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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of gonadotropin-releasing hormone and its receptor is a critical event in the endocrine regulation of reproduction. We have recently cloned the gene encoding for the human gonadotropin-releasing hormone receptor (hGnRHR). Partial sequence analysis revealed a structural organization consisting of three exons and two introns. Exon II contains only 219 bp and the remainder of the approximately 5 kb transcript is distributed between exons I and III. The complete coding region for the hGnRHR represented only 987 bp leaving an extensive 5' and 3' non-translated region and potentially additional exons unaccounted for. This report provides the complete sequence of exon I and III and demonstrates that further exons are unlikely to be contained within this gene. Sequencing of the 5' end of the gene revealed the presence of five consensus TATA sequences distributed within a 700 nucleotide region. Primer extension analysis detected multiple transcription initiation sites associated with this cluster of TATA sequences. Transcription of this region up to the most 5' initiation site was demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. The 5' non-translated region stretches between 703 and 1393 bp, depending on which initiation site is used. Several consensus cis-acting regulatory sequences were identified within the 5' end. These include, among others, sites for PEA-3, AP-1, and Pit-1. In addition, cAMP response element (CRE)-like and glucocorticoid/progesterone response element (GRE/PRE)-like sequences were found.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1995 Feb
PMID:The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals. 776 23

We have engineered GH3 cells with reporter genes under control of the growth hormone and prolactin promoters and measured protein production. The results indicate very low level production of reporter proteins from the cells regardless of the promoter used to drive expression. This was surprising in light of the observation that the cells still produced high levels of endogenous growth hormone and prolactin. Chinese hamster ovary (CHO) cells were engineered to express the Pit-1 transactivator. Transfection of reporter genes under control of the prolactin promoter demonstrated a clear enhancement of expression levels compared to the same promoter in parental CHO cells. Pit-1 expression is not sufficient, however, for high level, stable expression from the growth hormone promoter. These results indicate that the growth hormone and prolactin promoters are not sufficient for high level, stable expression even in normally permissive cells and suggest that Pit-1 alone is not sufficient for strong promoter activity from the integrated plasmids.
Mol Cell Endocrinol 1995 Feb
PMID:Endogenous and exogenous pituitary-specific promoters are differentially controlled. 776 27

The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH4C1 cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Oct
PMID:A sequence in the rat Pit-1 gene promoter confers synergistic activation by glucocorticoids and protein kinase-C. 785 49

The ability of Pit-1 to mediate transcriptional responses to cAMP has been explored. To test the ability of Pit-1 to mediate transcriptional responses to cAMP, an expression vector was prepared for a mutant Pit-1 in which the major sites of phosphorylation by the cAMP-dependent protein kinase were eliminated. Before using the mutant Pit-1 to study transcriptional regulation, we first examined the ability of the protein to be phosphorylated in vivo in response to cAMP. Transfection and in vivo labeling experiments confirmed that the mutant Pit-1 did not support cAMP-inducible phosphorylation. The ability of the wild type or mutant Pit-1 to mediate transcriptional responses to cAMP was assessed in cotransfection experiments using reporter genes containing either the proximal region of the rat PRL gene or seven copies of a Pit-1 binding site placed upstream of a minimal promoter. Surprisingly, the wild type and mutant Pit-1 expression vectors supported similar responses to cAMP. To further assess the ability of Pit-1 to mediate responses to cAMP, a GAL4-Pit-1 fusion gene was prepared. Although a GAL4-cAMP response element binding protein fusion gene was found to permit transcriptional responses to cAMP, the GAL4-Pit-1 gene was unresponsive. These findings demonstrate that although Pit-1 can facilitate the ability of the PRL promoter to respond to cAMP, phosphorylation of Pit-1 is not required for this response. It seems likely that additional factors that interact with Pit-1 binding sites are important for mediating transcriptional responses to cAMP.
Mol Endocrinol 1994 Nov
PMID:Pit-1 binding sites mediate transcriptional responses to cyclic adenosine 3',5'-monophosphate through a mechanism that does not require inducible phosphorylation of Pit-1. 787 13

The transcription factor Pit-1 has been shown to be important for both the developmental and homeostatic regulation of expression of the PRL and GH genes in pituitary cells. However, little is known about possible covalent modifications in Pit-1 that might mediate its transactivational properties. Previous studies showing that Pit-1 is a phosphorylation substrate for either protein kinase A or C, or their cellular inducers, led us to investigate whether phosphorylation of Pit-1 is required for its function in either basal or induced cellular activity of either the PRL or GH promoters. The transactivational properties of wild type Pit-1 were compared with those of Pit-1(A3), mutated in the three known phosphorylation sites. At saturating levels of Pit-1 expression vectors, activation of transient basal expression in HeLa cells of constructs (-1957)PRL-CAT or (-244)GH-CAT by RSV-Pit-1(A3) was, respectively, about 50% and 65% as strong as by RSV-Pit-1. Hence, phosphorylation at the sites mutated in Pit-1(A3) is not critically required for basal transactivation of either promoter but may modulate this activity. RSV-Pit-1 and RSV-Pit-1(A3) were equally effective in mediating estrogen receptor stimulation of (-1957)PRL-CAT expression in HeLa cells, thus revealing no phosphorylation requirement for the prerequisite for Pit-1 in estrogen receptor action on the PRL estrogen response element.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Nov
PMID:A Pit-1 phosphorylation mutant can mediate both basal and induced prolactin and growth hormone promoter activity. 787 13

Pit-1 is a pituitary-specific transcription factor with protein expression limited to thyrotrope, somatotrope, and lactotrope cells of the anterior pituitary gland. We have recently described a thyrotrope-specific variant isoform of Pit-1, called Pit-1T, which contains an additional 14 amino acids in the activation domain generated by an alternate 3'-splicing choice. Pit-1T, in the presence of Pit-1, stimulates the thyrotropin beta-subunit (TSH beta) promoter in a thyrotrope-derived cell that lacks all Pit-1 isoform proteins. Three laboratories have identified another Pit-1 splice variant, called Pit-1 beta, which contains an additional 26 amino acids in the activation domain that is generated by a similar 3'-alternate splice choice. Pit-1 beta has been shown to stimulate the GH promoter, but not the PRL or TSH beta promoters. In this report, we evaluate the effect of the three Pit-1 isoforms (Pit-1, Pit-1T, and Pit-1 beta) on the GH, PRL, and TSH beta promoters when introduced into different cell types. The combination of Pit-1 and Pit-1T had a synergistic stimulatory effect on the TSH beta promoter, but not on the PRL or GH promoters in a thyrotrope-derived cell line that lacks all Pit-1 protein isoforms (alpha TSH cells). When added to GH3 cells, which lack only the Pit-1T isoform, Pit-1T selectively stimulated the TSH beta promoter and not the GH or PRL promoters, suggesting that the thyrotrope-specific Pit-1T exhibits a promoter-specific effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Nov
PMID:The combination of Pit-1 and Pit-1T have a synergistic stimulatory effect on the thyrotropin beta-subunit promoter but not the growth hormone or prolactin promoters. 787 26

Expression of the human PRL (hPRL) gene in extrapituitary sites such as the uterus (decidualized endometrial stroma and myometrium) and cells of the hematopoietic lineage is directed by an alternative promoter which is located approximately 6 kilobases (kb) upstream of the pituitary-specific start site. In order to delineate the tissue-specific mechanisms governing the control of nonpituitary PRL gene expression, we have cloned and sequenced 3 kb 5'-flanking DNA of the upstream decidual/lymphoid (dPRL) promoter. Based on sequence homology we identified two binding motifs for Pit-1 and seven half-sites for glucocorticoid receptor/progesterone receptor (PR) binding. We focused our studies on the role of Pit-1 and of PR as potential transcriptional regulators, since the POU domain protein Pit-1 is essential in the control of pituitary PRL expression, and progesterone induces decidual transformation of the endometrial stroma, a differentiation process during which the decidual PRL gene is activated. We demonstrate in a variety of cell types, including lymphocytes and endometrial stroma, that Pit-1 is not involved in the regulation of dPRL promoter/reporter gene constructs carrying 3 kb 5'-flanking DNA. Our experiments also show that activated PR does not confer direct transcriptional control on the dPRL promoter. When we compared the activity of the transfected dPRL promoter in PRL-secreting and nonsecreting lymphoid cells, we found that the 3 kb 5'-flanking region of the dPRL promoter did not contain elements restricting expression to only those lymphocytes that produce PRL but allowed expression of fusion reporter genes irrespective of the status of the endogenous PRL gene. This was in sharp contrast to endometrial cells where 3 kb 5'-flanking DNA conferred strong transcriptional activation on the dPRL promoter in decidualized endometrial stromal cells actively secreting PRL, but did not allow transcription in undifferentiated non-PRL-secreting endometrial stromal cells. Activation of the dPRL promoter construct in these undifferentiated cells could however be induced by the addition of cAMP, in the absence of progesterone, suggesting that a signal transduced through the cAMP signaling pathway is a primary inducer of decidual PRL gene expression.
Mol Endocrinol 1994 Mar
PMID:Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma. 801 53

The pituitary-specific transcription factor Pit-1 is required for expression of the PRL gene. Transcription of the PRL gene in the anterior pituitary is both activated and repressed in response to neuroendocrine signals. The molecular events that mediate repression are unknown. Transplantation of GH3 pituitary tumor cells from culture to female Wistar-Furth rats resulted in repression of PRL gene expression. When the transplanted cells were returned to culture, PRL gene expression was rapidly activated. We used this model to study potential mechanisms by which PRL gene expression was silenced. In addition to the appropriate size Pit-1 proteins of 33 and 31 kilodaltons, smaller forms of the transcription factor, migrating at approximately 27 and 24 kilodaltons, were found in transplanted cells in which PRL gene expression was repressed. These smaller forms of Pit-1 protein were observed to disappear when transplanted cells were returned to culture, coincident with the activation of PRL gene expression. A transcript approximately 170 base pairs shorter than expected for that encoding full-length Pit-1 was detected in transplanted GH3 cell RNA by polymerase chain reaction. The shorter Pit-1 transcript was abundant only in GH3 cells after in vivo passage and was not readily detected in transplanted cells after as little as 12 h in culture. This shorter transcript was found to result from excision of sequence corresponding to exon IV and encodes a Pit-1 protein lacking 54 amino acids of the POU-specific domain. Gene transfer studies demonstrated this alternative form of Pit-1 inhibited PRL promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Mar
PMID:An alternatively spliced form of Pit-1 represses prolactin gene expression. 801 54

We have identified a form of the pituitary-specific POU protein Pit-1 that results from deletion of the POU-specific (POUs) domain by alternative RNA splicing. This natural variant of Pit-1 (called delta 4Pit-1) has revealed several aspects of the function of the POUs domain. The delta 4Pit-1 protein was characterized using a delta 4Pit-1-specific antiserum. Further, selection assays of random oligonucleotide pools identified binding site preferences for both wild type and delta 4Pit-1. Methylation interference, copper phenanthrolene, and missing contact analyses were used to compare the binding characteristics of the two forms of Pit-1 on a selected site. DNA binding affinity assays on several DNA elements revealed that the POUs domain contains a modular DNA binding activity affecting the DNA binding affinity of the entire POU domain on some, but not on other, DNA sites. Functional analysis on such DNA elements has revealed that the POUs domain is an essential, but nonmodular, component of the Pit-1 trans-activation domain dependent on its natural context within the Pit-1 protein.
Mol Endocrinol 1993 Dec
PMID:An alternative Pit-1 RNA splicing product reveals modular binding and nonmodular transcriptional activities of the POU-specific domain. 814 62

Pit-1, a member of the POU family of homeo-domain transcription factors, activates prolactin and GH gene expression but also has a role in pituitary cell differentiation and proliferation. Expression of Pit-1 may therefore be of central importance in the function and phenotype of human pituitary adenomas. We have found evidence that, in addition to Pit-1 mRNA, Pit-1-like immunoreactivity and DNA-binding activity are readily detectable in a series of human pituitary adenomas. Gel mobility shift assays using adenoma protein extracts with two Pit-1-binding sites from the human prolactin gene promoter demonstrated the formation of several DNA sequence-specific protein-DNA complexes; some of these could be accounted for by Oct-1-binding activity. Pit-1 activity was anticipated in prolactin- and GH-secreting adenomas, but was also detected in a proportion of endocrine-inactive (non-secreting) adenomas that did not express Pit-1 target genes. The data demonstrate the presence of Pit-1 in a range of pituitary adenomas. Different adenomas generated slightly differing patterns of DNA-binding activity, though Pit-1 mRNA and protein size appeared normal in all tumours so far examined.
J Mol Endocrinol 1993 Dec
PMID:Expression of Pit-1 and related proteins in diverse human pituitary adenomas. 814 36


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