RECEPTORS AND ACTIONS OF PEPTIDE HORMONES AND REGULATORY PROTEINS IN ENDOCRINE MECHANISMS
, Head, Section on Molecular Endocrinology
Chon-Hwa Tsai-Morris, PhD, Staff Scientist
Ying Zhang, PhD, Research Associate
Juying Dong, PhD, Postdoctoral Fellow1
Yuji Maeda, MD, PhD, Postdoctoral Fellow1
Ravi K. Gutti, PhD, Visiting Fellow
Junghoon Kang, PhD, Visiting Fellow
Mingjuan Liao, PhD, Visiting Fellow
Aamer Qazi, PhD, Visiting Fellow1
Hisashi Sato, MD, PhD, Visiting Fellow
Yili Xie, PhD, Visiting Fellow
We investigate the molecular basis of peptide hormone control of gonadal function, with particular emphasis on the structure and regulation of the luteinizing hormone receptor (LHR) and prolactin receptor (PRLR) genes and on the regulatory mechanism(s) involved in the progress of spermatogenesis and control of Leydig cell function. Our studies focus on the regulation of human LHR gene transcription (epigenetic or via nuclear orphan receptors, DNA methylation, second messengers) as well as on the multiple-promoter control of hPRLR gene transcription. We are elucidating the function of two inhibitory short forms of the prolactin receptors and their relevance to physiological regulation and breast cancer. We also investigate novel gonadotropin-regulated genes of relevance to the progression of testicular gametogenesis, Leydig cell function, and other endocrine processes.
Epigenetic control of luteinizing hormone receptor transcription
We previously demonstrated that the phosphatidylinositol 3-kinase/PKCzeta (PI3K/PKCV) cascade plays an essential role in the regulation of LHR gene transcription in human choriocarcinoma JAR cells and MCF-7 breast cancer cells. PI3K/PKCV-mediated Sp1 phosphorylation accounts for derepression of the LHR gene transcription induced by the HDAC inhibitor Trichostatin A (TSA). Blockade of PI3K or PKCV activity by specific inhibitors, kinase-deficient mutants, or small interfering RNA abolished the marked activation of LHR gene expression by TSA and by TSA-elicited Sp1 phosphorylation at Ser641. We observed that PKCV associated with Sp1 and that the association was enhanced by TSA. Sp1 phosphorylation at Ser641 was required for release of the pRB homologue p107 protein from the LHR gene promoter while p107 acted as a repressor of LHR gene transcription. Inhibition of PKCV activity largely reduced Sp1 phosphorylation, which in turn blocked p107 release and activation of the LHR gene promoter by TSA. Collectively, our findings have revealed a novel mechanism of TSA-regulated gene expression through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCV-induced Sp1 phosphorylation–dependent manner. This activation required changes in chromatin structure as a consequence of histone acetylation but independent of DNA methylation status. Furthermore, our findings supported the participation of protein phosphatase(s) in the control of LHR gene transcription, whereby a coordinate balanced between PI3K/PKCV (constitutive and/or induced activity) and phosphatase(s) could be critical for up- or downregulation of LHR gene expression by its effect on Sp1 phosphorylation status.
Our recent and current studies have revealed an important contribution of serine/threonine protein phosphatases PP2A and PP1 to TSA-induced activation of LHR gene transcription in a cell type–specific manner. While PP2A exhibited significant binding to the silenced LHR gene promoter under basal conditions in JAR cells, we observed only minimal binding of PP1. In contrast, PP1 but not PP2A was associated with repression of the LHR gene in MCF-7 cells. Parallel to the derepression of LHR gene expression induced by TSA in these cells, the inhibitor caused dose-dependent release of PP2A from the LHR promoter in JAR cells and of PP1 in MCF-7 cells. Blockade of PP2A activity in JAR cells by okadaic acid synergistically enhanced the TSA effect while overexpression of the PP2A catalytic subunit largely prevented both the promoter induction and Sp1 phosphorylation elicited by TSA. We found that PP2A associated with Sp1 at both its N- and C-termini of Sp1, where the phosphorylation site (at 641), which is critical for the PI3K/PKCz-mediated Sp1 phosphorylation induced by TSA, is located. Taken together, these results demonstrated the critical involvement of PP2A and PP1 in the derepression of the LHR gene by TSA through regulation of the Sp1 phosphorylation level. Our findings have demonstrated that TSA-induced changes in chromatin structure cause a cell-specific release of a phosphatase (PP2A or PP1) that is associated, respectively, with Sp1 directly or through HDAC. The result is that the phosphorylation of Sp1 mediated by the PI3K/ PKCz pathway (constitutively active) is favored, which in turn causes release of the p107 inhibitor from SP1 and the marked transcriptional activation of hLHR.
Dufau ML, Tsai-Morris C-H. The luteinizing hormone receptor. Contemporary endocrinology. In: Payne A, Hardy M, eds. The Leydig Cell in Health and Disease. Humana Press, 2007;227-52.
Zhang Y, Fatima N, Dufau ML. Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol 2005;25:7929-39.
Zhang Y, Liao M, Dufau ML. Phosphatidylinositol 3-kinase/protein kinase C zeta-induced phosphorylation of Sp1 and p107 repressor release have a critical role in histone deacetylase inhibitor-mediated derepression of transcription of the luteinizing hormone receptor gene. Mol Cell Biol 2006;26:6748-61.
Gonadotropin-regulated testicular genes
We previously identified a novel Gonadotropin-Regulated Testicular Helicase (GRTH/Ddx25). This enzyme, which is present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids, binds to mRNA species as an integral component of messenger RNP particles, with storage in chromatoid bodies located in the cytoplasm of spermatids (Tsai-Morris et al., Proc Natl Acad Sci USA 2004;101:6373). GRTH-targeted null male mice are sterile owing to spermatid arrest at step 8 of spermatogenesis, with marked diminution of chromatoid bodies and failure to elongate. The transcription of messages in spermatid steps 1 through 8 of the mice was not altered, but their translation was selectively abrogated. Our current studies are defining the function of GRTH/Ddx25 as an RNA-binding protein as well as its storage and translational functions during sperm progression.
We demonstrated differential localization of two GRTH protein species in subcellular compartments of germ cells. The species are the 56 kDa form and the phosphorylated 61 kDa form, which are localized primarily in, respectively, the nucleus and cytoplasm of germ cells. The post-transcriptional modification appears to be involved in cytoplasmic-related events induced by protein kinase A at threonine residue(s). We recently provided evidence for dual functional roles of GRTH as a component of mRNP in RNA export from nucleus to cytoplasm and in the translation of specific RNA transcripts at specific stages in germ cell development. The 56 kDa nuclear species interacted with CRM1 and participated in mRNA transport. The phosphorylated cytoplasmic 61kDa associated with polyribosomes and selectively regulated the translation of specific genes. In addition to facilitating the initiation of translation of its target genes, phosphorylation of GRTH might induce conformational changes to recruit protein(s) so as to allow access of mRNAs to chromatoid bodies for storage and/or degradation (Figure 5.2).
Figure 5.2
Model of GRTH action in male germ cell development. After translation in germ cells, the 56 kDa GRTH species is transported into the nucleus and selectively binds to a subset of nuclear RNAs as a component of mRNP complexes. Specific mRNAs associated with GRTH include PGK2, tACE, TP1 and TP2, and others. The unphosphorylated GRTH-bound mRNP particles are transported from the nucleus to the cytoplasm of germ cells via the CRM1 exporting signal pathway, where GRTH is phosphorylated at Thr residue(s). The 61 kDa phosphorylated GRTH species, through its interaction with actively elongating polyribosomes, presumably regulates target gene translation and could participate in the transport of mRNPs in and out of the chromatoid body, in the silencing and/or storage of genes in the chromatoid body, and/or in the degradation of messages through participation in the small interfering RNA pathway. Messages are released from the chromatoid body for translation at specific times during spermatogenesis.
We identified the N-terminal leucine-rich region as the nuclear export signal that participates in the CRM1-dependent nuclear export pathway. We identified a 14–amino acid GRTH sequence at residues 100–114 as the nuclear localization signal. GRTH selectively regulated the translation of specific genes, including histone 4 [H4] and high-mobility group protein [HMG2], in germ cells. In addition, GRTH participated in the nuclear export of RNA messages, including phosphoglycerate kinase 2 [PGK2], testicular angiotensin-converting enzyme (tACE), and transition proteins 1 and 2 [TP1 and TP2]. The cytoplasmic levels of these proteins are markedly reduced in GRTH null mice. Our studies demonstrated that GRTH/Ddx25 is a multifunctional RNA helicase that is an essential regulator of sperm maturation.
In collaboration with Eitsu Koh and Mikio Namiki, we identified two polymorphic forms of GRTH by genetic screening of GRTH/DDX25 in 100 fertile and 143 infertile Japanese men with non-obstructive azoospermia. We found heterozygous mutations in exon 8 (mis-sense) and exon 11 (sense). We identified the mis-sense mutation Arg242 His in exon 8 in 5.8 percent of infertile patients and 1 percent of normal subjects; we observed the silent mutation in 2 percent of infertile patients. Even though the mutant protein was efficiently expressed in COS-1 cells, only the 56 kDa nuclear/cytoplasmic non-phosphorylated species was present, whereas the 61 kDa species was absent.
Our findings highlight the relevance of the R242 residue for the post-transcriptional modification leading to generation of the 61 kDa species. The mis-sense mutation of the GRTH gene associated with expression of a protein of reduced basicity and the absence of the phospho-GRTH species could be relevant to some of the functional aspects of the protein that influence germ cell development and/or function.
Dufau ML, Tsai Morris C-H. Gonadotropin-regulated testicular helicase (GRTH/DDX25) an essential regulator of spermatogenesis. Trends Endocrinol Metab 2007;18:314-20.
Li J, Sheng Y, Tang P-Z, Tsai-Morris C-H, Dufau ML. Tissue-cell- and species-specific expression of gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) in gonads, adrenal and brain. Identification of novel forms in the brain. J Steroid Biochem Mol Biol 2006;98:207-17.
Sheng Y, Tsai-Morris C-H, Gutti, R, Maeda Y, Dufau ML. Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is a transport protein involved in gene-specific mRNA export and protein translation during spermatogenesis. J Biol Chem 2006;281:35048-56.
Tsai-Morris C-H, Koh E, Sheng Y, Maeda Y, Gutti R, Namiki M, Dufau ML. Polymorphism of the GRTH/DDX25 gene in normal and infertile Japanese men: a missense mutation associated with loss of GRTH phosphorylation. Mol Human Reprod 2007;13:887-92.
Prolactin receptor
We previously demonstrated that short forms with abbreviated cytoplasmic sequences (S1a and S1b) exert dominant negative effects on prolactin- (PRL) induced activation of transcription by the long form (LF). The LF homodimer is the only PRLR form able to activate the JAK2/STAT5 pathway, which is essential for PRL-induced transcription of milk protein genes, differentiation of normal epithelial cells, and initiation and maintenance of lactation. The inhibitory action of the short form (SF) results from its heterodimerization with the LF. Given the absence of essential cytoplasmic sequences in the SF heterodimer partner, which are essential for downstream activation, the subsequent signal transduction events via the JAK2/STAT signaling pathway are not operative and the transcription of relevant genes is reduced or abolished. We demonstrated the presence of pre-existing homodimers of the LF receptor in the absence of prolactin and concluded that PRL is a conformational modifier rather than a dimer inducer. We also observed the lack of hormone dependence in the formation of homo- and heterodimers. Assessment of biochemical surface biotinylation confirmed that hPRLR forms are expressed at the cell membrane. Furthermore, the SF does not influence the steady-state cell surface expression or half-life of the LF.
Bioluminescence resonance energy transfer analysis (BRET) demonstrated homo- and heterodimeric association of hPRLR variants in living cells (human embryonic kidney 293 cells). Detailed kinetic and dosage studies by BRET analysis revealed that prolactin did not affect the oligomerization of the PRLR variants. Thus, homo- and heterodimerization of PRLR variants was a ligand-independent process. However, ligand binding could alter dimerized PRLR conformation to facilitate downstream signal transduction. Site-directed point mutation of four conserved cysteine residues, which form intramolecular disulfide bonds (amino acids 36–46 and 75–86) within extracellular domain of the receptor and are necessary for ligand binding to the receptor, completely eliminated the prolactin-stimulated JAK phosphorylation that is normally observed in wild-type LFs and SFs. In contrast to the LF, the wild-type SF (S1b) displayed high constitutive JAK2 phosphorylation. Both wild-type and mutated S1b contain a typical box 1 in the cytoplasmic domain (adjacent to the membrane) as a JAK2 binding site. Given that we observed constitutive JAK phosphorylation only in S1b- and not in S1bX-transfected cells, the intramolecular disulfide bridge formed by these two pairs of Cys (36/46, 75/86) is required for the correct conformation to allow JAK binding to the box 1. This finding indirectly supports our earlier hypothesis that a subtle conformational change by the ligand is required to initiate JAK phosphorylation and consequent events. Furthermore, we observed no inhibitory action of wild-type S1b on LF-mediated b-casein promoter activity in the S1bX. BRET analysis demonstrated less heterodimeric association of the LF with S1bX than of the LF with wild-type S1b and an increase in homodimeric association of SFX homodimers. We concluded that the increased association and formation of homodimeric S1b cys-mutants leaves the LFs to form homodimers and for ligand-induced signaling.
We used the X-ray crystal structure of the extracellular domain of the HGH-liganded human prolactin receptor as a starting structure to simulate the hPRLR monomer and wild-type and mutant (4 Cys-Ser mutants) receptors in solution. We found that, compared with wild type, the mutant protein undergoes conformational changes and shows less bending of subdomain 1 of the ligand-binding region, which contains the two pairs of Cys (S1b versus S1bX: 70–90˚ versus 30–40˚). Similarly, we used the X-ray structure of the complex (oPL-rPRLR) as the starting structure to simulate the hPRLR dimers (wild-type and mutant) in solution. A groove of positive electric field, which is flanked by two negatively charged regions, could be a motif of ligand recognition, as the groove is occluded in the mutant dimer. This phenomenon might offer a structural explanation for why prolactin does not bind to the Cys mutant of PRLR. The intermonomer hydrogen bonds (HBs) in the wild type were confined to the C-terminal domain of the extracellular region (sub-domain 2) while, in the mutant, several residues in the N-terminus were also involved in intermonomer HBs; some of these residues might be responsible for locking the putative binding groove and may be the cause of the higher homodimerization signal observed with S1bX by BRET analysis. Our studies demonstrated that the conformational state provided by the intramolecular disulfide bridges of the prolactin receptor is essential for the inhibitory action of S1b on the prolactin-induced signaling mediated by the LF, accounting for the fact that the disulfide bridges are required for the constitutive activation of JAK-2 observed in the S1B form.
Dong J, Tsai-Morris C-H, Dufau ML. A novel estradiol/estrogen receptor a-dependent transcriptional mechanism controls expression of the human prolactin receptor. J Biol Chem 2006;281:18825-36.
Qazi AM, Tsai-Morris C-H, Dufau ML. Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization. Mol Endocrinol 2006;20:1912-23.
1 Left the NIH, former Postdoctoral Fellow
COLLABORATOR
Sergio A. Hassan, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
Eitsu Koh, MD, PhD, Kanazawa University, Kanazawa, Japan
Mikio Namiki, MD, PhD, Kanazawa University, Kanazawa, Japan
For further information, contact dufaum@mail.nih.gov.
