Melanocortin-4 receptor regulation of reproductive function in black rockfish (Sebastes schlegelii).

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor with multiple functions in mammals. However, the functions of MC4R in fish have not been investigated extensively. The purpose of this study was to determine potential regulation of reproduction by the MC4R. We cloned the black rockfish MC4R and analyzed its tissue distribution and function. The results showed that black rockfish mc4r cDNA consisted of 981 nucleotides encoding a protein of 326 amino acids. The quantitative PCR data showed that mc4r mRNA was primarily expressed in the brain, gonad, stomach and intestine. In the brain, mc4r was found to be primarily located in the hypothalamus. Both α-MSH and ß-MSH increased gnih expression and decreased sgnrh and cgnrh expression (P < 0.05). α-MSH and ß-MSH had opposite effects on kisspeptin expression. In contrast, α-MSH and ß-MSH increased the expression of cyp11, cyp19, 3ß-hsd and star. In summary, our study shows that MC4R in black rockfish might regulate reproductive function and that the effects of α-MSH and ß-MSH might differ.

Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses.

Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine(1), O-acetyl Serine(3)]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline(15)]-ß-MSH, together with [phosphoserine(15)]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.

The molecular genetics of the melanocortin pathway and energy homeostasis.

The CNS melanocortin pathway plays an important role in the control of body weight. Two papers in this issue of Cell Metabolism, Lee et al., 2006 and Biebermann et al., 2006, suggest that beta MSH--a product of POMC processing--plays an unanticipated role in this pathway in humans.

A POMC variant implicates beta-melanocyte-stimulating hormone in the control of human energy balance.

The melanocortin-4 receptor (MC4R) plays a critical role in the control of energy balance. Of its two pro-opiomelanocortin (POMC)-derived ligands, alpha- and beta-MSH, the majority of attention has focused on alpha-MSH, partly reflecting the absence of beta-MSH in rodents. We screened the POMC gene in 538 patients with severe, early-onset obesity and identified five unrelated probands who were heterozygous for a rare missense variant in the region encoding beta-MSH, Tyr221Cys. This frequency was significantly increased (p < 0.001) compared to the general UK Caucasian population and the variant cosegregated with obesity/overweight in affected family members. Compared to wild-type beta-MSH, the variant peptide was impaired in its ability to bind to and activate signaling from the MC4R. Obese children carrying the Tyr221Cys variant were hyperphagic and showed increased linear growth, both of which are features of MC4R deficiency. These studies support a role for beta-MSH in the control of human energy homeostasis.

A role for beta-melanocyte-stimulating hormone in human body-weight regulation.

Pro-opiomelanocortin (POMC) expressing neurons mediate the regulation of orexigenic drive by peripheral hormones such as leptin, cholecystokinin, ghrelin, and insulin. Most research effort has focused on alpha-melanocyte-stimulating hormone (alpha-MSH) as the predominant POMC-derived neuropeptide in the central regulation of human energy balance and body weight. Here we report a missense mutation within the coding region of the POMC-derived peptide beta-MSH (Y5C-beta-MSH) and its association with early-onset human obesity. In vitro and in vivo data as well as postmortem human brain studies indicate that the POMC-derived neuropeptide beta-MSH plays a critical role in the hypothalamic control of body weight in humans.

Spatiotemporal expression, distribution, and processing of POMC and POMC-derived peptides in murine skin.

In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides beta-endorphin, ACTH, beta-MSH, and alpha-MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, beta-endorphin, beta-MSH, and alpha-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin.

Staphylococcal exfoliative toxins cleave alpha- and beta-melanocyte-stimulating hormones.

The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains of Staphylococcus aureus and are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide beta-melanocyte-stimulating hormone (beta-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving alpha-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving beta-MSH.

Cloning of a proopiomelanocortin cDNA from the pituitary of the Australian lungfish, Neoceratodus forsteri: analyzing trends in the organization of this prohormone precursor.

The polypeptide hormone precursor, proopiomelanocortin (POMC), was cloned and sequenced from the pituitary of the Australian lungfish, Neoceratodus forsteri, the only surviving species of the oldest extant lineage of lungfish. The Australian lungfish POMC cDNA had an open reading frame that coded for a 255-amino acid precursor. A comparison of POMC sequences from the Australian lungfish and the African lungfish indicated that the deduced amino acid sequences for ACTH, beta-MSH, and beta-endorphin were over 90% identical. Furthermore, within the open reading frames of the two lungfish POMCs, there was 84% identity at the nucleotide level. Although a gamma-MSH-like region was detected in the Australian lungfish POMC cDNA, this sequence contained mutations that have been detected in the gamma-MSH sequences of some ray-finned fish and are not found in the gamma-MSH sequence of the African lungfish or those of tetrapods. In addition, the sequence of beta-endorphin in the two species of lungfish has amino acid motifs that are found in the beta-endorphin sequences of cartilaginous fish and ray-finned fish but not in tetrapods. However, maximum parsimony analysis of the entire POMC open reading indicated that the lungfish POMC sequences form a clade with two amphibian POMC sequences rather than with POMC sequences from ray-finned fish. This result is consistent with the accepted view that the sarcopterygians (lungfishes and tetrapods) are a monophyletic assemblage. Analysis of rates of divergence for various POMC sequences indicate that point mutations are accumulating in the lungfish POMC sequences at a slower rate than in either amphibian or mammalian POMC sequences. The phylogenetic implications of these observations are discussed.