Expression of proopiomelanocortin and its cleavage enzyme genes in Rana esculenta and Xenopus laevis gonads.

Proopiomelanocortin (POMC) is the precursor protein of different hormones and neuropeptides, and the POMC-derived peptides are produced through proteolytic cleavage. Prohormone convertase PC1 and PC2 are enzymes responsible for the cleavage of the POMC prohormone. The coexpression of POMC, PC1, and PC2 genes was previously described in the brain and the pituitary gland of Rana esculenta and Xenopus laevis, but no data are available for the gonad. The present work demonstrates a gonadal POMC convertase gene expression in Rana esculenta and Xenopus laevis.

Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1.

Prohormone convertases (PCs) are calcium-dependent serine endoproteases of the subtilisin/kexin family that play a key role in the posttranslational processing of precursors for biologically active peptides. In this study, we have characterized the cDNA encoding PC1 in the European green frog Rana ridibunda. A frog brain cDNA library was screened by using a heterologous probe at low stringency, and a 2.3-kb cDNA clone encoding PC1 was isolated. This cDNA encodes a 736-residue protein with a 26-amino-acid signal peptide. Comparative structural analysis revealed that frog PC1 exhibits a high degree of amino acid identity with its mammalian counterparts, in particular in the subtilisin-like catalytic domain. Northern blot analysis resolved two major transcripts of 3.0 kb and 5.0 kb that were expressed differentially in the brain and pituitary. In situ hybridization studies showed that, in the frog brain, the highest densities of PC1 mRNA are present in the amygdala, the hypothalamus, and the anterior preoptic area. High concentrations of PC1 mRNA also were found in the pars distalis and pars intermedia of the pituitary, whereas the pars nervosa was devoid of hybridization signal. The wide distribution of PC1 mRNA in the brain and pituitary suggests that, in frog, PC1 is involved in the processing of a number of hormone and neuropeptide precursors.

Characterization of the cDNA encoding the prohormone convertase PC2 and localization of the mRNA in the brain of the frog Rana ridibunda.

A number of precursors for neuropeptides have recently been cloned in amphibians, but little is known concerning the endoproteases responsible for the processing of these precursors. Here we report on the molecular cloning of the cDNA encoding the proprotein convertase PC2 and the distribution of the corresponding mRNA in the European green frog Rana ridibunda. The full cDNA structure (2125 bp) was obtained from the analysis of the PCR products combined with the sequence from a clone isolated from a frog pituitary cDNA library. The deduced amino acid sequence revealed that frog PC2 comprises 636 amino acid residues including a 22-residue signal peptide. RT-PCR analysis showed that PC2 is expressed not only in the brain and pituitary but also in various peripheral organs including the pancreas, stomach, intestine, liver, kidney and testis. In situ hybridization histochemistry revealed that, in the central nervous system, PC2 mRNA is widely distributed, the highest concentrations being found in the pallium, the anterior preoptic area, the hypothalamus and the medial amygdala. High levels of PC2 mRNA were also detected in the intermediate lobe of the pituitary. The overall distribution of PC2 mRNA in the frog brain is consistent with its involvement in the processing of a number of neuropeptide and hormone precursors.