The
Section on Molecular Endocrinology studies the structure-function properties
of gonadotropin and prolactin receptors, and the regulation of their
expression by homologous and heterologous hormones. The hormonal control
of gonadal function, including steroidogenic enzymes of the androgen
pathway and novel genes regulated by gonadotropins, is also investigated.
This work includes studies on the regulation of transcription of the
luteninizing hormone (LH) receptor and its expression in the testis
and ovary development. Current research includes studies on the mechanisms
of inhibition and stimulation of transcription of the LH receptor by
orphan receptors (EAR2, EAR3/COUP-TFI and TR4); the differences between
species; and the modulatory changes that occur during gonadotropin-induced
maturation of cultured granulosa cells and in Leydig cells at prepubertal
and pubertal stages. Other studies are concerned with the regulation
of the LH receptor and steroidogenic enzyme genes by gonadotropin, steroids
and second messengers, or by novel genes at the transcriptional level.
Recent
studies utilizing differential display analysis have identified a novel
gene, termed GRTH-DDX25 (Gonadotropin Regulated Testicular Helicase)
that is transcriptionally regulated by gonadotopin. This gene is a member
of the family of RNA helicases and is specifically expressed in Leydig
cells and meiotic cells (pachytene spermatocytes). GRTH shares conserved
core domains with all other members of the DEAD-box protein family of
RNA helicases. Apart from its genetic closeness to mouse DBP5 protein
(50% aa identity), the N-and C-terminal extensions of GRTH from the
signature sequences show little similarity to other members of the family.GRTH
is markedly up-regulated by hCG via cAMP-induced androgen formation
in Leydig cells, at hormone concentrations that cause down-regulation
of LH receptors and steroidogenic enzymes. Androgen produced by gonadotropin
stimulation exerts intracrine/autocrine actions on GRTH, and could influence
its transcription within the seminiferous tubule. GRTH functions as
a transcriptional activator, and could contribute to the control of
steroidogenesis, including the restoration of down-regulated functions.
It could also mediate paracrine regulation of androgen-dependent gene(s)
involved in the meiotic process, and may thus have a role in spermatogenesis.
Current studies are addressing the androgen control of the GRTH gene
at the transcriptional level. Also, a previously unidentified protein
that is constitutively present in Leydig cells and down-regulated by
gonadotropin was recently cloned and characterized as a gonadotropin-regulated
long chain acyl CoA synthetase (GRLACs). This protein, which is expressed
in the pubertal and adult Leydig cells of the rat testis and shares
sequence indentity with two conserved regions of the LACS and luciferase
families, displays low overall amino acid similarities with other members
of the LACS family (23-28%). The expressed GR-LACS protein present in
the cytoplasm of transfected cells displayed acyl-CoA synthetase activity
for long chain fatty acid substrates. In addition to the potential contributions
to energy production and testicular steoidogenesis, GR-LACS could provide
long-chain acyl-CoA esters with regulatory effects on enzyme ativity,
membrane function and gene expression. The function of GRTH and GR-LACS
will also be analyzed by the development of a null mouse model to evaluate
their roles in steroidogenesis and spermatogenesis.
Research
on the prolactin receptor has focused on its molecular structure and
the regulation of its transcription. Recent studies have included the
characterization of the upstream domains that drive transcription of
the receptor gene, and the identification of several independent promoters.
These include three in the rat -- one rat and gonad-specific, and five
in the human -- four
human-specific and one generic cross-species promoter, that are differentially
regulated. Further studies will analyze the control of prolactin receptor
gene transcription through its individual promoters. Two novel short
forms of the human prolactin receptor were recently found to have a
truncated cytoplasmic domain with unique C-termini that differ from
the short forms previously isolated in rodents. Both of the short human
species act as dominant negative forms for the long form of the receptor.
Ongoing studies include the evaluation of signal transduction pathways
involved in cellular actions of prolactin that are mediated by these
novel isoforms. The specific types of kinase activities that participate
in down-stream signaling, the identification of adapter sequences, and
the role of complex formation during receptor activation, are also under
investigation. In addition, the role of transactivation mechanism(s)
in the actions of prolactin through the short human receptor forms will
be evaluated.
