Regulation of pituitary-specific gene expression by constitutively active G proteins.
Item
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Title
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Regulation of pituitary-specific gene expression by constitutively active G proteins.
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Identifier
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AAI9530922
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identifier
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9530922
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Creator
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Tian, Jun.
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Contributor
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Adviser: Carter Bancroft
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Date
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1995
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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 | Biology, Animal Physiology | Biology, Genetics | Chemistry, Biochemistry
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Abstract
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The G proteins are a family of important intracellular signal transducers that play an essential role in linking diverse cell surface receptors to different intracellular signaling systems. Although the functions of G proteins in regulating many cellular functions has been intensely studied, virtually nothing is known about the role of G proteins in gene transcriptional regulation.;Transfection experiments with rat pituitary GH3 cells were employed to show that expression of a constitutively active mutant of an {dollar}\alpha{dollar}s subunit ({dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}){dollar} specifically stimulates expression of a co-transfected prolactin (PRL) gene promoter. Two copies of site 1P (Pit-1 binding site in prolactin promoter) direct a response to {dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} stimulation, implying that site 1P can serve as an independent cis response element for {dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} and that Pit-1 may be the transcription factor that mediates the {dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} action on the PRL promoter. Further studies of the intracellular signaling pathways through which {dollar}\alpha{lcub}\rm s{rcub}{dollar} stimulated PRL gene expression implicated protein kinase A (PKA), since co-expression of a dominant negative inhibitor of PKA blocked the {dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} stimulation of the PRL promoter activity. Similarly, a dominant negative inhibitor of CREB, k-CREB, also inhibited {dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} induction of the PRL promoter. In addition, CREB bound with low affinity to the CLE, a CRE-like element in the PRL proximal promoter which was shown previously to be required for basal expression and cAMP induction of the PRL promoter, implying that PKA action on the PRL promoter may be mediated by CREB.;Gq is generally thought to couple to TRH receptor whose activation stimulates PRL gene expression. To investigate the function of {dollar}\alpha{lcub}\rm q{rcub}{dollar} in regulating PRL gene expression, a constitutively active mutant of an {dollar}\alpha{dollar}q subunit ({dollar}\alpha{lcub}\rm q{rcub}\sp{lcub}*{rcub}){dollar} was used as described above. G-{dollar}\alpha{lcub}\rm q{rcub}\sp{lcub}*{rcub}{dollar} specifically stimulated the expression of a co-transfected prolactin or growth hormone gene promoter. Site 1P was observed to function as a heterlogous {dollar}\alpha{dollar}q response element. G-{dollar}\alpha{lcub}\rm q{rcub}\sp{lcub}*{rcub}{dollar} stimulation of PRL promoter activity was strongly inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, indicating that Raf may be involved in mediating the stimulation by {dollar}\alpha{dollar}q of the PRL gene.;To expand our understanding of the G-{dollar}\alpha{dollar}s regulation of gene expression, the action of constitutively active {dollar}\alpha{dollar}s on Pit-1 gene promoter was investigated. G-{dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} stimulated Pit-1 gene promoter activity. Mutation of the Pit-1 binding site in the Pit-1 gene promoter reduced the stimulation. Additionally, both the dominant negative PKA and CREB inhibitors described above inhibited {dollar}\alpha{lcub}\rm s{rcub}\sp{lcub}*{rcub}{dollar} stimulation of the Pit-1 gene promoter activity. These data suggest that the activated {dollar}\alpha{dollar}s subunit may employ PKA, CREB, and the Pit-1 binding site to stimulate Pit-1 gene promoter activity.;In addition to G protein {dollar}\alpha{dollar} subunits, the {dollar}\beta\gamma{dollar} dimer complex was recently recognized to be able to regulate cellular functions in higher eukaryotes. I found that {dollar}\gamma2,{dollar} but not {dollar}\beta2\ {lcub}\rm or{rcub}\ \beta2+\gamma2{dollar}, regulated the prolactin gene promoter activity. This study represents both the first study of transcriptional action of {dollar}\beta\gamma{dollar} in higher organisms, and the first indication of a independent function of the G protein {dollar}\gamma{dollar} subunit. In addition, a non-prenylated {dollar}\gamma2{dollar} mutant that was incapable of membrane targeting did not stimulate PRL promoter activity, indicating that the prenylated site of {dollar}\gamma2{dollar} is necessary for its transcriptional action.
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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.
