Transcription of the rat prolactin gene: Regulation by dopamine and the pituitary-specific protein Pit-1.
Item
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Title
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Transcription of the rat prolactin gene: Regulation by dopamine and the pituitary-specific protein Pit-1.
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Identifier
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AAI9510658
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identifier
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9510658
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Creator
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Fischberg, Daniel James.
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Contributor
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Adviser: Carter Bancroft
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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While dopamine is known to be the major tonic regulator of prolactin synthesis and secretion, little is known of the mechanism by which dopamine inhibits expression of the prolactin gene. A difficulty in studying dopaminergic repression of the prolactin gene has been the lack of an appropriate model system. Cloning of the D{dollar}\sb2{dollar} dopamine receptor has allowed the expression of this receptor in rat lactotrope cell lines which do not express the endogenous receptor such as the GH{dollar}\sb3{dollar} cell line. This system was used to address the mechanism by which dopamine represses transcription of the rat prolactin (rPRL) gene.;The ability of either isoform of the D{dollar}\sb2{dollar} receptor to mediate repression of the rPRL promoter was first confirmed using expression vectors containing cDNA's for the D{dollar}\sb{lcub}\rm 2A{rcub}{dollar} and D{dollar}\sb{lcub}\rm 2B{rcub}{dollar} receptors. It was then demonstrated that dopaminergic repression of the rPRL promoter in this system is mediated by one or more pertussis toxin-sensitive G proteins and, furthermore, that the proximal 187 base pairs of the prolactin promoter confer a full dopaminergic response. Over-expression of the catalytic subunit of the cAMP-dependent protein kinase (PKA) caused a nearly complete attenuation of dopaminergic repression, suggesting a possible role for PKA in mediating dopaminergic repression of the rPRL promoter.;The implication of both Pit-1 and PKA in the dopaminergic repression of the rPRL promoter led to studies described in Chapter 3 of the functional role of phosphorylation of Pit-1. The abilities of the wild type Pit-1 and a phosphorylation-deficient mutant to activate the rat prolactin and growth hormone (rGH) promoters were compared. The results of these experiments suggest that phosphorylation plays a modulatory role in regulating the activity of Pit-1 upon both the rPRL and rGH promoters. The magnitude of this modulatory role on the rPRL promoter is equivalent with the magnitude of dopaminergic repression of the rPRL promoter, consistent with the model that dopaminergic repression is mediated via inhibition of PKA-dependent phosphorylation of Pit-1.;In Chapter 4, the results of functional studies of isoforms of the pituitary-specific transcription factor Pit-1 are presented. While both Pit-1 and Pit-1a are capable of transactivating the rPRL and rGH promoters, {dollar}\Delta{dollar}4Pit-1, by itself, activates neither promoter. Additionally, the function of {dollar}\Delta{dollar}4Pit-1 as a repressor of the rPRL promoter has been confirmed in HeLa cells. Finally, preliminary evidence suggests that {dollar}\Delta{dollar}4Pit-1 may exert a permissive role in allowing expression of the rGH gene in somatomammotropes.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
