Secretory Protein Targeting in a Pituitary Cell Differential Transport of Foreign Secretory Proteins to Distinct Secretory Pathways Line: HSIAO-PING H. MOORE and REGIS

The mouse pituitary cell line, AtT-20, packages the adrenocorticotropic hormone (ACTH) in secretory vesicles and releases it when the cell is stimulated with secretagogues. These cells have the capacity, after transfection with the appropriate DNA, to package heterologous peptide hormones into the regulated secretory vesicles (Moore, H. P. H., M. D. Walker, F. Lee, and R. B. Kelly, 1983, Cell, 35:531-538). To test if other secreted proteins prefer a different route to the surface, we have transfected AtT-20 cells with DNAs coding for a fragment of a membrane protein, the vesicular stomatitis virus G protein from which the membrane spanning domain has been deleted (Rose, J. K., and J. E. Bergmann, 1982, Cell, 17:813-819). We found that the secreted vesicular stomatitis virus G proteins were not transported to the regulated secretory vesicles. Instead they preferentially exited the cell by the constitutive pathway previously found in these cells (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59). In contrast, human growth hormone transfected into the cells by the same procedure was transported to the regulated pathway with a similar efficiency as the endogenous hormone ACTH. Transport of the secreted G protein to the regulated pathway, if it occurs at all, is at least 30-fold less efficient than peptide hormones. We conclude that the transport machinery in AtT-20 cells must selectively recognize different secreted proteins and sort them into distinct secretory pathways. Most higher eucaryotic cells can secrete proteins into the extracellular space. It has been known for some time that secretion from a given cell type can be either regulated or constitutive (31). Regulated secretory cells store specialized secretory products at high concentrations in secretory vesicles, which accumulate in the cytoplasm until exocytosis is triggered by a stimulus. In constitutively secreting cells, newly synthesized protein is not stored but leaves the Golgi apparatus in short-lived membrane vesicles that fuse immediately with the plasma membrane in the absence of any extracellular signal. In both types of secretory cell, different secretory proteins can be found in the same secretory vesicle. It was therefore plausible to suggest that secreted proteins are not segregated from one another in the Golgi apparatus (7). Segregation of secretory proteins needed to be reconsidered, however, when the constitutive and the regulated secretory pathways were found to co-exist in the same cell. The pituitary cell line, AtT-20, has a regulated pathway in which the adreTHE JOURNAL OF CELL BIOLOGY . VOLUME 101 November 1985 1773-1781 © The Rockefeller University Press • 0021-9525/85/11/1773/09 $1.00 nocorticotropic hormone (ACTH) ~ precursor, proopiomelanocortin (POMC), is processed to mature ACTH, stored in regulated secretory vesicles, and released only when the cells are stimulated, for example, with 8-Br-cAMP. The AtT-20 cells use the constitutive pathway to externalize a viral membrane glycoprotein (gp70) made by these cells (12). Further evidence that there are two routes to the surface in endocrine cells was obtained using inhibitors (10, 17). Although membrane proteins preferentially take the constitutive route to the surface, secreted proteins examined thus far appear to use both pathways for externalization. Some newly synthesized hormones, for example, did not enter the Abbreviations used in this paper. ACTH, adrenocorticotropic hormone; CAT, chloramphenicol acetyltransferase; Endo H, endoglycosidase H; ER, endoplasmic reticulum; hGH, human growth hormone; POMC, proopiomelanocortin; RIA, radioimmunoassay; RSVLTR, Rous sarcoma virus long terminal repeats; SV40, Simian virus 40; TG, truncated G protein; VSV, vesicular stomatitis virus. 1773 on A ril 6, 2017 D ow nladed fom Published November 1, 1985