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Query: UNIPROT:P06889 (Mol)
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Recent studies using both normal and tumoral pituitary cell cultures have demonstrated that growth hormone (GH) and prolactin (PRL) secreting populations contain cells which release either one or both of these hormones. In order to determine whether these two cell types can be differentially regulated by hypothalamic factors we performed the following study employing plaque assays for GH and PRL. Using cultures of GH3 cells, a rat tumor cell line which contains both of these cell types, we found that the hypothalamic factors vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) when used together had a greater influence on plaque formation than when each was used individually. This suggested that cells were present in culture that responded to one peptide but not the other. Estradiol-treated cultures (which contain only dual-secreting cells) were then evaluated for VIP and TRH responsiveness and found to respond to TRH but not VIP. Finally, we assessed the peptide sensitivity of cultures that were exposed to a conjugate of VIP and the A-chain of ricin (a potent cytotoxin). In addition to eliminating VIP-responsive cells, this treatment markedly reduced the proportions of cells secreting GH-only while having no appreciable influence on dual-hormone secretors. When taken together, our findings indicate that single and dual secretors respond differently to at least two hypothalamic secretagogues and suggest that regulatory differences between these cell types may be important in the control of GH and PRL secretion.
Mol Cell Endocrinol 1992 Sep
PMID:Single and dual hormone secretors in GH3 cultures respond differently to hypothalamic factors. 144 81

We have applied the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis (SSCP) to detect activating mutations in the Gs alpha subunit gene, amplifying genomic DNA extracted from growth hormone (GH)- and GH/prolactin (PRL)-secreting human pituitary tumors. Of 15 tumors tested six contained mutations in the analyzed regions of the Gs alpha. SSCP analysis revealed band shift in exon 8 in four GH- and in one GH/PRL-secreting tumors, and in exon 9 in one GH/PRL-secreting tumor. Direct sequencing of PCR reaction products identified the mutations as R201-H, R201-S and R201-C in exon 8 and Q227-L in exon 9. These results show the efficacy of PCR/SSCP analysis in the detection of G protein mutations and extend the generalization that these sites are hot spots in tumor-inducing mutations.
Mol Cell Endocrinol 1992 Sep
PMID:Identification of G protein alpha subunit mutations in human growth hormone (GH)- and GH/prolactin-secreting pituitary tumors by single-strand conformation polymorphism (SSCP) analysis. 144 84

Casein gene expression is induced in the rabbit mammary gland by prolactin (PRL). alpha s1-casein is the major casein secreted into milk. In order to define the position of the DNA sequences involved in the control of rabbit alpha s1-casein gene regulation by PRL, chimeric genes were constructed between upstream regions of the rabbit alpha s1-casein gene and the chloramphenicol acetyl transferase (CAT) reporter gene. A series of 5'-deleted fusion genes was obtained by nuclease digestion of the alpha s1-casein gene upstream region. These gene constructs were transfected into rabbit primary mammary cells, or cotransfected in CHO cells with the plasmid coding for the rabbit mammary receptor (PRL-R). A regulatory region has been located between nt -3768 and -3155. This region enhances the prolactin induced promoter activity of the alpha s1-casein gene. It might possess or cooperate with prolactin responsive elements located further downstream in the alpha s1-casein gene.
Mol Cell Endocrinol 1992 Sep
PMID:A distal region enhances the prolactin induced promoter activity of the rabbit alpha s1-casein gene. 144 87

Previous studies have shown that basic fibroblast growth factor (bFGF) can modulate basal and luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated Leydig cell functions. It has not been ascertained whether these actions are due to direct or indirect effects on Leydig cells. To resolve this question, a multi-step procedure was used to isolate highly-purified Leydig cells from immature rats. 125I-bFGF binding studies were performed on cultured cells. Scatchard analysis of the data indicated a single binding site with an apparent Kd of 82 pM and a binding capacity of approximately 2800 sites per cell. Both bFGF and acidic FGF similarly were effective in displacing 125I-bFGF, suggesting that the receptor binds both bFGF and aFGF. However, neither hCG, follicle-stimulating hormone (FSH), insulin, insulin-like growth factor-1 (IGF-1), prolactin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) were effective competitors. When binding studies were conducted on cultured testicular interstitial cellular fractions that are normally discarded during Leydig cell purification, bFGF receptors were identified in these fractions. These results demonstrate that bFGF can have direct effects on Leydig cells through specific receptors; however, because other interstitial cell type(s) also have bFGF receptors, they stress the importance of using highly purified cells when evaluating bFGF actions on Leydig cells.
Mol Cell Endocrinol 1992 Oct
PMID:Evidence for basic fibroblast growth factor receptors in cultured immature Leydig cells. 145 39

A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1.2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.
J Mol Endocrinol 1992 Dec
PMID:Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries. 147 15

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11-33), the loop between the first disulfide bond and helix 2 (residues 54-74), the beginning of helix 3 (residues 109-112), the carboxyl half of helix 4 (residues 167-182), and the final carboxyl terminus segment of the molecule (residues 184-191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54-74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.
Mol Immunol 1992 Sep
PMID:Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis. 149 98

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.
Mol Biol Cell 1992 Jun
PMID:A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression. 149 70

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.
Mol Cell Biol 1992 Sep
PMID:Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation. 150 91

In GH4C1 rat pituitary cells, cell swelling stimulates prolactin (PRL) secretion by increasing Ca2+ influx through nifedipine-sensitive Ca2+ channels; however, the mechanism by which cell swelling opens Ca2+ channels is still unclear. To evaluate the role of protein kinase C (PKC) in this phenomenon, we studied the effect of down-regulating PKC by 12-h pretreatment with phorbol ester or by treatment with H-7, a protein kinase C inhibitor. Cell swelling induced by either 27% medium hyposmolarity or 80 mM isotonic urea caused a prompt rise in both [Ca2+]i and PRL secretion in otherwise untreated control GH4C1 cells. Removal of medium Ca2+ enhanced the osmotically induced cell swelling but prevented the increase in [Ca2+]i and PRL secretion. Both PKC down-regulation and H-7 suppressed the cell swelling-induced increases in [Ca2+]i concentration and PRL secretion, although they enhanced the induced cell volume expansion. Our data indicate that in GH4C1 cells PKC plays an important positive modulating role in the osmotic opening of plasmalemma Ca2+ channels, a critical component of the early transduction chain by which cell swelling causes PRL secretion in tumor-derived clonal pituitary cells.
Mol Cell Endocrinol 1992 Aug
PMID:Protein kinase C modulates cell swelling-induced Ca2+ influx and prolactin secretion in GH4C1 cells. 151 83

Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5' non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5'-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16 kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.
J Mol Endocrinol 1992 Apr
PMID:Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity. 151 20


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