REGULATION OF PROLACTIN GENE EXPRESSION

The overall goal is to understand the molecular events involved in
the tissue-specific, regulated expression of the prolactin gene.
The prolactin gene is regulated at the transcriptional level by a
number of hormones including estradiol, epidermal growth factor,
thyrotropin releasing hormone and dopamine. The proposed studied
seek to examine the mechanisms which permit the multihormonal
regulation of prolactin transcription. In particular attention
will be focused on intracellular regulators of prolactin gene
transcription, DNA sequences which are important for prolactin gene
transcription and on DNA-binding factors which interact with these
sequences. The specific aims are approaches include: 1) The role
of the catalytic subunit of the cAMP-dependent protein kinase in
mediating cAMP effects on prolactin gene transcription will be
examined by using a synthetic gene coding for the heat stable
inhibitor of the kinase 2) Structural features of the estrogen
response element of the rat prolactin gene which are necessary for
binding the estradiol receptor and for stimulation of transcription
will be examined through construction of synthetic estrogen
response elements and mutagenesis of the response element 3)
Features of the DNA binding domain of the estrogen receptor which
are important for interaction with the prolactin estrogen response
element will be explored through mutagenesis of the cloned receptor
cDNA. 4) Linker scanning mutagenesis will be used to dissect the
role of specific DNA sequences in the immediate 5' flanking region
and an upstream enhancer/estrogen response element of the prolactin
gene 5) DNA binding proteins which interact with specific prolactin
gene sequences will be identified by mobility shift assay, purified
by affinity chromatography and cDNAs cloned. These studies should
provide increased understanding of the general mechanisms involved
in the regulation of gene expression as well as the specific
regulation of this physiologically important hormone.