Molecualr Cloning, Functional Study And Regulation Of Steroid Hormones Of Two Distinct Prolactin Receptors In Seabream

By insilico anlysis, two prolactin receptors (PRLR) encoded by two different genes were identified in the fugu, zebrafish, medaka and three-spined stickleback fish genomes but not in the genomes of other vertebrates or invertebrates. Subsequently, two cDNA sequences corresponding to the two PRLRs (we call the conventional one PRLR1 and the newly identified as PRLR2) were identified in black seabream. Phylogenetic analysis of PRLR sequences in various vertebrates indicated that the co-existence of two PRLRs in a single species is a unique phenomenon in teleosts but not in other vertebrates or invertebrates. Both PRLRs in teleosts resemble the long form mammalian PRLRs. However, despite their overall structural similarities, the two PRLR subtypes in fish share very low amino acid similarities (about 30%), mainly due to differences in the intracellular domain. In particular, the Box 2 region and four fragments are missing in PRLR2, which make that that the PRLR2 is about 100 amino acids shorter than the PRLR1.Tissue distribution study by real-time PCR in black seabream (sb) revealed that both receptors (sbPRLRl and sbPRLR2) are widely expressed in different tissues. In gill, the expression level of sbPRLR2 is much higher than sbPRLRl, while in the intestine, the expression of sbPRLRl is higher than that of sbPRLR2. The expression levels of both receptors are relatively low in most other tissues, with sbPRLRl generally higher than sbPRLR2. The sbPRLRl and sbPRLR2 were functionally expressed in cultured HEK293 cells. Both receptors can activate theβ-casein and c-fos promoters, however, only sbPRLRl but not sbPRLR2 can activate the Spi 2.1 promoter upon receptor stimulation in a ligand specific manner. These results indicate that both receptors share some common functions but are distinctly different from each other in mobilizing post-receptor events. When using the salmon growth hormone and somatolactin to challenge the cells as the way of PRL, the results demonstrate that both hormone cannot stimulate any response ofβ-casein, c-fos or Spi 2.1 promters.The 5'-flanking regions of the sbPRLR1 and sbPRLR2 genes were cloned by genome walking and the promoter activities were studied in transient transfected GAKS cells in the absence or presence of different steroid hormones by using the constructs of pGL3-sbPRLR1 or pGL3-sbPRLR2. The sbPRLR1 gene promoter activity was activated by estradiol and cortisol but not by testosterone. In contrast, the sbPRLR2 gene promoter activity was inhibited by estradiol, cortisol and testosterone. The successive deletions of sbPRLR1 and sbPRLR2 promoters were inserted into pGL3 basic vector. Then the mutants were challenged with different steroid hormones. The results located the possible ERE, GRE and ARE in specific regions of each promoter.When challenged with different steroid hormones in vivo, the two PRLRs exhibited very different gene expression patterns in the seabream kidney. The sbPRLRl expression was up-regulated by estradiol and Cortisol, whereas testosterone had no significant effect. For sbPRLR2, its expression was down-regulated by estradiol and testosterone while Cortisol exerted no significant effect. The results of the promoter studies were in general agreement with the in vivo hormonal regulation of gene expression results.