Biosynthesis and post-translational processing of site-directed endoproteolytic cleavage mutants of Pro-CCK in AtT-20 cells.
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.
Vishnuvardhan
Daesety
D
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.
Beinfeld
Margery C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000494:pharmacology; D043402:Cathepsin A; D002766:Cholecystokinin / Q000737:chemistry* / Q000235:genetics*; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011498:Protein Precursors; D002766:Cholecystokinin; D002268:Carboxypeptidases; D043402:Cathepsin A