Processing of pro-opiomelanocortin in GH3 cells: inhibition by prohormone convertase 2 (PC2) antisense mRNA.
The processing of pro-opiomelanocortin (POMC) was examined in GH3 cells, a rat sommatomammotrope cell line, by transiently-transfecting the cells with mouse POMC cDNA. The peptide products were extracted, chromatographed on HPLC and identified by specific radioimmunoassay. POMC was processed to generate ACTH-related peptides, beta-endorphin and Lys-gamma 3- MSH, with complete disappearance of the POMC precursor. The ACTH-related molecules were identified as ACTH1-14, ACTH1-15, ACTH1-17, as well as ACTH1-39. GH3 cells which were not transfected with POMC cDNA did not contain endogenous POMC-related peptides. RT-PCR demonstrated that GH3 cells contain prohormone convertase 2 (PC2) mRNA but no PC1 mRNA. To determine if PC2 was the enzyme responsible for POMC processing in this cell line, GH3 cells were stably-transfected with PC2 antisense cDNA. A cell line was obtained which showed an absence of PC2 protein compared to control untransfected GH3 cells, indicating successful hybridization of PC2 antisense mRNA to the endogenous PC2 mRNA. When this cell line was then transiently-transfected with POMC cDNA, POMC was not processed. The results from these experiments suggest that PC2 alone can correctly process POMC to biologically active smaller peptides in vivo. Additionally, the GH3 cell line with and without incorporation of PC2 antisense cDNA can be used as a model system to study the role of PC2 in the post-translational processing of other prohormones and proproteins in vivo.
Friedman
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Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Cool
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Jayasvasti
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Louie
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Loh
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011333:Pro-Opiomelanocortin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045706:Proprotein Convertase 2; D011499:Protein Processing, Post-Translational; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D013381:Subtilisins / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D016372:RNA, Antisense; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin; D013381:Subtilisins; D045706:Proprotein Convertase 2