Proteolytic Processing of Pro-opiomelanocortin Occurs in Acidifying Secretory Granules of AtT-20 Cells

Abstract

Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as the pH probe suggested a correlation between POMC processing and acidic pH in the secretory granules. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely inhibited POMC processing and caused constitutive secretion of the unprocessed precursor. By contrast, chloroquine, a weak base that is known to neutralize acidic organelles, was unable to inhibit POMC processing. Electron microscopic analysis revealed that, in AtT-20 cells treated with bafilomycin A1, the trans-Golgi cisternae were dilated and few secretory granules were present in the cytoplasm. These observations suggest that acidic pH provides a favorable environment for proteolytic processing of POMC by PC1 but is not required, and that integrity of the trans-Golgi network and sorting of POMC into secretory granules are important for POMC processing.

Cloning and Expression of Vacuolar Proton-Pumping ATPase Subunits in the Follicular Epithelium of the Bullfrog Endolymphatic Sac

“…The deparaffinized sections were rinsed with distilled water and PBS. For single labeling of the A-and E-subunit proteins, immunofluorescence staining was performed essentially as described by Tanaka et al (1997). The sections were sequentially incubated with 1% bovine serum albumin-PBS, rabbit anti-A-subunit or E-subunit serum (1:4000), and indocarbocyanine (Cy3)-labeled donkey anti-rabbit IgG (Jackson).…”

“…Ultrathin sections were labeled with the double-immunogold labeling method (Tanaka et al, 1997). Briefly, two faces of the grids were incubated with different antibodies (rabbit anti-V-ATPase E-subunit, 1:4000; guinea pig anti-otoconin-22, 1:4000) and then with goat anti-rabbit Ig or goat anti-guinea pig IgG conjugated with different sizes of gold particles (10 and 5 nm, respectively) (BioCell; Cardiff, UK).…”

Generation of expression constructs that secrete bioactive αMSH and their use in the treatment of experimental autoimmune encephalomyelitis

“…After the initial cleavage of POMC that creates ACTH, the first 17 amino acids of the ACTH peptide are then liberated by pro-hormone convertase 2 (PC2) to form the backbone of what will become native aMSH ( Figure 1). 2,[4][5][6] The final steps in the production of native aMSH involve cleavage of ACTH1-17 to ACTH1-13, followed by amidation of the carboxyl terminus and acetylation of the amino terminus. The amidation modification requires a signal, located at amino acids 14-16 of ACTH (Gly, Lys, Lys; Figure 1).…”

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

“…1). Amidated POMC products are first detected in the TGN (22); pulsechase studies indicate that amidation of both 18-kDa fragment and JP is completed within 1 h of synthesis (37). An antibody specific for 18-kDa fragment-NH 2 and JP-NH 2 was generated by immunizing rabbits with synthetic D-Tyr-Pro-Glu-Pro-SerPro-Arg-Glu-NH 2 ; based on an ELISA, the affinity-purified NH 2 -specific antibody cross-reacted less than 10,000 times as well with synthetic Pro-Glu-Pro-Ser-Pro-Arg-Glu-Gly (23).…”