Progesterone mediates its anti-mitogenic and anti-apoptotic actions in rat granulosa cells through a progesterone-binding protein with gamma aminobutyric acidA receptor-like features. Peluso JJ et al. BOR 1998 Progesterone (P4) inhibits small granulosa cell (GC) mitosis and large GC apoptosis. These actions are steroid specific and dose dependent and are inhibited by the progesterone receptor (PR) antagonist, RU-486. However, these cells do not express the nuclear PR but rather an ill-defined P4-binding protein (P4BP). This binding protein could function as a receptor and mediate P4's actions in GCs. Therefore, a series of studies was designed to characterize this P4BP. First, an antibody directed against the ligand-binding site of the nuclear PR was used in a Western blot analysis. This analysis revealed the presence of a 60-kDa P4BP within ovarian and GC lysates as well as within an ovarian membrane preparation. This protein was not observed in lysates of cells derived from the ovarian surface epithelium. In addition, this P4BP was immunoprecipitated by an antibody to the alpha1 chain of the gamma aminobutyric acidA (GABA(A)) receptor, suggesting that the P4BP could be the ovarian GABA(A) receptor. Since activation of the rat ovarian GABA(A) receptor increases intracellular cAMP levels, GCs were cultured with control medium supplemented with either 8-bromo-cAMP (8-br-cAMP), P4, or muscimol (a GABA agonist). Increases in cAMP were detected by monitoring the cAMP-dependent phosphorylation of cAMP response element-binding protein (CREB). Phosphorylated CREB was not observed in control or P4-treated cultures, but it was detected in the majority of both small and large GCs exposed to either 8-br-cAMP or muscimol. Since activation of the GABA(A) receptor with muscimol increases phosphorylated CREB but P4 does not, this study indicates that P4 does not activate the ovarian GABA(A) receptor. However, both bicuculline, a GABA(A) receptor antagonist, and the antibody to PR inhibited P4's ability to prevent both insulin-dependent mitosis and apoptosis. Collectively, these studies suggest that P4 mediates its anti-mitotic and anti-apoptotic effects through this 60-kDa P4BP, which has GABA(A) receptor-like properties and is localized within the surface membrane of GCs.
Characterization of a putative membrane receptor for progesterone in rat granulosa cells. Peluso JJ et al. BOR 2001 Progesterone (P(4)) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P(4) receptor. It has been proposed that P(4) mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P(4) binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P(4) binding site of the nuclear P(4) receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P(4), proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P(4) in the presence or absence of nonlabeled P(4). This study demonstrated that the 60-kDa protein specifically binds P(4). Scatchard plot analysis revealed that (3)H-P(4) binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a K(d) of 360 nM. (3)H-P(4) binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P(4) inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P(4) binding protein functions as a low-affinity, high-capacity membrane receptor for P(4).
General function
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Cellular localization
Plasma membrane
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Insulin resistance causes impaired vasodilation and hypofibrinolysis in young women with polycystic ovary syndrome. Carmassi F et al. (2005) Insulin resistance, a novel cardiovascular risk factor, is often associated with increased plasminogen activator inhibitor-1 levels and impaired vasodilation. Insulin infusion in the forearm induces plasminogen activator inhibitor-1 and tissue plasminogen activator expression and endothelium-dependent vasodilation in normal subjects. The present study explores the relationship between insulin-induced vasodilatory and fibrinolytic properties of the endothelium in women with polycystic ovary syndrome, frequently affected by insulin resistance and early atherosclerosis. Metabolic, hormonal and fibrinolytic parameters were evaluated in 64 patients with polycystic ovary syndrome (19 insulin-resistant and 45 insulin-sensitive) and in 25 controls. In 16 women with polycystic ovary syndrome, 8 insulin-resistant and 8 insulin-sensitive, blood flow, plasminogen activator inhibitor-1 and tissue plasminogen activator expression were evaluated during insulin infusion into the forearm. Elevated basal plasminogen activator inhibitor-1 levels were found in women with polycystic ovary syndrome, correlating directly with insulin levels. Plasminogen activator inhibitor-1 expression increased during insulin infusion in all women with polycystic ovary syndrome, but was delayed and sustained in insulin-resistant patients (p<0.01). Vasodilatory response to insulin was blunted (p<0.01) and tissue plasminogen activator expression abolished in insulin-resistant patients (p<0.01). Our study demonstrates that women with polycystic ovary syndrome and insulin resistance show a blunted endothelial-dependent vasodilation. The impaired endothelial release of tissue-plasminogen activator and the sustained plasminogen activator inhibitor-1 release during insulin infusion suggest a hypofibrinolytic state in PCOS patients with insulin resistance. This hemodynamic and fibrinolytic derangement may contribute to the pathogenesis of early atherosclerosis in insulin resistance.//////////////////
Plasminogen Activator Inhibitor 1 RNA Binding Protein Interacts with Progesterone Receptor Membrane Component 1 to Regulate Progesterone's Ability to Maintain the Viability of Spontaneously Immortalized Granulosa Cells and Rat Granulosa Cells. Peluso JJ et al. Progesterone receptor membrane component 1 (PGRMC1) mediates the anti-apoptotic action of progesterone (P4). PGRMC1 interacts with plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1), but the functional significance of this interaction is unknown. To examine the function of PGRMC1-PAIRBP1 interaction, PAIRBP1 was depleted from spontaneously immortalized granulosa cells (SIGCs) and the effects on the expression and localization of PGRMC1 as well as P4's ability to bind to SIGCs and prevent apoptosis was assessed. Depleting PAIRBP1 enhanced cellular (3)H-P4 binding, did not alter the expression or cellular localization of PGRMC1 but attenuated P4's anti-apoptotic action. Transfection of a PGRMC1-GFP peptide mimic, which binds PAIRBP1 as demonstrated by in situ proximity assay, doubled the rate at which SIGCs undergo apoptosis compared to cells transfected with either the empty GFP expression vector or Pairbp1 siRNA. Moreover, P4 did not prevent these cells from undergoing apoptosis. Similar studies conducted with granulosa cells isolated from immature rats also showed that PGRMC1 interacts with PAIRBP1 and that transfection of PGRMC1-GFP peptide mimic accelerates the rate of granulosa cell apoptosis by four fold even in the presence of serum and P4. These studies support the concept that the interaction between PAIRBP1-PGRMC1 is an essential component of the mechanism through which P4 inhibits apoptosis. Surprisingly, PGRMC1-PAIRBP1 interaction is not required for P4 binding or the cellular localization of PGRMC1 but rather appears to couple PGRMC1 to downstream components of the P4-PGRMC1 signal transduction pathway.
A Tudor Domain Protein SPINDLIN1 Interacts with the mRNA-Binding Protein SERBP1 and Is Involved in Mouse Oocyte Meiotic Resumption. Chew TG 2013 et al.
Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.
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Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells. Peluso JJ et al. Endo 2004 Progesterone (P4) inhibits apoptosis of rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs), which were derived from rat granulosa cells. Defining the mechanism through which P4 mediates its action has been difficult because these cells do not express the classic nuclear P4 receptor. Previous studies have shown that a P4 receptor antibody, C-262, detects a 60-kDa protein that is involved in regulating P4's antiapoptotic action. Using a C-262 affinity column, this 60-kDa protein was isolated and sequenced by mass spectrometry. This analysis revealed that the C-262-detectable protein is an unnamed protein referred to as RDA288. This protein has several putative hyaluronic acid binding sites. Further hyaluronic acid antagonizes (3)H-P4 binding to SIGCs and mimics P4's action, whereas exogenous hyaluronic acid binding protein attenuates P4's actions. RT-PCR demonstrated that RDA288 mRNA was present in SIGCs, immature rat ovary, lung, and skeletal muscle but was not present in several other organs. Forced expression of RDA288 increased the capacity of SIGCs to bind and respond to P4. An antibody was also developed against RDA288. Using this antibody in a Western blot protocol, RDA288 expression was confirmed in both SIGCs and granulosa cells. An immunohistochemical study detected RDA288 in the cytoplasm and plasma membrane components of granulosa cells of antral follicles. Immunocytochemical studies on living nonpermeabilized SIGCs revealed that RDA288 was present on the extracellular surface of the plasma membrane. Finally, pretreatment with the RDA288 antibody blocked P4's antiapoptotic actions. Taken together, these data suggest that RDA288 plays a significant role in mediating P4's antiapoptotic action in granulosa cells.
Expression and Function of PAIRBP1 Within Gonadotropin-Primed Immature Rat Ovaries: PAIRBP1 Regulation of Granulosa and Luteal Cell Viability Peluso JJ, et al . BOR 2005
The protein, PAIRBP1 that was initially referred to as RDA288, is involved in mediating the anti-apoptotic action of progesterone (P4) in spontaneously immortalized granulosa cells (SIGCs). The present studies were designed to assess the expression and function of PAIRBP1 in the different cell types within the immature rat ovary. Western blot analysis detected PAIRBP1 within whole cell lysates of immature rat ovaries. Equine gonadotropin (eCG) induced a 3-fold increase in ovarian levels of PAIRBP1. Moreover, human chorionic gonadotropin (hCG), given 48 h after eCG, maintained these elevated levels for up to 4 days. Immunohistochemical analysis confirmed this and further demonstrated that interstitial, thecal and surface epithelial cells also expressed PAIRBP1. The level of PAIRBP1 in these cells was not influenced by gonadotropin treatment. In contrast eCG stimulated an increase in PAIRBP1 within the granulosa cells of the developing follicles. Treatment with hCG induced ovulation and ultimately the formation of corpora lutea (CL). High levels of PAIRBP1 expression were also observed within the luteal cells. Immunocytochemical studies on living, non-permeabilized granulosa and luteal cells revealed that some PAIRBP1 localized to the extracellular surface of these cells. The presence of PAIRBP1 on the extracellular surface was consistent with the observation that an antibody to PAIRBP1 attenuated P4's anti-apoptotic action in both granulosa and luteal cells. Although the PAIRBP1 antibody attenuated P4's action, it did not reduce the capacity of cells to specifically bind 3H-P4. Immunoprecipitation with the PAIRBP1 antibody pulled down the membrane P4 binding protein known as Progesterone Receptor Membrane Complex-1 (PGRMC1; rat homolog accession number AJ005837). Taken together, these findings suggest that gonadotropins regulate the expression of PAIRBP1 in granulosa and luteal cells and that PAIRBP1 plays an important role in mediating P4's anti-apoptotic action in these ovarian cell types. The exact mechanism of PAIRBP1's action remains to be elucidated but it may involve an interaction with PGRMC1.