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J. Biol. Chem., Vol. 268, Issue 6, 3903-3910, 02, 1993
WE Schoderbek, MS Roberson and RA Maurer
Transient transfection studies using gonadotrope-derived, alpha T3-1 cells
were used to determine the DNA sequences of the mouse glycoprotein hormone
alpha-subunit gene that mediate the transcriptional response to
gonadotropin releasing hormone (GnRH). The roles of phorbol esters and
cyclic AMP in mediating the GnRH response were also investigated. The
initial studies demonstrated that a construct containing approximately 500
base pairs of alpha-subunit flanking sequence was sufficient to mediate
responses to a GnRH agonist (GnRHa), phorbol myristate acetate (PMA) and a
cAMP analog. Responses to combinations of cAMP and GnRHa or cAMP and PMA
were approximately additive, whereas the response to the combination of
GnRHa and PMA was similar to that seen with either of the agents alone.
Cotransfection studies with an expression vector for the heat-stable
inhibitor of the cAMP-dependent protein kinase demonstrated that GnRHa and
PMA responses are not dependent on the cAMP-dependent kinase. Deletion
analysis indicated that sequences between -507 and -205 were involved in
mediating responses to GnRHa and PMA. To determine if this region alone
could support responses to these agents, the -507 to -205 region was linked
to a minimal promoter and tested in transient transfections. The results
demonstrated that this region supports responses to GnRHa, PMA, and cAMP.
Clustered point mutations of this region were used to further characterize
sequences involved in the GnRH response. Mutations in two regions, one at
positions -406 to -399 and one at positions -337 to - 330, resulted in
decreased responses to GnRH and PMA. There is no obvious sequence
similarity between the two regions that are required for the GnRH response.
An enhancer test demonstrated that multimers of the -416 to -385 region
were able to function as a GnRH-responsive element when linked to a minimal
promoter, although a single copy of this region was not sufficient to
permit a response to GnRH. In contrast, multimers of the -344 to -300
region did not permit a response to GnRH, but enhanced basal transcription.
These findings are consistent with the identification of a two-component
GnRH response unit, which probably involves the functional cooperation of
two different transcription factors. The observation that GnRH
responsiveness appears to co-localize with PMA responsiveness suggests that
GnRH effects on the alpha-subunit transcription are likely mediated by the
protein kinase C pathway.
Two different DNA elements mediate gonadotropin releasing hormone effects on expression of the glycoprotein hormone alpha-subunit gene
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
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