The Luteinizing Hormone Receptor (LHR): Characterization of regulatory mechanisms revealed that the human LHR (hLHR) expression is subject to regulation by a complex network and the participation of various signaling cascades through its promoter region at both genetic and epigenetic levels. The histone deacetylase HDAC1/2/mSin3A complex was shown to be recruited to the proximal Sp1 binding site to elicit chromatin-localized condensation and silencing of the LHR gene. The status of DNA methylation and histone acetylation in the LHR promoter region operates coordinately to orchestrate the LHR level. PI3K/PKCzeta signaling pathway was shown to be critical for trichostatin A (TSA)-induced LHR transcriptional activation through phosphorylation of Sp1 at Ser641 and release of p107, which interacts with Sp1. LRH transcriptional activation is initiated by changes in chromatin structure induced by TSA that causes the release of phosphatase PP2A in JAR cells or of PP1 in MCF-7 cells, which is associated with Sp1 directly or through HDAC 1/2, respectively, at the promoter. Other recent studies also demonstrated a critical role of the PKCalpha/Erk cascade in PMA-induced activation of LHR gene through Sp1 phosphorylation (other than Ser641 site), which causes release of the HDAC1/mSin3A repressor complex anchored at Sp1 sites. However, HDAC2 from the complex did not dissociate from the promoter, indicating the differential role of HDAC1/HDAC2. The phosphorylation induced by PKCalpha/ERK signaling disrupts the interaction between Sp1 and HDAC1 and, this causes release of HDAC1/mSin3A complex to activate hLHR. This is consistent with our finding that mSin3A functions as corepressor for HDAC1 but not HDAC2 in the regulation of LHR gene expression. In other studies we investigated the participation of PC4, a potential coactivator of Sp1, in the transcriptional control of LHR expression. Knockdown of PC4 expression with its specific siRNA significantly reduced LHR promoter activity in MCF-7 and JAR cancer cells. PC4 synergistically enhanced Sp1-activated gene expression, indicating a direct or indirect interaction of PC4 and Sp1. These studies have demonstrated that PC4 acts as a coactivator of Sp1 to modulate LHR transcription. Gonadotropin regulated genes: 1)Gonadotropin-regulated long chain fatty acid Acyl (GR-LACS), identified in our laboratory, is a member of the LACS family regulated by LH/hCG in the rat Leydig cell (LC). Its mouse/human homologs, lipidosin/bubblegum, have been suggested by others to participate in X-linked adrenoleukodystrophy (X-ALD), a disorder with accumulation of very Long Chain FA (VLCFA) in tissues and blood. A GRLACS/lipidosin null mice generated in our laboratory did not support its association with X-ALD. The studies revealed a role of GR-LACS in reducing the aging process of the LC, and its participation in gonadotropin-induced testicular desensitization of testosterone production that is observed in the adult animals. 2) Gonadotropin Regulated Testicular Helicase (GRTH/Ddx25), a multifunctional enzyme discovered in our laboratory, is an essential regulator of sperm maturation. GRTH null mice are sterile due to spermatid arrest and failure to elongate. Through its association with CRM-1, GRTH participates in the export of selected messages relevant to the progress of spermatogenesis from the nuclear to cytoplamic sites, including the Chromatoid Body (CB) of spermatids (equivalent to somatic P bodies, contains members of the RNA-induced silencing complex RISC-complex and is viewed as a storage/processing site of mRNAs during spermatogenesis) and polyribosomes where it participates in translation of proteins that are essential for spermatogenesis. Our recent studies investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the structure of the CB of spermatocytes in cell culture using immunofluorescence and electron microoscopy studies. GRTH resides in the nucleus, the cytoplasm and the CB. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. On other hand, proteins of the RISC complex that do not participate in mRNA transport and reside in the CB and cytoplasm were excluded from the CB and accumulated in the cytoplasm upon treatment with either inhibitor. This also occurred in spermatids of KO mice. The CB is changed from a large lobular-filamentous to a small condensed structure after treatment, resembling the CB of the GRTH-KO. GRTH did not interact with RISC members. Due to its export/transport function, GRTH is essential for the transport of messages to the CB, which governs the CB structure in spermatids, and to maintain systems that participate in mRNA storage and their processing during spermatogenesis. Prolactin receptor (PRLR): -Structure function: Our early studies demonstrated 1) the presence of long (LF) and short forms (SF) of PRL;2) the formation of homo- and heterodimers of these forms in the absence of hormone and the action of PRL as a conformational modifier that induces activation of the JAK2/STAT5 signaling through LF and of JAK2 signalling through SF;3) The short form of the PRLR (S1b), with an abbreviated cytoplasmic tail silences PRL-induced activation of gene transcription by the long-form. Mutation of any one of the two paired cysteines in S1b (S1bx) (residing in extracellular subdomain 1- D1) eliminated the inhibitory action of S1b. The constitutive JAK2 phosphorylation observed in S1b was not present in cells expressing S1bx, and JAK2 association (intracellular conserved proline-rich domain at the Box1 docking site for JAK2) was disrupted. BRET50 showed decreased LF/S1bx heterodimeric association and increased affinity in S1bx homodimerization, favouring dimerization and PRLR-induced signaling. Computer modeling based on PRLR crystal structure (extracellular) showed that minor changes in the tertiary structure of D1 upon S-S disruption propagated to the quaternary structure of the homodimer, affecting the dimerization interface. Wild- type dimers were stabilized only through interactions between H-bonds in D2 subdomains, while the mutant developed additional H-bonds that bridged D1s. These changes explain the higher homodimerization affinity of the mutant (S1bx) and provide the structural basis for its lack of inhibitory function. The PRLR conformation as stabilized by S-S bonds is required for the inhibitory action of S1b on PRL-induced LF-mediated function and JAK2 association/activity. -Transcriptional studies: Transcription of the human PRLR is governed by an estradiol (E2)/estrogen receptor-alpha mechanism independent of a estrogen responsive element. E2/ER-alpha through complex formation with SP1 and C/EBPbeta that associate to cognate elements leads to coactivator assembly and recruitment of TFIIB and Pol II. We have initiated the characterization of the physical interaction domains involved in the complex association among transfactors. This study provides evidence for direct association of components of the complex essential for PRLR transcription through defined interaction domains for the individual proteins.
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