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In Vitro and in Vivo Analysis of the Processing and Fate of the Peptide Products of the Short Proopiomelanocortin mRNA

Departments of Endocrinology and Chemical Endocrinology, St. Bartholomew's Hospital Medical College London, ECIA 7BE Great Britain

Address requests for reprints to: Dr. Adrian J. L. Clark, Endocrinology and Reproduction Research Branch, Room B1L 400, Building 10, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.

Abstract

Many peripheral tissues express the proopiomelanocortin (POMC) gene as an 800-base mRNA that lacks the 5' end of the 1200-base pituitary transcript. The missing region encodes the peptide signal sequence, and thus, it is unlikely that any translation product would be secreted. We have found that a RNA transcript equivalent to this short message, generated by transcription in vitro from a T7 polymerase promoter, is translatable in a rabbit reticulocyte lysate, generating peptides of 27.5, 22.5, and 15.5 kD. None of these peptides appears to be processed or protected from proteinase-K digestion by a microsomal membrane fraction. In vivo studies were undertaken by transfecting into GH₃ cells one of two expression vectors containing sequences that would produce either a full-length mRNA or a short (800-base) mRNA. The neomycin resistence gene was cotransfected with these plasmids, and 30 permanent cell lines were produced after selection in G418. Cell lines containing the full-length RNA secreted large quantities of ACTH and β-endorphin immunoreactivity, whereas those expressing the short transcript secreted neither of these peptides. However extractable peptide was present in this latter type of cell line, thereby suggesting that the 800-base mRNA was translated, and that no peptide reached the secretory vesicle. These findings raise important questions about the role of peripheral POMC gene expression.

FOOTNOTES

This work was supported in part by the Medical Research Council and the Joint Research Board of St. Bartholomew's Hospital.

Received for publication June 8, 1990. Revision received August 17, 1990. Accepted for publication August 21, 1990.

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