Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Sep;81(17):5340–5344. doi: 10.1073/pnas.81.17.5340

Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.

J G Hellerman, R C Cone, J T Potts Jr, A Rich, R C Mulligan, H M Kronenberg
PMCID: PMC391699  PMID: 6089196

Abstract

In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recombinant retrovirus encoding preproPTH, and this recombinant retrovirus was used to infect GH4 rat pituitary cells. Clonal lines of the infected GH4 cells contained copies of the recombinant provirus stably integrated via the long terminal repeats, and the expected RNA transcripts of proviral DNA accumulated in the cytoplasm, although no infectious virus was produced. The infected cells synthesized and processed preproPTH appropriately and secreted PTH in response to thyrotropin-releasing hormone, a secretagogue for GH4 cells. Use of recombinant retrovirus permits the introduction of DNA encoding normal and mutant secreted proteins into a number of cell types specialized for secretion. Analysis of the fate of the resultant proteins will help define the specific molecular interactions involved in transmembrane transport and processing of precursor proteins.

Full text

PDF
5340

Images in this article

5341Image
on p.5341
5342Image
on p.5342
5342Image
on p.5342
5342Image
on p.5342

Selected References

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

  1. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dannies P. S., Tashjian A. R., Jr Effects of thyrotropin-releasing hormone and hydrocortisone on synthesis and degradation of prolactin in a rat pituitary cell strain. J Biol Chem. 1973 Sep 10;248(17):6174–6179. [PubMed] [Google Scholar]
  3. Doehmer J., Barinaga M., Vale W., Rosenfeld M. G., Verma I. M., Evans R. M. Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2268–2272. doi: 10.1073/pnas.79.7.2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gething M. J., Sambrook J. Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nature. 1982 Dec 16;300(5893):598–603. doi: 10.1038/300598a0. [DOI] [PubMed] [Google Scholar]
  5. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  7. Gruss P., Khoury G. Expression of simian virus 40-rat preproinsulin recombinants in monkey kidney cells: use of preproinsulin RNA processing signals. Proc Natl Acad Sci U S A. 1981 Jan;78(1):133–137. doi: 10.1073/pnas.78.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Habener J. F., Amherdt M., Ravazzola M., Orci L. Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography. J Cell Biol. 1979 Mar;80(3):715–731. doi: 10.1083/jcb.80.3.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hendy G. N., Kronenberg H. M., Potts J. T., Jr, Rich A. Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7365–7369. doi: 10.1073/pnas.78.12.7365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  13. Kieffer J. D., Mirel R. D., Mover H., Erban J., Maloof F. Decreased thyroid function and high plasma prolactin levels in rats of the Buffalo strain. Endocrinology. 1978 May;102(5):1506–1510. doi: 10.1210/endo-102-5-1506. [DOI] [PubMed] [Google Scholar]
  14. Kronenberg H. M., Fennick B. J., Vasicek T. J. Transport and cleavage of bacterial pre-beta-lactamase by mammalian microsomes. J Cell Biol. 1983 Apr;96(4):1117–1119. doi: 10.1083/jcb.96.4.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laub O., Rutter W. J. Expression of the human insulin gene and cDNA in a heterologous mammalian system. J Biol Chem. 1983 May 25;258(10):6043–6050. [PubMed] [Google Scholar]
  16. Lomedico P. T. Use of recombinant DNA technology to program eukaryotic cells to synthesize rat proinsulin: a rapid expression assay for cloned genes. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5798–5802. doi: 10.1073/pnas.79.19.5798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  18. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  20. Milstein C., Brownlee G. G., Harrison T. M., Mathews M. B. A possible precursor of immunoglobulin light chains. Nat New Biol. 1972 Sep 27;239(91):117–120. doi: 10.1038/newbio239117a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  23. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mulligan R. C., Howard B. H., Berg P. Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome. Nature. 1979 Jan 11;277(5692):108–114. doi: 10.1038/277108a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pavlakis G. N., Hizuka N., Gorden P., Seeburg P., Hamer D. H. Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7398–7402. doi: 10.1073/pnas.78.12.7398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Potts J. T., Jr, Kronenberg H. M., Rosenblatt M. Parathyroid hormone: chemistry, biosynthesis, and mode of action. Adv Protein Chem. 1982;35:323–396. doi: 10.1016/s0065-3233(08)60471-4. [DOI] [PubMed] [Google Scholar]
  29. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  30. Robins D. M., Paek I., Seeburg P. H., Axel R. Regulated expression of human growth hormone genes in mouse cells. Cell. 1982 Jun;29(2):623–631. doi: 10.1016/0092-8674(82)90178-7. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Segre G. V., Tregear G. W., Potts J. T., Jr Development and application of sequence-specific radioimmunoassays for analysis of the metabolism of parathyroid hormone. Methods Enzymol. 1975;37:38–66. doi: 10.1016/s0076-6879(75)37005-5. [DOI] [PubMed] [Google Scholar]
  33. Shields A., Witte W. N., Rothenberg E., Baltimore D. High frequency of aberrant expression of Moloney murine leukemia virus in clonal infections. Cell. 1978 Jul;14(3):601–609. doi: 10.1016/0092-8674(78)90245-3. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Sveda M. M., Markoff L. J., Lai C. J. Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences. Cell. 1982 Sep;30(2):649–656. doi: 10.1016/0092-8674(82)90261-6. [DOI] [PubMed] [Google Scholar]
  36. Tashjian A. H., Jr, Lomedico M. E., Maina D. Role of calcium in the thyrotropin-releasing hormone-stimulated release of prolactin from pituitary cells in culture. Biochem Biophys Res Commun. 1978 Apr 14;81(3):798–806. doi: 10.1016/0006-291x(78)91422-5. [DOI] [PubMed] [Google Scholar]
  37. Walter P., Blobel G. Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature. 1982 Oct 21;299(5885):691–698. doi: 10.1038/299691a0. [DOI] [PubMed] [Google Scholar]
  38. Westendorf J. M., Schonbrunn A. Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture. Endocrinology. 1982 Feb;110(2):352–358. doi: 10.1210/endo-110-2-352. [DOI] [PubMed] [Google Scholar]
  39. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES