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Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA.
B T Bloomquist1, B A Eipper, R E Mains
1Neuroscience Department, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Molecular Endocrinology (Baltimore, Md.)
|December 1, 1991
Summary
Prohormone convertase 1 (PC1) plays a key role in peptide processing within the pituitary gland. Its expression is regulated by dopaminergic agents and glucocorticoids, impacting pro-opiomelanocortin (POMC) processing.
Area of Science:
- Molecular biology
- Neuroendocrinology
- Protease research
Background:
- Several endoproteases, including furin, PC1, and PC2, are homologous to yeast Kex2, but their roles in peptide posttranslational processing remain unclear.
- Understanding tissue-specific peptide processing requires detailed comparisons between physiological changes and biochemical/molecular studies.
Purpose of the Study:
- To clone rat pituitary cDNAs for PC1 and PC2 to facilitate comparative studies.
- To investigate the expression patterns and regulation of PC1 and PC2 in rat tissues and cell lines.
- To determine the role of PC1 in the posttranslational processing of peptides, particularly POMC.
Main Methods:
- Cloning of rat pituitary cDNAs for PC1 and PC2.
- Analysis of mRNA distribution using Northern blotting across various rat tissues and cell lines.
- Quantitative analysis of mRNA regulation in response to dopaminergic agents, CRH, and glucocorticoids.
- Functional analysis using antisense RNA to specifically decrease PC1 mRNA levels in AtT-20 cells.
- Biosynthetic labeling studies to assess POMC-derived peptide processing.
Main Results:
- Amino acid sequence homology confirms PC1, PC2, and furin as conserved mammalian subtilisin-like proteases.
- PC1 and PC2 mRNA expression is restricted to specific tissues, suggesting roles in tissue-specific processing, unlike the widespread furin mRNA.
- PC1 and PC2 mRNA levels in the neurointermediate pituitary are regulated by dopaminergic agents, paralleling POMC and other processing enzyme mRNAs.
- In AtT-20 cells, PC1 mRNA is co-regulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs by CRH and glucocorticoids.
- Reducing PC1 mRNA levels in AtT-20 cells via antisense RNA significantly blocks the normal processing of newly synthesized POMC-derived peptides.
Conclusions:
- PC1 and PC2 are distinct, highly conserved mammalian subtilisin-like proteases.
- The restricted expression and regulated expression of PC1 and PC2 suggest crucial roles in tissue-specific peptide processing.
- PC1 protein is implicated in endoproteolysis, potentially acting as a processing endoprotease or an activator of such enzymes, essential for proper POMC processing.