Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

We investigated whether granin aggregates could also be recovered from the TGN of another neuroendocrine cell line, the rat pituitary GH4C1 cells. These cells, like the related GH3B6 cell line, express prolactin and growth hormone (Tashjian, 1979) as well as CgB and SgII (Tougard et al., 1989; Scammell et al., 1990) . GH4C1 cells grown in the absence ofadded hormones contain a low number of secretory granules . Treatment of GH4C1 cells with a combination of EGF, estradiol, and insulin markedly increases the level of prolactin (Kiino and Dannies, 1982) , CgB and SgII (Scammell et al., 1990) , as well as that of secretory granules (Scammell et al., 1986) . As revealed by pulse labeling with r35 S]methionine ( Fig. 8 A, crease in SgII concentration in this compartment . Immunofluorescence of control and hormone-treated GH4Cl cells for SgII showed that, qualitatively, all ofthe cells were immunoreactive in both conditions, the immunostaining being observed in a perinuclear, Golgi-like pattern as well as in small punctate structures which were dispersed in the cytoplasm and close to the plasma membrane, and presumably were secretory granules ( Fig. 8 A, leftand right) . Quantitatively, however, hormone-treated cells showed an increase in SgII immunoreactivity, which was observed not only with respect to the secretory granule staining but, consistent with the increase in SgII synthesis, also in the Golgi area. This allowed us to use GH4C1 cells not only to extend our study on the aggregation of the granins in the TGN to other neuroendocrine cells, but also to investigate the possible role of protein concentration in this phenomenon. (Since prolactin is not sulfated in GH4C1 cells [data not shown], we could not include this protein in our analysis .) Hormone-treated GH4C1 cellswere pulse labeled for 4 min with FS]sulfate, and [uS]sulfate-labeled TGN vesicles were incubated with Triton X-100 in nonaggregative or aggregative milieu, as described above for PC12 cells. SgII, which was the major FS]sulfate-labeled band in these cells (data not shown), was found in the supernatant after incubation in nonaggregative milieu but was almost completely recovered in the pellet after incubation in aggregative milieu ( Fig. 8 B , bottom) . As shown by protein staining ofthe corresponding gel ( Fig. 8 B, top) , the protein pattern was not significantly affected by the incubation milieu .