Biosynthesis and post-translational processing of site-directed endoproteolytic cleavage mutants of Pro-CCK in AtT-20 cells

Biochemistry. 2002 Jan 15;41(2):570-8. doi: 10.1021/bi015566u.


Site-directed mutagenesis in which individual cleavage site P1 amino acids were changed to Ala was performed to delineate their importance in the processing of pro-CCK in mouse pituitary tumor AtT-20 cells. Individual substitution of cleavage sites on pro-CCK, viz., CCK 58 cleavage site R/A to A/A, CCK 33 cleavage site R/K to A/K, CCK 22 cleavage site K/N to A/N, and CCK 8 cleavage site R/D to A/D, did not inhibit pro-CCK expression or the production of some form of amidated CCK. Wild-type CCK cDNA expression in these cells results in production and secretion of CCK 8 and CCK 22. Substitution of the 58R/A cleavage site with A/A produces only CCK 33; 33A/K and 22A/N produce only CCK 8, whereas 8A/D produces CCK 12 and some CCK 22. Where the GRR residues on the C-terminus of CCK 8 were mutated to GAA, no amidated CCK was produced. Significant amounts of the pro-CCK, C-terminal peptide S9S was found in the medium of cells transfected with GAA mutant cDNA, indicating that this pro-CCK was cleaved at the GAA site probably by a nonprohormone convertase enzyme. Further analysis of the cells expressing the GAA mutant demonstrated that it is not extensively cleaved at other sites to produce CCK 8 GAA or larger peptides. In the mutant where the entire pro-CCK, C-terminal S9S was deleted, CCK 8 is processed and secreted normally. Thus, the cleavage at the C-terminal GRR site is essential for subsequent cleavages, and modification of other cleavage sites (58, 33, 22, and 8) has a major impact on pro-CCK processing. These results suggest that there is a temporal order of cleavages, and the structure of pro-CCK has a strong influence on where and whether pro-CCK is processed.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Carboxypeptidases / pharmacology
  • Cathepsin A
  • Cholecystokinin / chemistry*
  • Cholecystokinin / genetics*
  • Chromatography
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Precursors / chemistry*
  • Protein Precursors / genetics*
  • Protein Processing, Post-Translational*
  • Protein Structure, Tertiary
  • Radioimmunoassay
  • Rats
  • Transfection
  • Tumor Cells, Cultured


  • DNA, Complementary
  • Protein Precursors
  • Cholecystokinin
  • Carboxypeptidases
  • Cathepsin A