| In order to explore the role of prolactin (PRL) and prolactin receptor (PRLR) during metamorphosis of amphibians, both cDNAs from Rana chensinensis were identified. Furthermore, their expression patterns were also analyzed in different tissues.1. A PRL cDNA consisting of a 645-bp open reading frame (ORF) named rcPRL (GenBank accession No. KP260562) was obtained encoding 214 amino acid (aa) residues with a 27 aa signal peptide. The molecular weight of rcPRL was around 24313.6 Da with a pI of 6.07; besides, we obtained a PRLR cDNA of 2120-bp from R. chensinensis, named rcPRLR (GenBank accession no. KJ490947). The rcPRLR sequence was composed of a 29-bp 5’-UTR,237-bp 3’-UTR and 1854-bp ORF encoding 617 aa residues. The deduced protein contained a signal peptide of 26 aa followed by a 212 aa ECD, a 22 aa TMD, and a 357 aa ICD. The molecular weight of rcPRLR was around 69298.6 Da with a pI of 5.59.2. Three pairs of conserved Cys residues were found in rcPRL. However, the first pair located in the signal peptide sequence, which were removed during the formation of the mature protein. Therefore, only the second and the third pairs formed two disulfide bonds. Moreover, the relative position of the last 4 Cys residues was highly conserved throughout the vertebrates. Prediction of secondary structure showed that rcPRL contained 4 a-helices. In addition, alignment of PRLs from various species showed that rcPRL had two conserved motifs as the same with others, motif 1 and 2. They were composed of 34 aa and 18 aa, respectively; In rcPRLR, some conserved motifs were found, including two pairs of Cys residues at positions 38,48,77 and 88, a WS motif, three potential N-linked glycosylation sites at positions 61,114 and 134 in ECD, Box 1 and 2 in ICD. All these features corresponded to the long form PRLR of mammals. ECD was more conservative than ICD:the identity values ranged from 48% to 92% in the ECD and 38% to 95% in the ICD separately.3. The multi-alignments of PRL and PRLR aa sequences from R. chensinensis and other species were performed by DNAman software. It is worth mentioning that the relations of identity between rcPRL and other vertebrate PRLs resemble those of identity between rcPRLR and other vertebrate PRLRs:the overall identity of both rcPRL and rcPRLR is high with amphibian, low with birds and reptiles, and lower with mammals, which suggests that PRL may have coevolved with PRLR in R. chensinensis.4. In this paper, a His-212 within ECD (i.e His-188 in mature human PRLR) is well conserved in most species investigated. However, no His was found at the same position in anurans including Rana chensinensis. This may imply that receptor recognition in R. chensinensis and other anurans is not pH-dependent. As far as we know, this is the first time that the attention is paid on the relationship between anuran PRLR recognition and pH.5. A conserved DSGRGS motif, which is closely linked to the ubiquitin-dependent degradation of long form PRLR, was found in most species except Trichosurus vulpecula and Myotis davidii. Thus, the degradation of rcPRLR is probably also ubiquitin-dependent.6. By RT-PCR, rcPRL mRNA in the brain, rcPRLR mRNA in the liver, skin, tail and kidney of tadpoles was detected. The rcPRL in brain was continuously expressed from pro-metamorphosis to late metamorphosis. The expressional trend of rcPRLR in liver indicated that liver was not a principal target site of PRL before metamorphosis, the high expression of rcPRLR in skin and tail may be involved in osmoregulation and regression respectively, while the rcPRLR expressional changes in kidney were in connection with metamorphic changes. The expression pattern of rcPRL and rcPRLR supported the point that PRL was not equivalent to JH (juvenile hormone)during metamorphosis. |
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