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Accumulation of Synaptosomal-Associated Protein of 25 kDa (SNAP-25) and Other Proteins Associated with the Secretory Pathway in GH₄C₁ Cells Upon Treatment with Estradiol, Insulin, and Epidermal Growth Factor¹

Department of Pharmacology, Yale University School of Medicine (M.S.L., Y.L.Z., Z.S., H.R.), New Haven, Connecticut 06510; and Samuel Lunenfeld Research Institute, Mount Sinai Hospital (A.J., J.R.), Toronto, Ontario, Canada J5G 1X5

Address all correspondence and requests for reprints to: Dr. Priscilla Dannies, Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06511.

Treatment of rat pituitary GH₄C₁ cells with estradiol, insulin, and epidermal growth factor induces secretory granule accumulation, PRL storage, and stabilization of ICA512, a membrane protein associated with secretory granules. In these investigations we found that the same treatment induced accumulation over 2-fold of other proteins in the secretory pathway, including synaptosomal-associated protein of 25 kDa (SNAP-25), synaptotagmin III, synaptobrevin, synaptophysin, and cyclophilin B, and did not affect accumulation of others, including synaptotagmin I, calnexin, and glucose-regulated protein 94. The induction of proteins was not a coordinate event, because epidermal growth factor alone maximally stimulated SNAP-25 accumulation, but not that of synaptotagmin III. Induction of SNAP-25 accumulation occurred without an increase in its synthesis, and induction of cyclophilin B occurred without an increase in its messenger RNA accumulation, suggesting that accumulation may be caused by stabilization of the proteins. SNAP-25 immunofluorescence was located in the cytoplasm and on the plasma membrane and sometimes was heavily concentrated in protrusions from the cell surface, especially in hormone-treated cells. Frequenin immunofluorescence was also sometimes concentrated in intense patches, but did not colocalize with SNAP-25. Growth hormone and prolactin immunofluorescence was not found in the protrusions and sometimes did not colocalize with each other when they were present in the same cell. Hormone treatment of GH₄C₁ cells therefore induces accumulation of specific proteins in all parts of the secretory pathway and causes morphological changes in addition to accumulating secretory granules.

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