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K Haraguchi, T Saito, M Kaneshige, T Endo, and T Onaya


To understand the functional significance of the carboxy-terminal half of the intracellular region of the human TSH receptor (hTSHR), a mutant hTSHR lacking amino acids from the carboxyterminal to His726 was constructed.

Wild type hTSHR cDNA and truncated hTSHR cDNA were subcloned into a eukaryotic expression vector, pRc/CMV, and transfected into Chinese hamster ovary cells to obtain cell lines which stably expressed hTSHRs at high levels. This allowed us to observe highly efficient coupling of hTSHR and adenylyl cyclase as well as desensitization and internalization of hTSHR.

Despite the differences in potential phosphorylation sites and internalization signals, dose-dependent stimulation of adenylyl cyclase by TSH, TSH-dependent desensitization and the rate of hTSHR internalization were similar for wild type and truncated hTSHRs. We conclude that the carboxy-terminal half of the intracellular region of hTSHR does not have a major functional role in TSH-dependent signal transduction.

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S Kobayashi, H Shibata, I Kurihara, K Yokota, N Suda, I Saito, and T Saruta

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.

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M Tanaka, M Suzuki, T Kawana, M Segawa, M Yoshikawa, M Mori, M Kobayashi, N Nakai, and T R Saito

In addition to the known four alternative first exons E11, E12, E13 and E14 of the rat prolactin receptor (PRL-R) gene, a novel first exon, E15, was identified by cDNA cloning of the 5′-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E15 and its 5′- or 3′-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E15 revealed that E15 is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E15 was preferentially expressed in the liver, brain and kidney. Expression profiles of E12-, E13- and E15-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E12-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E15-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E12-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of β-oestradiol. On the contrary, the levels of E15-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E12-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E15-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E13-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.