NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells

Abstract

Rat PC12 pheochromocytoma cells respond to Nerve Growth Factor (NGF) by ceasing to undergo mitosis and acquiring neuronal characteristics, including outgrowth of neurites and electrical excitability. We have found that NGF treatment results in no consistent qualitative and only a few minor quantitative changes in the two-dimensional electrophoresis pattern of 14C-amino acid-labeled proteins synthesized by the cells. On the other hand, NGF stimulates the incorporation of radiolabeled fucose or glucosamine into several components, including one of apparent molecular weight 230,000 daltons on one- and two-dimensional SDS-polyacrylamide gels. This component is removed by mild trypsinization of intact cells and therefore appears to be a glycoprotein (named here NILE) at least partially exposed on the cell surface. Stimulation of NILE glycoprotein labeling can first be detected after 2 days of exposure to NGF and increases progressively with time of treatment. This change is not solely a consequence of the cessation of cell division caused by NGF, since it does not occur in nondividing PC12 populations prepared by cytosine arabinoside treatment. NGF stimulates labeling of NILE glycoprotein even when attachment and process outgrowth are prevented by growing the cells in spinner suspension cultures. The relative rate of labeling attained under these conditions is less, however, than in NGF-treated, substrate-attached cells. Stimulation of NILE glycoprotein labeling by NGF is selectively blocked (as is neurite outgrowth) by camptothecin, an RNA synthesis inhibitor, and thus may require transcription. Despite their similarities in apparent size and exposure on cell surfaces, the NILE and LETS glycoproteins are shown to be immunologically distinct.