Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing.

Open Access
Publisher Website
Google Scholar
Abstract
A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT‐20D16v) that normally expresses pro‐opiomelanocortin but not proenkephalin. The plasmid was introduced by co‐transformation with the G418‐selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT‐20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT‐20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met‐enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro‐opiomelanocortin, the precursor to ACTH, beta‐endorphin and melanocyte stimulating hormones. The release of both ACTH and Met‐enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.