulin. We have investigated the involvement of a newly identified inositol-containing glycolipid in signal transduction for the actions of NGF. NGF stimulates the rapid generation of a species of diacylglycerol that is labeled with [3H]myristate but not with [3H]arachidonate. NGF stimulates [3H]myristate- or [32P]phosphate-labeled phosphatidic acid production over the same time course. Although NGF alone has no effect on the turnover of inositol phospholipids, it does stimulate the hydrolysis of glycosylphosphatidylinositol. The NGF-dependent cleavage of this lipid is accompanied by an increase in the accumulation of its polar head group, an inositol phosphate glycan, which is generated within 30-60 sec of NGF treatment. In an unresponsive PC-12 mutant cell line, neither the diacylglycerol nor inositol phosphate glycan response is detected. A possible role for the NGF-stimulated diacylglycerol is suggested by the inhibition of NGF-dependent c-fos induction by staurosporin, a potent inhibitor of protein kinase C. These results suggest that, like insulin, some of the cellular effects of NGF may be mediated by the phospholipase C-catalyzed hydrolysis of glycosylphosphatidylinositol.

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Lipoprotein lipase (LPL) plays a critical role in the metabolism of plasma lipoproteins. In 3T3-L1 adipocytes, insulin elicits the rapid release of LPL through mechanisms that are independent of energy metabolism and protein synthesis. Some of the metabolic actions of insulin may be mediated by the activation of a specific phospholipase that hydrolyzes a glycosyl phosphatidylinositol (PI) molecule. The insulin-sensitive glycosyl-PI is structurally similar to the glycolipid membrane anchor of a number of proteins. LPL appears to be anchored to the 3T3-L1 cell surface by glycosyl-PI, and its rapid release by insulin may be due to activation of a glycosyl-PI-specific phospholipase C.

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The molecular events involved in the cellular actions of insulin remain unexplained. Some of the acute actions of the hormone may be due to the intracellular generation of a chemical substance which modulates certain enzyme activities. Such an enzyme-modulating substance has been identified as an inositol phosphate-glycan, produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) precursor. This precursor glycolipid is structurally similar to the glycosyl-phosphoinositide membrane protein anchor. The exposure of fat, liver or muscle cells to insulin results in the hydrolysis of glycosyl-PtdIns, giving rise to the inositol phosphate glycan and diacylglycerol. This hydrolysis reaction is catalysed by a glycosyl-PtdIns-specific phospholipase C. This enzyme has been characterized and purified from a plasma membrane fraction of liver. This reaction also results in the acute release of certain glycosyl-PtdIns-anchored proteins from the cell surface. Elucidation of the functional role of glycosyl-phosphoinositides in the generation of second messengers or the release of proteins may provide further insights into the pleiotropic nature of insulin action.

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(Ca2+ + Mg2+)-ATPase activator protein associated with human erythrocyte membranes could be extracted with EDTA under isotonic condition at pH 7.6. No activator was released, however, using isotonic buffer alone. Like calmodulin, the activator in the EDTA extract migrated as a fast moving band on polyacrylamide gel electrophoresis. It was also heat-stable, was capable of stimulating active calcium transport and could stimulate (Ca+ + Mg2+)-ATPase to the same extent. When chromatographed on a Sephacryl S-200 column, it was eluted in the same position as calmodulin and a membrane associated (Ca2+ + Mg2+)-ATPase activator prepared according to Mauldin and Roufogalis (Mauldin, D. and Roufogalis, B. D. (1980) Biochem. J. 187, 507-513). Furthermore, both Mauldin and Roufogalis protein and the activator in the EDTA extract exhibited calcium-dependent binding to a fluphenazine-Sepharose affinity column. On the basis of these data, it is concluded that the activator protein released from erythrocyte membranes by EDTA is calmodulin. A further pool of the ATPase activator could be released by boiling but not by Triton X-100 treatment of the EDTA-extracted membranes. This pool amounted to 8.9% of the EDTA-extractable pool.

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