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. 1992 May 2;117(4):717–728. doi: 10.1083/jcb.117.4.717

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules

PMCID: PMC2289459  PMID: 1577852

Abstract

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.

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Selected References

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  1. Arvan P., Kuliawat R., Prabakaran D., Zavacki A. M., Elahi D., Wang S., Pilkey D. Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. J Biol Chem. 1991 Aug 5;266(22):14171–14174. [PubMed] [Google Scholar]
  2. Barr P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell. 1991 Jul 12;66(1):1–3. doi: 10.1016/0092-8674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  3. Beaudry G. A., Bertelsen A. H. Secreted alpha amidating enzymes are generated by specific posttranslational processing of precursors containing transmembrane domains. Biochem Biophys Res Commun. 1989 Sep 15;163(2):959–966. doi: 10.1016/0006-291x(89)92315-2. [DOI] [PubMed] [Google Scholar]
  4. Brodsky F. M. Living with clathrin: its role in intracellular membrane traffic. Science. 1988 Dec 9;242(4884):1396–1402. doi: 10.1126/science.2904698. [DOI] [PubMed] [Google Scholar]
  5. Burgess T. L., Craik C. S., Matsuuchi L., Kelly R. B. In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules. J Cell Biol. 1987 Aug;105(2):659–668. doi: 10.1083/jcb.105.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chevrier D., Fournier H., Nault C., Zollinger M., Crine P., Boileau G. Expression of porcine pro-opiomelanocortin in mouse neuroblastoma (Neuro2A) cells: targeting of the foreign neuropeptide to dense-core vesicles. Mol Cell Endocrinol. 1991 Aug;79(1-3):109–118. doi: 10.1016/0303-7207(91)90101-w. [DOI] [PubMed] [Google Scholar]
  7. Chu W. N., Baxter J. D., Reudelhuber T. L. A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin. Mol Endocrinol. 1990 Dec;4(12):1905–1913. doi: 10.1210/mend-4-12-1905. [DOI] [PubMed] [Google Scholar]
  8. Devi L., Gupta P., Douglass J. Expression and posttranslational processing of preprodynorphin complementary DNA in the mouse anterior pituitary cell line AtT-20. Mol Endocrinol. 1989 Nov;3(11):1852–1860. doi: 10.1210/mend-3-11-1852. [DOI] [PubMed] [Google Scholar]
  9. Dickerson I. M., Peden K. W., Mains R. E. Metallothionein-I promoter-directed expression of foreign proteins in a mouse pituitary corticotrope tumor cell line. Mol Cell Endocrinol. 1989 Jul;64(2):205–212. doi: 10.1016/0303-7207(89)90147-0. [DOI] [PubMed] [Google Scholar]
  10. Eipper B. A., Green C. B., Campbell T. A., Stoffers D. A., Keutmann H. T., Mains R. E., Ouafik L. Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM). J Biol Chem. 1992 Feb 25;267(6):4008–4015. [PubMed] [Google Scholar]
  11. Eipper B. A., Mains R. E., Glembotski C. C. Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc Natl Acad Sci U S A. 1983 Aug;80(16):5144–5148. doi: 10.1073/pnas.80.16.5144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eipper B. A., Park L. P., Dickerson I. M., Keutmann H. T., Thiele E. A., Rodriguez H., Schofield P. R., Mains R. E. Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis. Mol Endocrinol. 1987 Nov;1(11):777–790. doi: 10.1210/mend-1-11-777. [DOI] [PubMed] [Google Scholar]
  13. Eipper B. A., Perkins S. N., Husten E. J., Johnson R. C., Keutmann H. T., Mains R. E. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression. J Biol Chem. 1991 Apr 25;266(12):7827–7833. [PubMed] [Google Scholar]
  14. Eipper B. A., Stoffers D. A., Mains R. E. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu Rev Neurosci. 1992;15:57–85. doi: 10.1146/annurev.ne.15.030192.000421. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Guest P. C., Ravazzola M., Davidson H. W., Orci L., Hutton J. C. Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans. Endocrinology. 1991 Aug;129(2):734–740. doi: 10.1210/endo-129-2-734. [DOI] [PubMed] [Google Scholar]
  17. Hosaka M., Nagahama M., Kim W. S., Watanabe T., Hatsuzawa K., Ikemizu J., Murakami K., Nakayama K. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J Biol Chem. 1991 Jul 5;266(19):12127–12130. [PubMed] [Google Scholar]
  18. Husten E. J., Eipper B. A. The membrane-bound bifunctional peptidylglycine alpha-amidating monooxygenase protein. Exploration of its domain structure through limited proteolysis. J Biol Chem. 1991 Sep 15;266(26):17004–17010. [PubMed] [Google Scholar]
  19. Huttner W. B., Tooze S. A. Biosynthetic protein transport in the secretory pathway. Curr Opin Cell Biol. 1989 Aug;1(4):648–654. doi: 10.1016/0955-0674(89)90029-x. [DOI] [PubMed] [Google Scholar]
  20. Hutton J. C. Subtilisin-like proteinases involved in the activation of proproteins of the eukaryotic secretory pathway. Curr Opin Cell Biol. 1990 Dec;2(6):1131–1142. doi: 10.1016/0955-0674(90)90167-d. [DOI] [PubMed] [Google Scholar]
  21. Katopodis A. G., Ping D., May S. W. A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation. Biochemistry. 1990 Jul 3;29(26):6115–6120. doi: 10.1021/bi00478a001. [DOI] [PubMed] [Google Scholar]
  22. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  23. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lapps W., Eng J., Stern A. S., Gubler U. Expression of porcine cholecystokinin cDNA in a murine neuroendocrine cell line. Proteolytic processing, sulfation, and regulated secretion of cholecystokinin peptides. J Biol Chem. 1988 Sep 15;263(26):13456–13462. [PubMed] [Google Scholar]
  25. Lindberg I. The new eukaryotic precursor processing proteinases. Mol Endocrinol. 1991 Oct;5(10):1361–1365. doi: 10.1210/mend-5-10-1361. [DOI] [PubMed] [Google Scholar]
  26. MacDonald M. R., Takeda J., Rice C. M., Krause J. E. Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, alpha-, beta-, and gamma-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants. J Biol Chem. 1989 Sep 15;264(26):15578–15592. [PubMed] [Google Scholar]
  27. 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]
  28. Mains R. E., Glembotski C. C., Eipper B. A. Peptide alpha-amidation activity in mouse anterior pituitary AtT-20 cell granules: properties and secretion. Endocrinology. 1984 May;114(5):1522–1530. doi: 10.1210/endo-114-5-1522. [DOI] [PubMed] [Google Scholar]
  29. Mains R. E., May V. The role of a low pH intracellular compartment in the processing, storage, and secretion of ACTH and endorphin. J Biol Chem. 1988 Jun 5;263(16):7887–7894. [PubMed] [Google Scholar]
  30. Matsuuchi L., Buckley K. M., Lowe A. W., Kelly R. B. Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells. J Cell Biol. 1988 Feb;106(2):239–251. doi: 10.1083/jcb.106.2.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. May V., Eipper B. A. Long term culture of primary rat pituitary adrenocorticotropin/endorphin-producing cells in serum-free medium. Endocrinology. 1986 Apr;118(4):1284–1295. doi: 10.1210/endo-118-4-1284. [DOI] [PubMed] [Google Scholar]
  32. Meiniel A., Molat J. L., Meiniel R. Complex-type glycoproteins synthesized in the subcommissural organ of mammals. Light- and electron-microscopic investigations by use of lectins. Cell Tissue Res. 1988 Aug;253(2):383–395. doi: 10.1007/BF00222295. [DOI] [PubMed] [Google Scholar]
  33. Miller S. G., Moore H. P. Regulated secretion. Curr Opin Cell Biol. 1990 Aug;2(4):642–647. doi: 10.1016/0955-0674(90)90105-n. [DOI] [PubMed] [Google Scholar]
  34. Moore H. H., Kelly R. B. Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature. 1986 May 22;321(6068):443–446. doi: 10.1038/321443a0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
  37. Nagahama M., Nakayama K., Murakami K. Effects of propeptide deletion on human renin secretion from mouse pituitary AtT-20 cells. FEBS Lett. 1990 May 7;264(1):67–70. doi: 10.1016/0014-5793(90)80766-c. [DOI] [PubMed] [Google Scholar]
  38. Noel G., Keutmann H. T., Mains R. E. Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin. Mol Endocrinol. 1991 Mar;5(3):404–413. doi: 10.1210/mend-5-3-404. [DOI] [PubMed] [Google Scholar]
  39. Noel G., Mains R. E. The ordered secretion of bioactive peptides: oldest or newest first? Mol Endocrinol. 1991 Jun;5(6):787–794. doi: 10.1210/mend-5-6-787. [DOI] [PubMed] [Google Scholar]
  40. Payne G. S., Schekman R. Clathrin: a role in the intracellular retention of a Golgi membrane protein. Science. 1989 Sep 22;245(4924):1358–1365. doi: 10.1126/science.2675311. [DOI] [PubMed] [Google Scholar]
  41. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  42. Pelham H. R. Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. EMBO J. 1988 Apr;7(4):913–918. doi: 10.1002/j.1460-2075.1988.tb02896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Perkins S. N., Eipper B. A., Mains R. E. Stable expression of full-length and truncated bovine peptidylglycine alpha-amidating monooxygenase complementary DNAs in cultured cells. Mol Endocrinol. 1990 Jan;4(1):132–139. doi: 10.1210/mend-4-1-132. [DOI] [PubMed] [Google Scholar]
  44. Perkins S. N., Husten E. J., Eipper B. A. The 108-kDA peptidylglycine alpha-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation. Biochem Biophys Res Commun. 1990 Sep 28;171(3):926–932. doi: 10.1016/0006-291x(90)90772-f. [DOI] [PubMed] [Google Scholar]
  45. Reaves B. J., Dannies P. S. Is a sorting signal necessary to package proteins into secretory granules? Mol Cell Endocrinol. 1991 Aug;79(1-3):C141–C145. doi: 10.1016/0303-7207(91)90085-7. [DOI] [PubMed] [Google Scholar]
  46. Rivas R. J., Moore H. P. Spatial segregation of the regulated and constitutive secretory pathways. J Cell Biol. 1989 Jul;109(1):51–60. doi: 10.1083/jcb.109.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schnabel E., Mains R. E., Farquhar M. G. Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells. Mol Endocrinol. 1989 Aug;3(8):1223–1235. doi: 10.1210/mend-3-8-1223. [DOI] [PubMed] [Google Scholar]
  48. Sevarino K. A., Stork P. Multiple preprosomatostatin sorting signals mediate secretion via discrete cAMP- and tetradecanoylphorbolacetate-responsive pathways. J Biol Chem. 1991 Oct 5;266(28):18507–18513. [PubMed] [Google Scholar]
  49. Sevarino K. A., Stork P., Ventimiglia R., Mandel G., Goodman R. H. Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity. Cell. 1989 Apr 7;57(1):11–19. doi: 10.1016/0092-8674(89)90167-0. [DOI] [PubMed] [Google Scholar]
  50. Sossin W. S., Fisher J. M., Scheller R. H. Sorting within the regulated secretory pathway occurs in the trans-Golgi network. J Cell Biol. 1990 Jan;110(1):1–12. doi: 10.1083/jcb.110.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stoffers D. A., Green C. B., Eipper B. A. Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium. Proc Natl Acad Sci U S A. 1989 Jan;86(2):735–739. doi: 10.1073/pnas.86.2.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stoffers D. A., Ouafik L., Eipper B. A. Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation. J Biol Chem. 1991 Jan 25;266(3):1701–1707. [PubMed] [Google Scholar]
  53. Stoller T. J., Shields D. The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells. J Cell Biol. 1989 May;108(5):1647–1655. doi: 10.1083/jcb.108.5.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Takahashi K., Okamoto H., Seino H., Noguchi M. Peptidylglycine alpha-amidating reaction: evidence for a two-step mechanism involving a stable intermediate at neutral pH. Biochem Biophys Res Commun. 1990 Jun 15;169(2):524–530. doi: 10.1016/0006-291x(90)90362-q. [DOI] [PubMed] [Google Scholar]
  55. Thiele E. A., Eipper B. A. Effect of secretagogues on components of the secretory system in AtT-20 cells. Endocrinology. 1990 Feb;126(2):809–817. doi: 10.1210/endo-126-2-809. [DOI] [PubMed] [Google Scholar]
  56. Tooze J., Kern H. F., Fuller S. D., Howell K. E. Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells. J Cell Biol. 1989 Jul;109(1):35–50. doi: 10.1083/jcb.109.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tooze J., Tooze S. A. Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells. J Cell Biol. 1986 Sep;103(3):839–850. doi: 10.1083/jcb.103.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Virca G. D., Northemann W., Shiels B. R., Widera G., Broome S. Simplified northern blot hybridization using 5% sodium dodecyl sulfate. Biotechniques. 1990 Apr;8(4):370–371. [PubMed] [Google Scholar]

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