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. 1992 Jun 1;89(11):4977–4981. doi: 10.1073/pnas.89.11.4977

Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.

S Ohagi 1, J LaMendola 1, M M LeBeau 1, R Espinosa 3rd 1, J Takeda 1, S P Smeekens 1, S J Chan 1, D F Steiner 1
PMCID: PMC49211  PMID: 1594602

Abstract

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.

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Selected References

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

  1. Andrews P. C., Brayton K., Dixon J. E. Precursors to regulatory peptides: their proteolytic processing. Experientia. 1987 Jul 15;43(7):784–790. doi: 10.1007/BF01945356. [DOI] [PubMed] [Google Scholar]
  2. Barr P. J., Mason O. B., Landsberg K. E., Wong P. A., Kiefer M. C., Brake A. J. cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues. DNA Cell Biol. 1991 Jun;10(5):319–328. doi: 10.1089/dna.1991.10.319. [DOI] [PubMed] [Google Scholar]
  3. Behr J. P., Demeneix B., Loeffler J. P., Perez-Mutul J. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6982–6986. doi: 10.1073/pnas.86.18.6982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benjannet S., Rondeau N., Day R., Chrétien M., Seidah N. G. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3564–3568. doi: 10.1073/pnas.88.9.3564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breathnach R., Benoist C., O'Hare K., Gannon F., Chambon P. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4853–4857. doi: 10.1073/pnas.75.10.4853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Docherty K., Steiner D. F. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol. 1982;44:625–638. doi: 10.1146/annurev.ph.44.030182.003205. [DOI] [PubMed] [Google Scholar]
  7. Efrat S., Linde S., Kofod H., Spector D., Delannoy M., Grant S., Hanahan D., Baekkeskov S. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9037–9041. doi: 10.1073/pnas.85.23.9037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuller R. S., Brake A. J., Thorner J. Intracellular targeting and structural conservation of a prohormone-processing endoprotease. Science. 1989 Oct 27;246(4929):482–486. doi: 10.1126/science.2683070. [DOI] [PubMed] [Google Scholar]
  9. Fuller R. S., Brake A., Thorner J. Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1434–1438. doi: 10.1073/pnas.86.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Funk C. D., Hoshiko S., Matsumoto T., Rdmark O., Samuelsson B. Characterization of the human 5-lipoxygenase gene. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2587–2591. doi: 10.1073/pnas.86.8.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  13. Julius D., Brake A., Blair L., Kunisawa R., Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell. 1984 Jul;37(3):1075–1089. doi: 10.1016/0092-8674(84)90442-2. [DOI] [PubMed] [Google Scholar]
  14. Jung Y. K., Kunczt C. J., Pearson R. K., Dixon J. E., Fricker L. D. Structural characterization of the rat carboxypeptidase-E gene. Mol Endocrinol. 1991 Sep;5(9):1257–1268. doi: 10.1210/mend-5-9-1257. [DOI] [PubMed] [Google Scholar]
  15. Kiefer M. C., Tucker J. E., Joh R., Landsberg K. E., Saltman D., Barr P. J. Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. DNA Cell Biol. 1991 Dec;10(10):757–769. doi: 10.1089/dna.1991.10.757. [DOI] [PubMed] [Google Scholar]
  16. Knowles B. B., Howe C. C., Aden D. P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497–499. doi: 10.1126/science.6248960. [DOI] [PubMed] [Google Scholar]
  17. Mitchell P. J., Wang C., Tjian R. Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell. 1987 Sep 11;50(6):847–861. doi: 10.1016/0092-8674(87)90512-5. [DOI] [PubMed] [Google Scholar]
  18. Mizuno K., Nakamura T., Ohshima T., Tanaka S., Matsuo H. Characterization of KEX2-encoded endopeptidase from yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1989 Feb 28;159(1):305–311. doi: 10.1016/0006-291x(89)92438-8. [DOI] [PubMed] [Google Scholar]
  19. Nelson C., Shen L. P., Meister A., Fodor E., Rutter W. J. Pan: a transcriptional regulator that binds chymotrypsin, insulin, and AP-4 enhancer motifs. Genes Dev. 1990 Jun;4(6):1035–1043. doi: 10.1101/gad.4.6.1035. [DOI] [PubMed] [Google Scholar]
  20. Nishi M., Chan S. J., Nagamatsu S., Bell G. I., Steiner D. F. Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5738–5742. doi: 10.1073/pnas.86.15.5738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roebroek A. J., Schalken J. A., Leunissen J. A., Onnekink C., Bloemers H. P., Van de Ven W. J. Evolutionary conserved close linkage of the c-fes/fps proto-oncogene and genetic sequences encoding a receptor-like protein. EMBO J. 1986 Sep;5(9):2197–2202. doi: 10.1002/j.1460-2075.1986.tb04484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rowley J. D., Diaz M. O., Espinosa R., 3rd, Patel Y. D., van Melle E., Ziemin S., Taillon-Miller P., Lichter P., Evans G. A., Kersey J. H. Mapping chromosome band 11q23 in human acute leukemia with biotinylated probes: identification of 11q23 translocation breakpoints with a yeast artificial chromosome. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9358–9362. doi: 10.1073/pnas.87.23.9358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schalken J. A., Roebroek A. J., Oomen P. P., Wagenaar S. S., Debruyne F. M., Bloemers H. P., Van de Ven W. J. fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas. J Clin Invest. 1987 Dec;80(6):1545–1549. doi: 10.1172/JCI113240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sehgal A., Patil N., Chao M. A constitutive promoter directs expression of the nerve growth factor receptor gene. Mol Cell Biol. 1988 Aug;8(8):3160–3167. doi: 10.1128/mcb.8.8.3160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Seidah N. G., Gaspar L., Mion P., Marcinkiewicz M., Mbikay M., Chrétien M. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Biol. 1990 Jul-Aug;9(6):415–424. doi: 10.1089/dna.1990.9.415. [DOI] [PubMed] [Google Scholar]
  27. Seidah N. G., Marcinkiewicz M., Benjannet S., Gaspar L., Beaubien G., Mattei M. G., Lazure C., Mbikay M., Chrétien M. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol. 1991 Jan;5(1):111–122. doi: 10.1210/mend-5-1-111. [DOI] [PubMed] [Google Scholar]
  28. Seidah N. G., Mattei M. G., Gaspar L., Benjannet S., Mbikay M., Chrétien M. Chromosomal assignments of the genes for neuroendocrine convertase PC1 (NEC1) to human 5q15-21, neuroendocrine convertase PC2 (NEC2) to human 20p11.1-11.2, and furin (mouse 7[D1-E2] region). Genomics. 1991 Sep;11(1):103–107. doi: 10.1016/0888-7543(91)90106-o. [DOI] [PubMed] [Google Scholar]
  29. Smeekens S. P., Avruch A. S., LaMendola J., Chan S. J., Steiner D. F. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):340–344. doi: 10.1073/pnas.88.2.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smeekens S. P., Steiner D. F. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem. 1990 Feb 25;265(6):2997–3000. [PubMed] [Google Scholar]
  31. Thomas L., Leduc R., Thorne B. A., Smeekens S. P., Steiner D. F., Thomas G. Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5297–5301. doi: 10.1073/pnas.88.12.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]

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