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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 brainPRL-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.
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.
