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K Sakaguchi, T Ohkubo, T Sugiyama, M Tanaka, H Ushiro, and K Nakashima


Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen.

Journal of Endocrinology (1994) 143, 383–392

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N Nakao, Y Higashimoto, T Ohkubo, H Yoshizato, N Nakai, K Nakashima, and M Tanaka

Growth hormone receptor (GHR) cDNA and gene of the Japanese flounder (Paralicthys olivaceus) were cloned and their molecular structures were characterized. The 641 amino acid sequence predicted from the cDNA sequence showed more than 75% overall sequence similarity with GHRs of other teleosts such as turbot and goldfish, and contained common structural features of vertebrate GHRs. The extracellular domain of flounder GHR had three pairs of cysteines and an FGEFS motif with a replacement E to D. The cytoplasmic domain contained two conserved motifs referred to as box 1 and box 2. The flounder GHR gene was cloned by PCR using primers designed from the sequence of the GHR cDNA. The GHR gene was composed of 10 exons. The sequence of exon 1 corresponded to the 5'-untranslated region of the cDNA, and exons 2-6 encoded most parts of the extracellular domain. The transmembrane domain was found in exon 7, and the intracellular domain was encoded in exons 8-10. Exon 10 also encoded the 3'-untranslated region. Comparison of the flounder GHR gene with the human GHR gene shows that the flounder gene contains no exons corresponding to exon 3 of the human GHR gene, and that the region corresponding to exon 10 in the human GHR gene is encoded by exons 9 and 10 in the flounder GHR gene. These findings indicate that the flounder GHR gene diverged from those of mammalian and avian GHR genes, especially in the organization of the exons encoding the cytoplasmic domain. In addition to the regular form of GHR mRNA, a 3'-truncated form lacking the region derived from exons 9 and 10 was detected as a minor species in the liver by RT-PCR and by RNase protection assay. RT-PCR analysis showed that both the regular and the 3'-truncated GHR mRNAs are expressed in a wide range of flounder tissues with the highest levels being found in the liver. The 5'-flanking region of the flounder GHR gene was cloned by inverse PCR, and three transcription start points were identified with similar frequency by RNase protection assay.