Posttranslational processing of transfected mouse pro-adrenocorticotropin/endorphin in rat growth hormone-secreting tumor cells.
Pro-ACTH/endorphin (PAE; also POMC) is a hormone precursor that contains potential sites for endoproteolytic cleavage, glycosylation, phosphorylation, acetylation, sulfation, and amidation. Different sets of these sites are used in different neuroendocrine tissues. To learn more about the factors influencing the posttranslational processing of peptide hormone precursors, the cDNA for mouse PAE was cloned into a metallothionein expression vector. Two lines of rat GH-secreting tumor cells (GH3 and GC) were transfected with the resulting expression plasmid, and stable PAE-producing clones were isolated. Both GH3 and GC cells gave rise to clones in which about 75% of PAE was endoproteolytically processed. Mol wt distributions of PAE peptides were similar in cell extracts and medium, indicating that processing was not the result of extracellular proteases. The identity of immunoprecipitated molecules was confirmed by analysis of tryptic digests by reverse phase HPLC. Amidated forms of joining peptide were detected in the transfected cells by immunocytochemistry, adding evidence that GH-producing tumor cells can perform endoproteolysis, exoproteolysis, and alpha-amidation of the PAE precursor. One of the two forms of joining peptide from cell extracts was also shown to comigrate during reverse phase HPLC with authentic alpha-amidated mouse joining peptide. Comparisons of Northern analyses and peptide synthetic rates suggested that PAE mRNA was used for synthesis of PAE more efficiently in corticotropes than in the transfected GC cells.
Cullen
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mains
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D000818:Animals; D015152:Blotting, Northern; D002850:Chromatography, Gel; D013006:Growth Hormone / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D010455:Peptides / Q000032:analysis; D011333:Pro-Opiomelanocortin / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D014162:Transfection; D014364:Tryptophan; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000502:physiology*
D010455:Peptides; D011333:Pro-Opiomelanocortin; D014364:Tryptophan; D013006:Growth Hormone