Expression of three proopiomelanocortin subtype genes and mass spectrometric identification of POMC-derived peptides in pars distalis and pars intermedia of barfin flounder pituitary

Abstract

Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-α-MSH-A/B (amino acid sequence of α-MSH-A is identical to that of α-MSH-B), β-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide ( N-POMC)-A were identified in the PD. Moreover, Des-Ac-α-MSH-A/B, α-MSH-A/B, β-MSH-A and -B, N-β-lipotropin-A, CLIP-A, N-Ac-β-END-A 1–41 (C-terminally truncated form of N-Ac-β-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-α-MSH-A, CLIP-A, and β-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of α-MSH-A/B and N-Ac-β-END-A 1–41. These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.