Proopiomelanocortin (POMC) is a precursor protein of multiple peptide hormones such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), endorphin (END), etc. The major tissue that biosynthesizes POMC is the pituitary gland, where POMC is produced in the corticotrophs of the pars distalis (PD) and in the melanotrophs of the pars intermedia (PI). In these cells, POMC is differentially cleaved through tissue-specific proteolysis to generate functional peptides. In corticotrophs, relatively larger peptides such as ACTH are the final products, whereas in melanotrophs, relatively smaller peptides such as α-MSH are generated. In addition to cleavage, POMC and POMC-derived peptides undergo several posttranslational modifications such as acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation. Therefore, the POMC system is undoubtedly multifunctional, i.e., in addition to the generation of several peptides, various modifications could diversify the biological functions of POMC-derived peptides.
Adrenocorticotropic hormone and MSH are collectively called melanocortin (MC). Their receptor is called the MC receptor (MCR), for which five subtypes (MC1R to MC5R) have thus far been identified. The receptors for END are opioid receptors. Both receptors are G protein-coupled receptors (GPCRs) with seven transmembrane domains. MCRs are widely distributed throughout animal bodies indicating that POMC-derived peptides have a variety of biological functions. Moreover, posttranslational modification sometimes alters either the binding affinity between the hormonal peptides and their receptors, or the downstream intracellular signal transduction. It is thought that the complex POMC network is made up of a variety of peptides with additional modifications and receptor subtypes distributed in many different tissues and organs. Therefore, POMC could be a useful model for investigating posttranslational modifications in endocrine systems.
Posttranslational processing of POMC in mammals is well understood. Mammalian POMC is composed of three major segments, N-POMC, ACTH, and β-LPH. These segments are divided by the dibasic amino acid residues Arg and Lys, which act as cleavage signals, and contain one MSH sequence whose common sequence is His-Phe-Arg-Trp. The END sequence is always located at the C-terminal end of the β-LPH segment. Therefore, mammalian POMC is described as the 3MSH/1END type. We have investigated the molecular cloning of POMC in non-mammalian species such as birds, reptiles, and fish, including teleosts, cartilaginous fish, lobe-finned fish, and agnathans. Based on the results, we showed the variation in the molecular organization of POMC; the POMC structures are not always the 3MSH/1END type. Moreover, we found that different POMCs are generated in the PD and PI of the most primitive vertebrates, the lampreys, whereas identical POMCs are generated in these lobes in other vertebrates. In addition, we also identified POMC-derived peptides from the pituitary glands in non-mammalian vertebrates.
Herein, we compare the posttranslational modifications of POMC in the corticotrophs and melanotrophs in vertebrates such as birds, reptiles, and fish, which are largely based on the results of our investigations. The results for mammalian and amphibian POMCs will also be summarized. Moreover, we also propose a heteromer hypothesis that would explain the interesting activities of α-MSH in relation to its posttranslational modifications, namely the presence or absence of acetyl groups at the N-terminus.
Mammalian POMC is the 3MSH/1END type. Posttranslational processing of POMC in the cells of AtT 20/D16v (mouse pituitary epithelial-like tumor cell line) in addition to the PD and PI cells of the pituitary gland has been extensively investigated in mammals such as rodents, including rat and mouse, artiodactyls, including ox and sheep, and humans by peptide isolation/purification and amino acid sequence analysis or by biosynthetic labeling, immunoprecipitation, and sequence analysis. The results showed that the products from POMC in the PD and PI vary depending on the presence of prohormone convertase 1 and 2 (PC1 and PC2). In the PD, where PC1 is present, pro-γ-MSH, joining peptide (JP), ACTH, and β-LPH are generated; however, in the PI, where PC1 and PC2 are present, pro-γ-MSH is further cleaved to adrenal mitogenic hormone (AMH) and γ-MSH, ACTH is cleaved to generate α-MSH and corticotropin-like intermediate lobe peptide (CLIP), and β-LPH is cleaved to generate N-β-LPH, β-MSH, and β-END. α-MSH is produced by way of an intermediate (ACTH 1–17), and then mature α-MSH is generated after further processing, including removal of C-terminal residues by carboxypeptidase E, formation of a C-terminal amide by peptidyl α-amidating mono-oxygenase, and N-acetylation by POMC N-acetyltransferase. Some amount of β-END also undergoes N-terminal acetylation. As adult human pituitary glands lack PI and are only composed of anterior lobes containing the PD and pars tuberalis, POMC is predominantly processed into pro-γ-MSH, JP, ACTH, and β-LPH.
Similar to human pituitary glands, adult avian pituitary glands are composed of only the PD. The ostrich (Struthio camelus) is a non-flying bird from which several POMC-derived peptides have been isolated, including ACTH, β-LPH, β-END, γ-LPH, and pro-γ-MSH, which lacks the C-terminal segment of γ 3-MSH, and thus is shorter than the pro-γ-MSH as shown in Figure 1. The occurrence of ACTH, γ-LPH, and β-END was further confirmed in a single frozen ostrich pituitary slice through matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS). Based on the results of peptide identification, we cloned the POMC cDNA from ostrich pituitary and determined its sequence. Sequence comparison of these isolated peptides with the POMC cDNA sequence suggests that all the dibasic sequences are cleaved to produce the peptides. Therefore, the major products in ostrich corticotrophs are pro-γ-MSH, ACTH, and β-LPH, although a substantial amount of β-LPH is further cleaved into γ-LPH and β-END. The generation of a substantial amount of β-END in the ostrich pituitary gland is different from what was observed in the human pituitary, in which β-LPH is a predominant form.
Similar to other tetrapods, snake and alligator POMCs contain α-MSH, β-MSH, γ-MSH, and β-END. These data together with those for gecko and turtle POMC indicate that reptile POMCs are consistently the 3MSH/1END type. Interestingly, the γ-MSH segment in snake POMC has a mutation in the essential His-Phe-Arg-Trp sequence, and the Phe and Arg residues are deleted (Figure 2). It is conceivable that an ancestor of snake γ-MSH had weak functional constraints and lacked biological significance during evolution. In contrast, analyses of whole snake and alligator pituitary glands by MALDI-TOF MS revealed several peptides, such as desacetyl (Des-Ac)-α-MSH, β-MSH, β-END, etc., are generated by posttranslational processing as predicted by the locations of the dibasic sequence processing sites. These results revealed interesting features of the posttranslational processing that generates γ-MSH and β-MSH with reference to the snake POMC as described below.
The γ-MSH segment of snake POMC is characterized by the change in the essential sequence from His-Phe-Arg-Trp to His-Trp. Based on this, the term γ-MSH-like sequence was assigned. Moreover, the amino acid residues flanking this segment are Gln-Lys and Lys-Ser at N-terminal and C-terminal sides, respectively. These characteristics suggest that the γ-MSH-like sequence is non-functional and is not liberated from the precursor protein by proteolytic cleavage. This hypothesis was supported by the identification of an N-POMC peptide consisting of AMH and γ-MSH-like sequences. The γ-MSH-like sequence seems to be a so-called vestige. The snake POMC is assigned as a 3MSH/1END type on the basis of its overall molecular organization. However, taking its probable lack of a functional γ-MSH into consideration, its direction in evolution is perhaps toward a 2MSH/1END type.
The amino acid sequence of the alligator γ-MSH is identical to the γ-MSH sequences of the leopard gecko, mud turtle, and birds. The detection of AMH and JP indicates that alligator POMC is cleaved at Arg 75 and Arg 89–Arg 90 by posttranslational processing; therefore, γ-MSH or γ 3-MSH must also be liberated. However, these peptides were not detected. Non-detection of γ 3-MSH suggests that a carbohydrate side chain is probably linked to the C-terminal region of γ 3-MSH via an N-glycosylation site at alligator prePOMC 91–93. In contrast, γ-MSH seems not to be liberated from POMC, or in other words, Arg 89–Arg 90 are not functional processing signals in alligator POMC.
Despite the consistent presence of the β-MSH sequence in all vertebrate POMCs, β-MSH is not always liberated. In ostriches, β-LPH and γ-LPH peptides, both containing β-MSH, have been detected, whereas the β-MSH peptide has not been detected.
