Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Aug 1;101(2):639–645. doi: 10.1083/jcb.101.2.639

The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer

PMCID: PMC2113688  PMID: 2991303

Abstract

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br- cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler M., Wong B. S., Sabol S. L., Busis N., Jackson M. B., Weight F. F. Action potentials and membrane ion channels in clonal anterior pituitary cells. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2086–2090. doi: 10.1073/pnas.80.7.2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baratti J., Maroux S., Louvard D. Effect of ionic strength and calcium ions on the activation of trypsinogen by enterokinase. A modified test for the quantitative evaluation of this enzyme. Biochim Biophys Acta. 1973 Oct 10;321(2):632–638. doi: 10.1016/0005-2744(73)90206-4. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blundell T. L., Cutfield J. F., Cutfield S. M., Dodson E. J., Dodson G. G., Hodgkin D. C., Mercola D. A., Vijayan M. Atomic positions in rhombohedral 2-zinc insulin crystals. Nature. 1971 Jun 25;231(5304):506–511. doi: 10.1038/231506a0. [DOI] [PubMed] [Google Scholar]
  6. Burgess T. L., Kelly R. B. Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan. J Cell Biol. 1984 Dec;99(6):2223–2230. doi: 10.1083/jcb.99.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craik C. S., Choo Q. L., Swift G. H., Quinto C., MacDonald R. J., Rutter W. J. Structure of two related rat pancreatic trypsin genes. J Biol Chem. 1984 Nov 25;259(22):14255–14264. [PubMed] [Google Scholar]
  8. Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
  9. Durnam D. M., Palmiter R. D. Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem. 1981 Jun 10;256(11):5712–5716. [PubMed] [Google Scholar]
  10. Gumbiner B., Kelly R. B. Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):318–322. doi: 10.1073/pnas.78.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gumbiner B., Kelly R. B. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell. 1982 Jan;28(1):51–59. doi: 10.1016/0092-8674(82)90374-9. [DOI] [PubMed] [Google Scholar]
  12. Huber R., Kukla D., Bode W., Schwager P., Bartels K., Deisenhofer J., Steigemann W. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. J Mol Biol. 1974 Oct 15;89(1):73–101. doi: 10.1016/0022-2836(74)90163-6. [DOI] [PubMed] [Google Scholar]
  13. Karin M., Andersen R. D., Herschman H. R. Induction of metallothionein mRNA in HeLa cells by dexamethasone and by heavy metals. Eur J Biochem. 1981 Sep 1;118(3):527–531. doi: 10.1111/j.1432-1033.1981.tb05551.x. [DOI] [PubMed] [Google Scholar]
  14. Karin M., Haslinger A., Holtgreve H., Cathala G., Slater E., Baxter J. D. Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA. Cell. 1984 Feb;36(2):371–379. doi: 10.1016/0092-8674(84)90230-7. [DOI] [PubMed] [Google Scholar]
  15. Kelly R. B., Buckley K. M., Burgess T. L., Carlson S. S., Caroni P., Hooper J. E., Katzen A., Moore H. P., Pfeffer S. R., Schroer T. A. Membrane traffic in neurons and peptide-secreting cells. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):697–705. doi: 10.1101/sqb.1983.048.01.073. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lingappa V. R., Devillers-Thiery A., Blobel G. Nascent prehormones are intermediates in the biosynthesis of authentic bovine pituitary growth hormone and prolactin. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2432–2436. doi: 10.1073/pnas.74.6.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  19. MacDonald R. J., Stary S. J., Swift G. H. Two similar but nonallelic rat pancreatic trypsinogens. Nucleotide sequences of the cloned cDNAs. J Biol Chem. 1982 Aug 25;257(16):9724–9732. [PubMed] [Google Scholar]
  20. Mains R. E., Eipper B. A. Biosynthesis of adrenocorticotropic hormone in mouse pituitary tumor cells. J Biol Chem. 1976 Jul 10;251(13):4115–4120. [PubMed] [Google Scholar]
  21. Mains R. E., Eipper B. A. Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells. J Cell Biol. 1981 Apr;89(1):21–28. doi: 10.1083/jcb.89.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martial J. A., Hallewell R. A., Baxter J. D., Goodman H. M. Human growth hormone: complementary DNA cloning and expression in bacteria. Science. 1979 Aug 10;205(4406):602–607. doi: 10.1126/science.377496. [DOI] [PubMed] [Google Scholar]
  23. Moore H. P., Walker M. D., Lee F., Kelly R. B. Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. Cell. 1983 Dec;35(2 Pt 1):531–538. doi: 10.1016/0092-8674(83)90187-3. [DOI] [PubMed] [Google Scholar]
  24. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  25. Rose J. K., Bergmann J. E. Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell. 1982 Oct;30(3):753–762. doi: 10.1016/0092-8674(82)90280-x. [DOI] [PubMed] [Google Scholar]
  26. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  27. Stroud R. M., Kay L. M., Dickerson R. E. The structure of bovine trypsin: electron density maps of the inhibited enzyme at 5 Angstrom and at 2-7 Angstron resolution. J Mol Biol. 1974 Feb 25;83(2):185–208. doi: 10.1016/0022-2836(74)90387-8. [DOI] [PubMed] [Google Scholar]
  28. Tartakoff A., Greene L. J., Palade G. E. Studies on the guinea pig pancreas. Fractionation and partial characterization of exocrine proteins. J Biol Chem. 1974 Dec 10;249(23):7420–7431. [PubMed] [Google Scholar]
  29. Tartakoff A., Vassalli P., Détraz M. Comparative studies of intracellular transport of secretory proteins. J Cell Biol. 1978 Dec;79(3):694–707. doi: 10.1083/jcb.79.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Timpl R., Rohde H., Robey P. G., Rennard S. I., Foidart J. M., Martin G. R. Laminin--a glycoprotein from basement membranes. J Biol Chem. 1979 Oct 10;254(19):9933–9937. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES