Prolactin and secretogranin-II, a marker for the regulated pathway, are secreted in parallel by pituitary GH4C1 cells.
The granins are a family of tyrosine-sulfated secretory proteins. Two members of this family, chromogranin-B (CgB) and secretogranin-II (SgII), are found in GH4C1 cells, a pituitary cell line that secretes PRL and GH. We have compared the spontaneous and regulated secretion of CgB and SgII with that of PRL in GH4C1 cells and have assessed the importance of granin sulfation on granin and PRL processing and secretion. CgB and SgII were identified by metabolic labeling with [35S]SO4, which was predominantly incorporated into two bands of 105,000 (CgB) and 84,000 (SgII) mol wt. The secretion of [35S]SgII and [35S]PRL from GH4C1 cells simultaneously labeled with 35S-labeled SO4 and methionine showed similar kinetics over 60 min, suggesting that the two proteins are similarly processed. CgB, SgII, and PRL were released in parallel after 10-min treatment with secretagogues (high K+ and BAY K8644, 8-bromo-cAMP, a phorbol ester, and TRH). Hypertonicity and substitution of chloride with isethionate, which inhibit stimulated PRL release, reduced the amount of CgB and SgII released in response to secretagogues, but not basally. Cells were labeled with [35S]SO4 with or without 10 mM chlorate, which inhibits sulfation by more than 90%, and media and cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and immunoblotting using an antibody directed against the N-terminus of SgII. Chlorate reduced [35S]SO4 labeling of CgB and SgII, but had little effect on immunoreactive SgII in cells or media. Inhibiting sulfation with chlorate did not change the amount of PRL or GH synthesized and secreted by GH4C1 cells, basally or in response to secretagogues, or the induction of PRL storage by insulin, estrogen, and epidermal growth factor. The results show that granins are released from GH4C1 cells in parallel with GH and PRL under basal and stimulated conditions, and that sulfation is not essential for normal packaging and processing of these secretory proteins. The data suggest a model in which PRL, CgB, and SgII are sorted to the regulated pathway and released from this pathway basally as well as under stimulated conditions.
Hinkle
P M
PM
Department of Pharmacology, University of Rochester Medical Center, New York 14642.
Scammell
J G
JG
Shanshala
E D
ED
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002704:Chlorates / Q000494:pharmacology; D002864:Chromogranins; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010911:Pituitary Neoplasms; D011388:Prolactin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D002704:Chlorates; D002864:Chromogranins; D003470:Culture Media; D011506:Proteins; D013431:Sulfates; D013462:Sulfur Radioisotopes; D011388:Prolactin; D008715:Methionine