The Secretory Granule Protein 7B2 is Secreted in Parallel with Proopiomelanocortin and its End‐Products by the Mouse Corticotroph Tumour Cells AtT‐20

Abstract

First isolated in porcine pituitary glands the protein 7B2 subsequently proved to be a specific biochemical marker of the secretory granules. Likewise 7B2 was detected in almost all normal and tumoral endocrine tissues. Unexpectedly, several authors failed to demonstrate its presence in rat and human adrenocorticotrophin (ACTH)-secreting cells. In order to definitely establish whether this cell type also produces 7B2 we chose the mouse pituitary corticotroph tumour cell line AtT-20 as a model. Serial dilutions of the mouse culture medium generated displacement curves parallel to that of the standard in a specific 7B2 RIA directed against the human 7B2(23-39) fragment. Under basal secretory conditions immunoreactive 7B2 accumulated in the culture medium in parallel with proopiomelanocortin (POMC) and its fragments N-terminal-joining peptide (NT-JP), joining peptide (JP), beta-lipotropin (beta-LPH), and beta-endorphin (beta-end), although at a much lower (approximately 100-fold) molar concentration. As expected mouse corticotroph cells responded to the stimulatory action of cyclic AMP (3.5 mM) with a preferential increase in the release of POMC end-products, JP and beta-end, which was accompanied by a parallel increase in immunoreactive 7B2 release.

Maintained PC1 and PC2 Expression in the AtT-20 Variant Cell Line 6T3 Lacking Regulated Secretion and POMC: Restored POMC Expression and Regulated Secretion after cAMP Treatment

“…It was interesting to note that 7B2 mRNA levels were only partially reduced (Fig. 4), even though like POMC and CgB, it is normally targeted to the regulated pathway of secretion (Marcinkiewicz et al, 1987;Vieau et al, 1991). Finally, we note that the CgB expression is almost two-fold higher in 10T2 cells as compared to AtT-20 cells.…”

“…AtT-20 cells have a regulated pathway of secretion and dense-core secretory granules (Mains and Eipper, 1976). They also express various proteins that are known to be targeted to a regulated pathway of secretion, such as proopiomelanocortin (POMC) (Burgess and Kelly, 1987), chromogranin B (CgB) (Weidenmann and Huttner, 1989), and 7B2 (Vieau et al, 1991;Seidah et al, 1994 Kelly, 1991). These cells are deficient (10T2) or completely lack (6T3) adrenocorticotropic hormone (ACTH) immunoreactivity, sulfated proteins characteristic of the regulated pathway, and dense-core secretory granules, but retain constitutive secretion.…”

The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

“…Biosynthesis ofthe %21-kDa 7B2 protein occurs through carboxyl-terminal processing of a --27-kDa precursor protein (10,11) and only the cleaved form of 7B2 is released (10). Secretion of the 7B2 cleavage product could be regulated (10,12,13), establishing that, like PC1/PC3 and PC2, the polypeptide 7B2 is in the regulated secretory pathway. The 7B2 protein is highly conserved and widely distributed in the central nervous system and endocrine tissues (14)(15)(16) and was generated by PCR using specific primers; the 5' primer corresponded to nucleotides 107-129 with a BamHI site introduced at the 5' end and the 3' primer consisted of nucleotides 538-562 with an introduced 5' stop codon and 5' HindIII site.…”