Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family

The primary structure of the rat liver prolactin receptor has been deduced from a single complementary DNA clone. The sequence begins with a putative 19 amino acid signal peptide followed by the 291 amino acid receptor that includes a single 24 amino acid transmembrane segment. In spite of the fact that the prolactin receptor has a much shorter cytoplasmic region than the growth hormone receptor, there is strong localized sequence identity between these two receptors in both the extracellular and cytoplasmic domains, suggesting that the two receptors originated from a common ancestor.     

Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of rat growth hormone receptor gene expression

A cDNA encoding the growth hormone (GH) receptor was cloned from rat liver. Both the nucleotide and translated amino acid sequence share greater than 70% similarity with the GH receptors from rabbit and human. An RNA probe was generated from this sequence for use in a solution hybridization assay to quantitate GH receptor mRNA expression in rat tissues. Expression was detected in 9/12 tissues examined, with the highest levels observed in the liver. Expression in liver, kidney, heart and muscle was developmentally regulated, being low at birth and rising to adult levels in 5 weeks. No difference was observed between hepatic expression in males and females, although livers from pregnant rats had elevated levels. Hypophysectomy and GH treatment did not affect hepatic GH receptor mRNA levels.     

Rat corticotropin-releasing hormone gene: sequence and tissue-specific expression

The rat corticotropin releasing hormone (CRH) gene has been isolated and characterized by DNA sequence analysis. The gene exhibits a structural organization similar to that of the human CRH gene. The nucleotide sequence encoding the entire rat CRH precursor is located on the second exon, while exon I encodes the 5'-untranslated region of the mRNA. Analysis of the nucleotide sequence homology between the human and rat CRH genes reveals several highly conserved regions including the CRH peptide-encoding sequence and the 5'-flanking sequence. RNA blot analysis demonstrates that CRH mRNA can be observed in numerous regions of the rat brain as well as the spinal cord, adrenal gland, pituitary, and testis.     

Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.     

The effects of 5-azacytidine on the expression of the rat growth hormone gene. Methylation modulates but does not control growth hormone gene activity

The role of DNA methylation in the expression of the rat growth hormone (rGH) gene was assessed by using a hypomethylating agent, 5-azacytidine, and the iso-schizomeric restriction enzymes MspI and HpaII. 5-Azacytidine increased rGH mRNA 3-8-fold in GH3D6 cells, a subclone of rat pituitary tumor cell lines that expresses one-tenth to one-fifteenth the GH expressed by two other clones, GH3 and GC. The effect was also detected at the level of pre-mRNA. The effect was independent of glucocorticoids and thyroid hormones and was found to be inheritable. The DNA methylation pattern generated by the isoschizomeric restriction enzymes indicated that the HpaII sites in the rGH gene were mostly methylated in GH3D6 cells but mostly unmethylated in GC cells. After treatment with 5-azacytidine, about 22% of these HpaII sites in GH3D6 cells became unmethylated. Thus, DNA methylation correlates inversely with the expression of the rGH gene in these cell lines. However, three other observations indicate that factors in addition to DNA methylation control rGH expression. First, in GC cells, even though most of the HpaII sites are unmethylated, the gene is not fully expressed. Second, in rat hepatoma cells, which do not express GH at all, the GH gene is less methylated than that in GH3D6 cells. Third, within the sensitivities of the assay methods, 5-azacytidine has no effect on the GH gene when it is completely silent. Taken together, the findings indicate that DNA methylation modulates but does not control GH gene expression. It is tempting to speculate that DNA methylation can influence expression only when the gene is committed to express.     

Identification of four new members of the rat prolactin/growth hormone gene family.

The rodent prolactin (PRL)/growth hormone (GH) gene family currently consists of at least 14 distinct genes that are expressed mainly in pituitary, uterus, and/or placenta. We report here the identification of novel four members from rat with significant homology to PRL. The encoding proteins are not homologs of other known members of this hormone family. The four new cDNAs were assigned to PRL family based on sequence homology and were referred to as PRL-like protein-I (PLP-I), PLP-J, PLP-K, and PLP-L, following the current naming order of rodent PLP family, where PLP-H is the most recent gene. They encode amino acids with 211-228 amino acids, and 34-38% identity with PRL. All have one or two N-linked glycosylation sites. Among the examined rat tissues by Northern blot analysis, only PLP-I was expressed in testis. Our results indicate that the rodent PRL/GH gene family is large with at least 18 distinct genes.     

Growth hormone pretranslationally regulates the sexually dimorphic expression of the prolactin receptor gene in rat liver

The female-specific expression of the rat liver PRL receptor (PRL-R) gene was investigated by Northern analysis of hypophysectomized rats after two alternative human GH treatments that were to mimic either 1) the continuous female-specific or 2) the discontinuous male-specific serum GH patterns. The former (female-specific) pattern was shown to result in a dramatic increase in PRL-R mRNA in both males and females, while the latter (male-specific) pattern failed to evoke this response. A similar inductive effect in hypophysectomized females was shown after continuous administration of bovine GH and was found to constitute an approximately 60-fold increase in PRL-R mRNA levels. This effect by bovine GH, which, unlike the human isoform, is devoid of lactogenic properties, thus indicates the somatogenic origin of the signal resulting in this inductive response. These observations in conjunction with previous data obtained for other GH-regulated nonreceptor genes are interpreted to support the proposal of GH serum patterns being an early signal in a more general mechanism for pretranslational regulation of sex-specific gene expression. In contrast to GH, only a slight elevation of PRL-R mRNA was evoked by the ligand ovine PRL, while coadministration of ovine PRL with bovine GH failed to enhance the mRNA level found with bovine GH alone. The detection of previously unreported PRL-R mRNAs in liver of approximately 3.0, 3.8, and 5 kilobases in addition to the major 2.2-kilobase form was also evident after continuous GH administration.