Yamamoto T et al. (2000), Cloning of a cDNA for Xenopus prolactin recepto...

XB-ART-10930

Dev Growth Differ 2000 Apr 01;422:167-74. doi: 10.1046/j.1440-169x.2000.00496.x.

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Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile.

Yamamoto T , Nakayama Y , Tajima T , Abe S , Kawahara A .

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A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.

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Species referenced: Xenopus laevis
Genes referenced: prl.1 prl.2 prlr

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	Fig. 1. Nucleotide and deduced amino acid sequences of Xenopus prolactin receptor (PRL-R). The transmembrane region is underlined with a solid line. Two pairs of cysteine residues in the extracellular domain are circled. Potential N-linked glycosylation sites are underlined with dashed lines. A WSXWS sequence is surrounded by a stippled box. A motif of proline-rich box 1 is indicated by an open box. Tyrosine residues are double-underlined. An asterisk indicates the termination codon. The nucleotide sequence presented here was deposited into the DDBJ, EMBL and GenBank nucleotide sequence databases with accession number AB030443.
	Fig. 2. Competitive binding of [125I] rat prolactin (PRL) to Xenopus prolactin receptor (PRL-R) expressed in COS-7 cells. The membrane fraction was prepared from COS-7 cells transfected with a plasmid bearing Xenopus PRL-R cDNA (see Materials and Methods). Aliquots of the membrane fraction were incubated with a constant amount of the labeled ligand (30 000 c.p.m.) and various concentrations of unlabeled rat and ovine PRL and growth hormone (GH). Binding levels are expressed as a percentage of the maximal specific binding observed in the absence of competitor. (d) rat PRL; (j) ovine PRL; (s) rat growth hormone (GH); (h) ovine GH.
	Fig. 3. Distribution of Xenopus prolactin receptor (PRL-R) messenger ribonucleic acid (mRNA) in adult tissues. Total RNA (each 2 μg) from various tissues of the juvenile frogs (6–12 months after metamorphosis) was loaded on 1.2% agarose-formaldehyde gel. The gel was stained with ethidium bromide (lower panel) and used for detecting PRL-R mRNA by northern blot hybridization (upper panel). Asterisks show the positions of ribosomal RNA (28S and 18S rRNA). The main hybridization signal was detected at approximately 8 kb in length (arrow). A faint signal was also seen at approximately 3 kb (arrowhead) in some tissues. In the skin RNA, no 8 kb signal was detected but extra signals different from those described earlier were observed.
	Fig. 4. Tissue distribution of prolactin receptor (PRL-R) messenger ribonucleic acid (mRNA) at premetamorphic stage (a) and metamorphic climax stage (b). The RNA was prepared from the indicated tissues of tadpoles at stages 53 and 62. Total RNA (each 2 μg) was loaded on 1.2% agarose-formaldehyde gels. The gels were stained with ethidium bromide (lower panel) and used for detecting PRL-R mRNA (upper panel) by northern blot hybridization. Asterisks show the positions of ribosomal RNA (28S and 18S rRNA).
	Fig. 5. Developmental changes in prolactin receptor (PRL-R) messenger ribonucleic acid (mRNA) expression in whole body (a) and in the indicated organs (b). Total RNA (each 3 μg) from the whole body and indicated organs at the indicated developmental stages were loaded on 1.2% agarose-formaldehyde gels. The gels were stained with ethidium bromide. Asterisks show the positions of ribosomal RNA (28S and 18S rRNA). The hybridization signal (3 kb) in whole body RNA was detectable at earlier stages than stage 32 by long exposure to X-ray film.