A pituitary tumor cell line (AcT-20) from the mouse packages mature ACTH into secretory vesicles but also leaks the longer precursor form, proopiomelanocortin (POMC) into the medium by a non-regulated or constitutive pathway. Human proinsulin, human growth hormone and rat trypsinogen, encoded by transfected DNA also take two pathways to the surface but other proteins such as the endogenous laminin or the truncated viral G protein, encoded by transfected DNA, only exit constitutively. Since the truncated viral G protein can be targeted to the secretory vesicle by fusing it with human growth hormone, we propose that peptide hormones, and trypsinogen, share a common sorting domain responsible for correct targeting to the secretory vesicle. To identify such domains we plan to delete by oligonucleotide mutagenesis candidate domains from rat trypsinogen, taking advantage of the known three-dimensional structure of rat trypsinogen and other proteases. To examine the properties of the constitutive pathway, DNA encoding immunoglobulin light and heavy chains will be transfected into AtT-20 cells. Since immunoglobulins must assemble before secretion, we hope to gain insight into rules of protein assembly that will facilitate the engineering of proteins capable of secretion by mammalian cells. The immediate goal is to link fragments of peptide hormone to immunoglobulin chains, and re-direct them to secretory vesicles. To allow us to begin a molecular explanation of protein secretion we have developed an AtT-20 variant into the cytoplasm of which macromolecules can be delivered with high efficiency using red blood cell fusion. Antibodies to vesicle and cytoskeletal components will be introduced to try to inhibit secretory vesicle formation, and vesicle movement to the cell surface. Evidence that secretory vesicles show a selective association with cytoskeletal elements will be sought. The ability to study secretory vesicle movement, secretory vesicle formation and exocytosis in a cell line that can be readily transfected with appropriate expression vectors, and whose cytoplasm can be readily modified by red blood cell microinjection gives an unusually rich opportunity to study the nature of protein secretion and its disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK033937-04
Application #
3232340
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1984-04-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Cleves, A E; Cooper, D N; Barondes, S H et al. (1996) A new pathway for protein export in Saccharomyces cerevisiae. J Cell Biol 133:1017-26
Day, R; Benjannet, S; Matsuuchi, L et al. (1995) Maintained PC1 and PC2 expression in the AtT-20 variant cell line 6T3 lacking regulated secretion and POMC: restored POMC expression and regulated secretion after cAMP treatment. DNA Cell Biol 14:175-88
Sauer, M K; Kelly, R B (1995) Conjugation rescue of exocytosis mutants in Tetrahymena thermophila indicates the presence of functional intermediates in the regulated secretory pathway. J Eukaryot Microbiol 42:173-83
Grote, E; Hao, J C; Bennett, M K et al. (1995) A targeting signal in VAMP regulating transport to synaptic vesicles. Cell 81:581-9
Green, S A; Setiadi, H; McEver, R P et al. (1994) The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes. J Cell Biol 124:435-48
Turkewitz, A P; Kelly, R B (1992) Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody. Dev Genet 13:151-9
Green, S A; Kelly, R B (1992) Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells. J Cell Biol 117:47-55
Matsuuchi, L; Gold, M R; Travis, A et al. (1992) The membrane IgM-associated proteins MB-1 and Ig-beta are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line. Proc Natl Acad Sci U S A 89:3404-8
Grimes, M; Kelly, R B (1992) Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells. J Cell Biol 117:539-49
Grimes, M; Kelly, R B (1992) Sorting of chromogranin B into immature secretory granules in pheochromocytoma (PC12) cells. Ann N Y Acad Sci 674:38-52

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