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PMID: 1577852 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

The Journal of cell biology ·Vol. 117 ·No. 4 ·1992-05-00 ·Pages 717-28

Milgram SL, Johnson RC, Mains RE

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.

MeSH Terms
Base Sequence Cells, Cultured Cytoplasmic Granules/metabolism Gene Expression In Vitro Techniques Membrane Proteins/metabolism Mixed Function Oxygenases/metabolism Molecular Sequence Data Multienzyme Complexes Protein Processing, Post-Translational RNA, Messenger/genetics Recombinant Proteins Regulatory Sequences, Nucleic Acid Solubility Transfection
Chemicals
Membrane Proteins Multienzyme Complexes RNA, Messenger Recombinant Proteins Mixed Function Oxygenases peptidylglycine monooxygenase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Milgram S L
Neuroscience Department, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Johnson R C
Mains R E
References (58)
58 references, click to expand
  1. Effect of secretagogues on components of the secretory system in AtT-20 cells.
    Endocrinology. 1990 Feb;126(2):809-17 PMID: 1967578
  2. Peptide alpha-amidation activity in mouse anterior pituitary AtT-20 cell granules: properties and secretion.
    Endocrinology. 1984 May;114(5):1522-30 PMID: 6714154
  3. Effects of propeptide deletion on human renin secretion from mouse pituitary AtT-20 cells.
    FEBS Lett. 1990 May 7;264(1):67-70 PMID: 2186927
  4. 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):524-30 PMID: 2357219
  5. Stable expression of full-length and truncated bovine peptidylglycine alpha-amidating monooxygenase complementary DNAs in cultured cells.
    Mol Endocrinol. 1990 Jan;4(1):132-9 PMID: 2325663
  6. Sorting within the regulated secretory pathway occurs in the trans-Golgi network.
    J Cell Biol. 1990 Jan;110(1):1-12 PMID: 2295680
  7. 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):404-13 PMID: 1653894
  8. The ordered secretion of bioactive peptides: oldest or newest first?
    Mol Endocrinol. 1991 Jun;5(6):787-94 PMID: 1656241
  9. An analysis of vertebrate mRNA sequences: intimations of translational control.
    J Cell Biol. 1991 Nov;115(4):887-903 PMID: 1955461
  10. 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):109-18 PMID: 1936537
  11. Is a sorting signal necessary to package proteins into secretory granules?
    Mol Cell Endocrinol. 1991 Aug;79(1-3):C141-5 PMID: 1936531
  12. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.
    J Biol Chem. 1991 Apr 25;266(12):7827-33 PMID: 1902227
  13. Protein discharge from immature secretory granules displays both regulated and constitutive characteristics.
    J Biol Chem. 1991 Aug 5;266(22):14171-4 PMID: 1860833
  14. Mammalian subtilisins: the long-sought dibasic processing endoproteases.
    Cell. 1991 Jul 12;66(1):1-3 PMID: 2070411
  15. Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation.
    J Biol Chem. 1991 Jan 25;266(3):1701-7 PMID: 1988445
  16. Regulated secretion.
    Curr Opin Cell Biol. 1990 Aug;2(4):642-7 PMID: 2252589
  17. 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):926-32 PMID: 2222453
  18. 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):6115-20 PMID: 2207061
  19. 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):383-95 PMID: 3409290
  20. The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.
    J Cell Biol. 1989 May;108(5):1647-55 PMID: 2565905
  21. Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment.
    EMBO J. 1988 Apr;7(4):913-8 PMID: 3402439
  22. A C-terminal signal prevents secretion of luminal ER proteins.
    Cell. 1987 Mar 13;48(5):899-907 PMID: 3545499
  23. Intracellular targeting and structural conservation of a prohormone-processing endoprotease.
    Science. 1989 Oct 27;246(4929):482-6 PMID: 2683070
  24. Biosynthetic protein transport in the secretory pathway.
    Curr Opin Cell Biol. 1989 Aug;1(4):648-54 PMID: 2516740
  25. Metallothionein-I promoter-directed expression of foreign proteins in a mouse pituitary corticotrope tumor cell line.
    Mol Cell Endocrinol. 1989 Jul;64(2):205-12 PMID: 2792562
  26. 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):15578-92 PMID: 2768279
  27. Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells.
    J Cell Biol. 1989 Jul;109(1):35-50 PMID: 2745555
  28. Long term culture of primary rat pituitary adrenocorticotropin/endorphin-producing cells in serum-free medium.
    Endocrinology. 1986 Apr;118(4):1284-95 PMID: 3004908
  29. Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells.
    J Cell Biol. 1988 Feb;106(2):239-51 PMID: 2828380
  30. 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):7887-94 PMID: 2836400
  31. Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis.
    Mol Endocrinol. 1987 Nov;1(11):777-90 PMID: 3153462
  32. Multiple preprosomatostatin sorting signals mediate secretion via discrete cAMP- and tetradecanoylphorbolacetate-responsive pathways.
    J Biol Chem. 1991 Oct 5;266(28):18507-13 PMID: 1680862
  33. Clathrin, adaptors, and sorting.
    Annu Rev Cell Biol. 1990;6:151-71 PMID: 2177341
  34. A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin.
    Mol Endocrinol. 1990 Dec;4(12):1905-13 PMID: 2082189
  35. The new eukaryotic precursor processing proteinases.
    Mol Endocrinol. 1991 Oct;5(10):1361-5 PMID: 1775127
  36. 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):17004-10 PMID: 1894599
  37. Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans.
    Endocrinology. 1991 Aug;129(2):734-40 PMID: 1855471
  38. Subtilisin-like proteinases involved in the activation of proproteins of the eukaryotic secretory pathway.
    Curr Opin Cell Biol. 1990 Dec;2(6):1131-42 PMID: 2099807
  39. 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):12127-30 PMID: 1905715
  40. The biosynthesis of neuropeptides: peptide alpha-amidation.
    Annu Rev Neurosci. 1992;15:57-85 PMID: 1575450
  41. Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM).
    J Biol Chem. 1992 Feb 25;267(6):4008-15 PMID: 1740449
  42. In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.
    J Cell Biol. 1987 Aug;105(2):659-68 PMID: 3040770
  43. 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):13456-62 PMID: 2843521
  44. Re-routing of a secretory protein by fusion with human growth hormone sequences.
    Nature. 1986 May 22-28;321(6068):443-6 PMID: 3012361
  45. 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):839-50 PMID: 3017997
  46. Expression and posttranslational processing of preprodynorphin complementary DNA in the mouse anterior pituitary cell line AtT-20.
    Mol Endocrinol. 1989 Nov;3(11):1852-60 PMID: 2575215
  47. Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity.
    Cell. 1989 Apr 7;57(1):11-9 PMID: 2564811
  48. The biogenesis of lysosomes.
    Annu Rev Cell Biol. 1989;5:483-525 PMID: 2557062
  49. Spatial segregation of the regulated and constitutive secretory pathways.
    J Cell Biol. 1989 Jul;109(1):51-60 PMID: 2545730
  50. Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells.
    Mol Endocrinol. 1989 Aug;3(8):1223-35 PMID: 2550814
  51. Clathrin: a role in the intracellular retention of a Golgi membrane protein.
    Science. 1989 Sep 22;245(4924):1358-65 PMID: 2675311
  52. Secreted alpha amidating enzymes are generated by specific posttranslational processing of precursors containing transmembrane domains.
    Biochem Biophys Res Commun. 1989 Sep 15;163(2):959-66 PMID: 2783131
  53. 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):735-9 PMID: 2911604
  54. Living with clathrin: its role in intracellular membrane traffic.
    Science. 1988 Dec 9;242(4884):1396-402 PMID: 2904698
  55. 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):531-8 PMID: 6317196
  56. Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells.
    J Cell Biol. 1981 Apr;89(1):21-8 PMID: 6262331
  57. 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):5144-8 PMID: 6576381
  58. Simplified northern blot hybridization using 5% sodium dodecyl sulfate.
    Biotechniques. 1990 Apr;8(4):370-1 PMID: 1692715
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.117.4.717
Published
1992-05-00
Pages
717-28
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2289459
Subset
IM
Grants
NIDA NIH HHS · DA-00097 · United States
NIDDK NIH HHS · DK-32948 · United States
External Links
PubMed source DOI: 10.1083/jcb.117.4.717 PMC Full Text View journal details
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