Nobuhiro Nakao

Insulin receptor (IR) and lGF-I receptor (IGF-IR) are structurally arid functionally related and belong to the tyrosine kinase receptor family. In teleosti such as salmonids and turbot, occurrence of multiple IR and IGF-IR members has been reported. but the structures of a complete set of both IR and IGF-IR members in a single teleost species have not yet been characterized. In this study, we cloned arid analysed four distinct cDNA clones for IR arid IGF-IR members from the liver and kidney of the Japanese flounder (Paralichthys olivaceus). Deduced amino acid sequence analyses and phylogenetic analysis have revealed that two of them (flR-1 and flR-2) belong to IR, members arid the other two (flGF-IR-1 and flGF-IR-2) are IGF-IRs. flR-1 arid fIR-2 comprised 1369 and 1368 amino acid residues respectively, and flGF-IR-1 and flGF-IR-2 comprised 1412 and 1418 residues respectively. All the receptor proteins contained cysteine-rich domains in their alpha-subunits, and conserved each transmembrane arid tyrosine kinase domains in their beta-subunits. The amino acid sequences of flRs arid flGF-IRs showed more than 90% sequence identity with turbot IR arid IGF-IR respectively. When compared with their mammalian homologues. flGF-IR-1 and flGF-IR-2 proteins contained large insertions at their C-termini, as was observed in the corresponding region of turbot IGF-IR. Occurrence of multiple species of iuRNA for each IR and IGF-IR was suggested by Northern blot analyses. A ribonuclease protection assay revealed diverse expressions of four receptor mRNAs in a wide range of tissues , including heart, liver. ovary, testis, brain, gill arch, kidney, skeletal muscle, intestine, stomach, spleen and eye of the flounder.

One of the main function of growth hormone (GH) is to stimulate the synthesis of insulin-like growth factor-I (IGF-I) in the liver and other tissues. In order to clarify the molecular mechanisms of the GH and IGF-I actions on the growth of fish, molecular analysis of GH receptor (GH-R) and insulin/IGF-I receptors (IR/IGF-IRs) of Japanese flounder was performed. A cDNA, fGH-R, was cloned from the liver and sequenced. Two pairs of cysteine and prolin-rich Box-1 motif were conserved in the deduced amino acid sequence of fGH-R but the overall sequence was considerably diverged from those of other animal species. fGH-R mRNA was predominantly expressed in the liver. In addition to fGH-R cDNA, four distinct IR/IGF-IR cDNAs, fIR-1, fIR-2, fIGF-IR-1 and fIGF-IR-2 were cloned from the liver and kidney of Japanese flounder. Cysteine-rich domain in alpha-subunit and tyrosine kinase domain in p-subunit were conserved in each amino acid sequence of IR/IGF-IRs. Each IR/IGF-IR mRNA were expressed in a wide range of tissues in Japanese flounder.

Ghrelin is a novel growth hormone-releasing peptide isolated from rat stomach. In the present study, we report expression of a ghrelin gene-derived transcript (GGDT) in the mouse testis. Analysis of GGDT cDNA revealed that the 68 bp sequence at the 5'-end was unique and the remaining 252 bp sequence was identical with the sequence encoded by exons 4 and 5 of mouse ghrelin gene. The 5'-unique sequence encoded 12 amino acid residues being in-frame with the C-terminal 42 amino acid sequence of mouse ghrelin. The 54-amino-acid polypeptide encoded by GGDT contained no apparent signal peptide, sequence but possessed a nuclear localization signal-like sequence. Ghrelin mRNA was extensively expressed in the stomach, while GGDT was expressed only in the testis. The 5'-unique sequence of GGDT was identified between exons 3 and 4 of the ghrelin gene, indicating that GGDT was generated by alternative usage of the 68 bp exon as the testis-specific first exon. The GGDT expression in the testis was initiated and increased after 2 weeks of postnatal period. These results indicate that the expression of GGDT is regulated in testis-specific and developmental stage-specific manners. (C) 2001 Elsevier Science B.V. All rights reserved.

In diadromous and euryhaline teleosts, it has been established that prolactin (PRL) is a major hormone regulating the maintenance of water and electrolyte homeostasis by acting on its receptor (PRLR) expressed in the osmoregulatory organs. To investigate the major physiological role of PRL in a marine teleost, cDNA for the Japanese flounder (Paralichtys olivaceus) prolactin receptor (fPRLR) has been cloned and characterized. The predicted fPRLR is composed of 636 amino acids conserving common structural features, such as the WSXWS motif and box 1, that are observed in the members of the cytokine receptor superfamily. By Northern blot analysis, 3.5-kb transcripts for fPRLR were clearly detected in the gill, kidney, and intestine. By RNase protection assay, similarly high levels of mRNA expression were detected in these osmoregulatory organs and lower expression levels were seen in the brain for both males and females. Interestingly, a distinct expression level of fPRLR mRNA was observed in the testis, but not in the ovary. The present results suggest that PRL may play an important role in the control of water and electrolyte balance through PRLR expressed in the osmoregulatory organs in the marine teleost the Japanese flounder as well as in other teleosts. Furthermore, PRL may differentially regulate gonadal functions in males and females of Japanese flounder. (C) 2001 Academic Press.

Ghrelin is a growth hormone-releasing peptide recently discovered in the stomach of rat and human as an endogenous ligand for growth hormone-secretagogue receptor. In the present study, a full-length cDNA for mouse ghrelin has been cloned from the stomach using the oligo-capping and rapid amplification methods, and the organization of its gene and promoter has been analyzed. The mouse ghrelin cDNA was 521 bp long, consisting of 44 bp 5'-noncoding region, 354 bp coding region encoding a pre-proghrelin composed of 117 amino acid residues and 123 bp 3'-noncoding region. The genomic sequence analysis has revealed that the mouse ghrelin gene consists of 5 exons and 4 introns. The first exon was revealed to be only 19 bp long presented at the noncoding region of cDNA. The identical 19 bp sequence was also found as the first exon at the 5'-end of full-length rat ghrelin cDNA obtained from the stomach. A TATA box-like sequence, TATATAA was localized 24 bp upstream of the transcription start site of the mouse ghrelin gene. The sequence of the 5'-promoter region of mouse ghrelin gene including the TATA-like sequence and short exon I was highly homologous to that of reported human ghrelin gene. These findings suggest that the structure of the promoter region including the short noncoding first exon and its transcriptional regulation are conserved among the mammalian ghrelin genes.

Cloning and sequencing of the chicken prolactin receptor (PRLR) gene segment from the transmembrane domain to the box 2 motif revealed the presence of the two testis-specific first exons, TSE-1 and TSE-2, encoding the unique 5'-end sequences of the reported and newly identified multiple 5'-truncated PRLR transcripts containing only the cytoplasmic domain in the testis. TSE-1 was located downstream of the exon encoding the transmembrane domain and TSE-2 presented downstream of the exon encoding the box 1 motif. These findings indicate that the box 1-containing 5'-truncated transcripts are generated by the utilization of TSE-1 as the first exon with distinct splicing donor sites to the box 1-containing exon, and that the utilization of TSE-2 as the first exon and its splicing to the box 2-containing exon results in the generation of the box 1-lacking transcript. Three transcription initiation sites for the box 1-containing 5'-truncated transcripts and two transcription initiation sites for the box 1-lacking transcript were detected by the RNase protection assays. Reverse transcription-polymerase chain reaction analysis showed that the expression levels of all these 5'-truncated PRLR transcripts are simultaneously increased during sexual maturation, accompanying the decrease of the amount of the canonical full-length transcript for PRLR. (C) 2000 Elsevier Science B.V. All rights reserved.