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APPL1, APPL2, Akt2 and FOXO1a interact with FSHR in a potential signaling complex
Abstract
A number of signaling proteins have been demonstrated to interact with follicle stimulating hormone (FSH) receptor (FSHR), including APPL1, 14-3-3tau and Akt2. To further define the repertoire of proteins involved in FSH-induced signal transduction, several signaling and adapter proteins were examined for the ability to associate with FSHR. This report shows that, in addition to APPL1, FSHR interacts with FOXO1a and APPL2. Moreover, APPL1 and APPL2 associate with one another via the N-terminus of APPL1, presumably via the Bin-Amphiphysin-Rvs (BAR) domain. The interactions between FSHR and APPL2 and between FSHR and FOXO1a evidently are distinct since FOXO1a does not associate with either APPL1 or with APPL2. Though APPL1 and APPL2 show some similarity in primary sequence, APPL1 associates with Akt2, whereas APPL2 does not. This is the first documented difference in function between APPL1 and APPL2. These results suggest that FSHR, APPL1, APPL2, Akt2 and FOXO1a are organized into distinct scaffolding networks in the cell. Accordingly, the spatial organization of signaling and adapter proteins with FSHR likely facilitates and finely regulates the signal transduction induced by FSH.
... APPL1 lacks catalytic activity; however, it is composed of various membrane and protein interacting domains, functioning as an endosomal marker in addition to its ability to integrate between different trafficking and signaling pathways from the endomembrane [65,70]. FSHR has been shown to form complexes with APPL1, via intracellular loop 1, IL1, Lys393, Leu394, and Phe399, which facilitates FSHinduced PI3K/Akt activation, IP 3 production, Ca 2+ release, and the nuclear exclusion of forkhead transcription factor 1 (FOXO1a) [18,52,[71][72][73]. Given that FSHR associates with both APPL1 and FOXO1a and that APPL1 interacts with AKT [72], an interplay between FSHR-FOXO1a complex with an active FSHR-APPL1-Akt complex, which leads to the phosphorylation of FOXO1a and the abrogation of apoptosis, has been proposed [18,72,74]. ...
... FSHR has been shown to form complexes with APPL1, via intracellular loop 1, IL1, Lys393, Leu394, and Phe399, which facilitates FSHinduced PI3K/Akt activation, IP 3 production, Ca 2+ release, and the nuclear exclusion of forkhead transcription factor 1 (FOXO1a) [18,52,[71][72][73]. Given that FSHR associates with both APPL1 and FOXO1a and that APPL1 interacts with AKT [72], an interplay between FSHR-FOXO1a complex with an active FSHR-APPL1-Akt complex, which leads to the phosphorylation of FOXO1a and the abrogation of apoptosis, has been proposed [18,72,74]. In granulosa cells, FSH stimulation results in rapid FOXO1a phosphorylation and extrusion from the nucleus and control of lipid biosynthetic pathways [72,75]. ...
... FSHR has been shown to form complexes with APPL1, via intracellular loop 1, IL1, Lys393, Leu394, and Phe399, which facilitates FSHinduced PI3K/Akt activation, IP 3 production, Ca 2+ release, and the nuclear exclusion of forkhead transcription factor 1 (FOXO1a) [18,52,[71][72][73]. Given that FSHR associates with both APPL1 and FOXO1a and that APPL1 interacts with AKT [72], an interplay between FSHR-FOXO1a complex with an active FSHR-APPL1-Akt complex, which leads to the phosphorylation of FOXO1a and the abrogation of apoptosis, has been proposed [18,72,74]. In granulosa cells, FSH stimulation results in rapid FOXO1a phosphorylation and extrusion from the nucleus and control of lipid biosynthetic pathways [72,75]. ...
Gonadotropin hormones and their receptors play a central role in the control of male and female reproduction. In recent years, there has been growing evidence surrounding the complexity of gonadotropin hormone/receptor signalling, with it increasingly apparent that the Gαs/cAMP/PKA pathway is not the sole signalling pathway that confers their biological actions. Here we review recent literature on the different receptor-receptor, receptor-scaffold and receptor-signaling molecule complexes formed and how these modulate and direct gonadotropin hormone-dependent intracellular signal activation. We will touch upon the more controversial issue of extragonadal expression of FSHR and the differential signal pathways activated in these tissues, and lastly, highlight the open questions surrounding the role these gonadotropin hormone receptor complexes, and how this will shape future research directions.
... The FSHR is connected via conformational selectivity to a nonlinear and complex signaling network. This network can involve either several G protein subtypes, including the G s , G i , and G q/11 proteins (25)(26)(27)(28)(29)(30), interaction with other receptors [e.g., the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR)] (24, 31-33) and proteins [e.g., b-arrestins, adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL)1, forkhead box transcription factor O (FOXO)1a (also known as forkhead homolog in rhabdomyosarcoma), and 14-3-3t] (34)(35)(36)(37)(38)(39), or heterodimerization with the LHCGR (26,(40)(41)(42). These mechanisms have been reported to regulate a number of intertwined signaling pathways, including engagement of distinct kinases [such as PKA, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), protein kinase B/Akt, p70S6 kinase (p70S6K), and ERK1/ 2] (1,5,14,22,24,27,43,44) (Fig. 2) (45). ...
... In addition to signaling through G proteins, the FSHR also directly interacts with a number of partners that elicit particular signaling mechanisms. These partners include, among others, the APPL1 adapter, the abovementioned FOXO1a, and 14-3-3t (34,35,37,38,(84)(85)(86). The APPL1 adapter is an important regulator of signaling and trafficking events within cells and is expressed in many tissues, including the testis and the ovary (87). ...
... APPL1 may govern signal specificity and trafficking by interacting with PI3K and Akt (88) that in turn phosphorylates FOXO1a, as well as with the small GTPase Rab5 (89), a mediator of fusion of clathrin-coated vesicles with early endosomes (90). Studies employing a yeast interaction trap identified APPL1 as an important interacting protein with the FSHR (35,37,38); furthermore, it has been found that linking between this adapter and the FSHR occurs at the IL1, specifically at Lys393, Leu394, and Phe399 (39) (Fig. 1). In HEK293 cells stably expressing the FSHR, APPL1 and APPL2 appear to be associated with each other via the NH 2 terminus of APPL1 in an FSH-independent manner (35,37,38). ...
... The PTB domain is close to the C terminus, ensuring an easy binding structure for its binding partners [18]. This domain interacts with a diverse set of receptors, including netrin-1 receptor DCC [2], nerve growth factor (NGF) receptor TrkA [9,19], follicle-stimulating hormone (FSH) receptor (FSHR) [20,21], epidermal growth factor (EGF) receptor (EGFR) [22], and adiponectin receptors AdipoR1 and AdipoR2 [4,23]. The PTB domain also interacts with signaling proteins, such as AKT [1,21,24], PI3K subunits [1,25], βCTF [7], and TRAF2 [26], demonstrating its importance in various signaling pathways. ...
... This domain interacts with a diverse set of receptors, including netrin-1 receptor DCC [2], nerve growth factor (NGF) receptor TrkA [9,19], follicle-stimulating hormone (FSH) receptor (FSHR) [20,21], epidermal growth factor (EGF) receptor (EGFR) [22], and adiponectin receptors AdipoR1 and AdipoR2 [4,23]. The PTB domain also interacts with signaling proteins, such as AKT [1,21,24], PI3K subunits [1,25], βCTF [7], and TRAF2 [26], demonstrating its importance in various signaling pathways. ...
... In many cases, they play different or even opposite roles to each other. For example, as a binding partner of APPL1 in the BAR domain [3,21] stimulated glucose uptake [42] and adiponectin-induced fatty acid oxidation in muscle cells [8]. A detailed overview of the roles of APPL1 and APPL2 in insulin and adiponectin signaling will be provided in the following sections. ...
... Although the structure of the FSH-FSHRHB(Fan and Hendrickson, 2005) revealed that the FSHR ectodomain may form weakly associated dimers, with each molecule bearing one FSH molecule, later studies based on combined biochemical and biophysical approaches directly demonstrated that the FSHR self-associates early during receptor biosynthesis and that it can be identified as dimers or oligomers at the plasma membrane(Thomas et al., 2007).Further, this study demonstrated that the carboxyl-terminus of the receptor is clipped before trafficking to the plasma membrane(Thomas et al., 2007), a finding that partially explained the paucity of studies on FSHR association because of the difficulties in constructing either functional epitope-tagged FSHRs or FSHR fluorescent fusion proteins at this particular domain. More recently, Mazurkiewicz and colleagues(Mazurkiewicz et al., 2015), overcame this problem by constructing functional chimeras, in which the carboxyl-terminus domain of the human WT FSHR was replaced with that of the LHR (which in contrast to the FSHR is not clipped during its biosynthesis(Thomas et al., 2007)) fused with fluorescent proteins (FP). ...
... Although the structure of the FSH-FSHRHB(Fan and Hendrickson, 2005) revealed that the FSHR ectodomain may form weakly associated dimers, with each molecule bearing one FSH molecule, later studies based on combined biochemical and biophysical approaches directly demonstrated that the FSHR self-associates early during receptor biosynthesis and that it can be identified as dimers or oligomers at the plasma membrane(Thomas et al., 2007).Further, this study demonstrated that the carboxyl-terminus of the receptor is clipped before trafficking to the plasma membrane(Thomas et al., 2007), a finding that partially explained the paucity of studies on FSHR association because of the difficulties in constructing either functional epitope-tagged FSHRs or FSHR fluorescent fusion proteins at this particular domain. More recently, Mazurkiewicz and colleagues(Mazurkiewicz et al., 2015), overcame this problem by constructing functional chimeras, in which the carboxyl-terminus domain of the human WT FSHR was replaced with that of the LHR (which in contrast to the FSHR is not clipped during its biosynthesis(Thomas et al., 2007)) fused with fluorescent proteins (FP). ...
... Although the structure of the FSH-FSHRHB(Fan and Hendrickson, 2005) revealed that the FSHR ectodomain may form weakly associated dimers, with each molecule bearing one FSH molecule, later studies based on combined biochemical and biophysical approaches directly demonstrated that the FSHR self-associates early during receptor biosynthesis and that it can be identified as dimers or oligomers at the plasma membrane(Thomas et al., 2007).Further, this study demonstrated that the carboxyl-terminus of the receptor is clipped before trafficking to the plasma membrane(Thomas et al., 2007), a finding that partially explained the paucity of studies on FSHR association because of the difficulties in constructing either functional epitope-tagged FSHRs or FSHR fluorescent fusion proteins at this particular domain. More recently, Mazurkiewicz and colleagues(Mazurkiewicz et al., 2015), overcame this problem by constructing functional chimeras, in which the carboxyl-terminus domain of the human WT FSHR was replaced with that of the LHR (which in contrast to the FSHR is not clipped during its biosynthesis(Thomas et al., 2007)) fused with fluorescent proteins (FP). Fluorescence correlation spectroscopy as a freely diffusing homodimer (Fig. 3A-C). ...
Follitropin, or follicle-stimulating hormone (FSH) receptor (FSHR), is a G protein-coupled receptor belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although its primary location is the gonad, the FSHR has also been reported in extragonadal tissues including bone, placenta, endometrium, liver, and blood vessels from a number of malignant tumors. The recently resolved crystal structure of FSH bound to the entire FSHR ectodomain has been instrumental in more clearly defining the role of this domain in ligand binding and receptor activation. Biochemical, biophysical and structural data also indicate that the FSHR exists as a higher order structure, and that it may heterodimerize with its closely related receptor, the luteinizing hormone receptor; this association may have physiological implications during ovarian follicle maturation given that both receptors coexist in the same cell. FSHR heterodimerization is unique to the ovary since in the testes, gonadotropin receptors are expressed in separate compartments. FSHR self-association appears to be required for receptor coupling to multiple effectors and adaptors, for the activation of multiple signaling pathways and the transduction of asymmetric signaling, and for negative and positive receptor cooperativity. It also provides a mechanism through which the glycosylation variants of FSH may exert distinct and differential effects at the target cell level. Given its importance in regulating activation of distinct signaling pathways, functional selectivity at the FSHR is briefly discussed, as well as the potential implications of this particular functional feature on the design of new pharmacological therapies in reproduction.
... Several studies provide evidence showing that the endosomes are involved in the Akt isoform and substrate specificity. Adaptor protein containing PH-domain, PTB domain, and leucine zipper motif 1 (APPL1), an endosomal protein, was reported to physically associate with Akt2 in a complex and can be reciprocally coimmunoprecipitated (114)(115). In zebrafish, Akt was found to specifically co-localize with its substrate GSK-3β but not TSC2 in APPL1-containing endosomes (116). ...
... In another example, the endosomal proteins APPL (adaptor protein containing PH domain, PTB domain, and leucine zipper motif) 1 and APPL2, which reside in an endosomal population devoid of EEA1 (191), regulate signaling by interacting with downstream effectors, such as the protein kinase Akt (114)(115)(116)(191)(192)(193). It has been ...
... Recent studies have shown that the endosomal proteins APPL1 and APPL2 may help to determine the Akt1 and Akt2 isoform specificity. For example, Akt2, but not Akt1, was found to be localized in APPL-containing endosomes (114)(115); and APPL1-containing endosomes mediate Akt substrate specificity by dynamically interacting with specific Akt substrates (116). Previously, our lab has identified a new endosomal protein WDFY2, which defines a distinct endosomal population lacking the canonical markers EEA1 and Rab5 (146,183). ...
Excess food consumption and/or lack of exercise have dramatically contributed to the prevalence of overweight (BMI≥25) and obesity (BMI≥30) in modern society. The obesity epidemic has been linked to the rise in type 2 diabetes. In recent years, evidence has pointed to a close association between mitochondrial dysfunction in white adipose tissue (WAT) and insulin resistance, a key feature of type 2 diabetes. In order to dissect the cause and effect relationship between WAT mitochondrial dysfunction and insulin resistance, we established an in vitro cell line system to investigate this issue. We artificially introduced mitochondrial dysfunction in 3T3-L1 adipocytes by depleting the mitochondrial transcription factor A (Tfam) during adipogenesis, without changing the overall adipocyte differentiation program. We found that these Tfam-depleted 3T3-L1 adipocytes showed symptoms of insulin resistance, evidenced by impaired insulin stimulated GLUT4 translocation and glucose uptake. This result suggested that mitochondrial dysfunction could be a primary contributor to insulin resistance in fat tissue. However, the exact mechanism underlying this finding remains unclear.
As part of a comprehensive understanding of insulin signaling in fat cells, we also investigated the involvement of the endosomal protein WDFY2 in the regulation of Akt isoform-specific effect on glucose uptake. In 3T3-L1 adipocytes, both Akt1 and Akt2 isoforms are expressed, but only Akt2 plays an indispensible role in insulin-stimulated GLUT4 translocation and glucose uptake. Previous studies implied that endosomal proteins may take a part in determining Akt substrate specificity. Here we found that WDFY2 preferentially co-localized with Akt2 and that knockdown of WDFY2 inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes, suggesting that endosomes are involved in this regulation. The effect of WDFY2 knockdown on insulin-stimulated glucose uptake worked through the down-regulation of Akt2, but not Akt1, protein level. We concluded that, endosomal protein WDFY2, by preferentially interacting with Akt2, regulates insulin signaling in glucose uptake in 3T3-L1 adipocytes. Our findings may help to develop specific therapeutic interventions for treatment of insulin resistance and type 2 diabetes.
... They have a common domain organization: an N-terminal BAR (Bin/Amphiphysin/RVS167) domain, followed by a central PH domain and a C-terminal PTB domain. APPL BAR domains can either homodimerize or heterodimerize (2)(3)(4)(5), a common feature of the BAR superfamily (6), which is necessary for induction, sensing, and maintenance of curved membrane structures (6). The BAR domain and PH domain act as a functional unit together (2,(7)(8)(9), laying the structural basis for endosomal targeting and binding of active form of small GTPase Rab5 (1,2). ...
... APPL proteins interact with various transmembrane receptors including AdipoR1, AdipoR2 (10), EGF receptor (11), FSH receptor (3), and NGF receptor (12,13), and recruit a series of downstream interacting partners such as Rab5 (2), AMPK (10,14), Akt (2), PI3K (3), FOXO1a (3), PKC (15), GIPC1 (12), and so on. Therefore, they provide a common platform for the centralized communication of these signaling components and allow for the cross-talks among multiple signaling pathways (16). ...
The APPL (adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif) family consists of two isoforms, APPL1 and APPL2. By binding to curved plasma membrane, these adaptor proteins associate with multiple transmembrane receptors and recruit various downstream signaling components. They are involved in the regulation of signaling pathways evoked by a variety of extracellular stimuli, such as adiponectin, insulin, FSH (follicle stimulating hormone), EGF (epidermal growth factor). And they play important roles in cell proliferation, apoptosis, glucose uptake, insulin secretion and sensitivity. However, emerging evidence suggests that APPL1 and APPL2 perform different or even opposite functions and the underlying mechanism remains unclear. As APPL proteins can either homodimerize or heterodimerize in vivo, we hypothesized that heterodimerization of APPL proteins might account for the mechanism. By solving the crystal structure of APPL1-APPL2 BAR-PH heterodimer, we find that the overall structure is crescent-shaped with a longer curvature radius of 76 Å, compared to 55 Å of the APPL1 BAR-PH homodimer. However, there is no significant difference of the curvature between APPL BAR-PH heterodimer and APPL2 homodimer. The data suggest that the APPL1 BAR-PH homodimer, APPL2 BAR-PH homodimer and APPL1/APPL2 BAR-PH heterodimer may bind to endosomes of different sizes. Different positive charge distribution is observed on the concave surface of APPL BAR-PH heterodimer than the homodimers, which may change the affinity of membrane association and subcellular localization. Collectively, APPL2 may regulate APPL1 function through altering the preference of endosome binding by heterodimerization.
... APPL1 was first identified as an interacting protein of Akt2 via its PTB domain [34,35], and its role in partitioning Akt2 into endosomes is well established [36][37][38][39][40]. Therefore, to test for the involvement of Akt2 in mPR signaling, we first knocked down endogenous Akt2 mRNA specifically without affecting Akt1 mRNA (S3C Fig). ...
... It associates with both the insulin [68] and adiponectin [31] receptors to mediate metabolic and vascular homeostasis [32,69]. In addition, APPL1 interacts with GPCRs [40] and mediates physical and signaling compartmentalization to subsets of signaling endosomes [67]. Here we show that APPL1 interacts with mPRs to mediate their downstream signaling. ...
The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRβ is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRβ signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRβ into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.
... Some of the motifs involved in these complex signaling networks are shown in Figure 1. For example interaction of the FSHR with the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL), has been mapped to the IL-1, specifically to K393, L394, and F399 (144,145). The adapter APPL1 may regulate signal specificity and trafficking through the interaction with PI3K and Akt, which is followed by FOXO1a phosphorylation, leading to abrogation of apoptosis (145); in addition, this adaptor is also involved in FSHR-mediated Ca 2+ signaling and other functions (84,146). ...
... For example interaction of the FSHR with the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL), has been mapped to the IL-1, specifically to K393, L394, and F399 (144,145). The adapter APPL1 may regulate signal specificity and trafficking through the interaction with PI3K and Akt, which is followed by FOXO1a phosphorylation, leading to abrogation of apoptosis (145); in addition, this adaptor is also involved in FSHR-mediated Ca 2+ signaling and other functions (84,146). Meanwhile, association of the FSHR with the 14-3-3τ protein has been mapped to the IL2, overlapping the above mentioned ERW motif (138, 147); 14-3-3 proteins are involved in several cell processes and play an important role in modulating signaling pathways through interacting with activated signaling proteins (148). ...
The follicle-stimulating hormone receptor (FSHR) plays a crucial role in reproduction. This structurally complex receptor is a member of the G-protein coupled receptor (GPCR) superfamily of membrane receptors. As with the other structurally similar glycoprotein hormone receptors (the thyroid-stimulating hormone and luteinizing hormone-chorionic gonadotropin hormone receptors), the FSHR is characterized by an extensive extracellular domain, where binding to FSH occurs, linked to the signal specificity subdomain or hinge region. This region is involved in ligand-stimulated receptor activation whereas the seven transmembrane domain is associated with receptor activation and transmission of the activation process to the intracellular loops comprised of amino acid sequences, which predicate coupling to effectors, interaction with adapter proteins, and triggering of downstream intracellular signaling. In this review, we describe the most important structural features of the FSHR intimately involved in regulation of FSHR function, including trafficking, dimerization, and oligomerization, ligand binding, agonist-stimulated activation, and signal transduction.
... APPL1 is identified as an immediately downstream signaling protein of adiponectin receptors and serves as an essential mediator in both adiponectin and insulin signaling. APPL1 regulates follicle stimulating hormone (FSH)-mediated PI3kinase/Akt pathway (Nechamen et al., 2007). Furthermore, APPL1 plays a critical role in cell survival and differentiation during myogenesis and osteoclastogenesis (Bae et al., 2010;Schenck et al., 2008;Wen et al., 2010). ...
... APPL1 is required for cell survival in forebrain, neural tube and olfactory placodes during zebrafish embryogenesis (Schenck et al., 2008). APPL2 has 54% identity with APPL1 in sequences, and interact with PI3K/Akt pathway (Nechamen et al., 2007;Wang et al., 2009). Lines of evidence indicate that APPL2 plays essential roles in cell proliferation and embryonic development (Miaczynska et al., 2004;Schenck et al., 2008), and could be a survival signal for human glioma cells (Pyrzynska et al., 2013). ...
Metabolic disorders like diabetes and obesity are commonly companied with neurological diseases and psychiatric disorders. Accumulating evidences indicated that cellular metabolic factors affect adult neurogenesis and have modulating effects on neurodegenerative disorders and psychiatric diseases. Adult neurogenesis contains multiple steps including proliferation of neural stem cells, lineage commitments of neural progenitor cells, maturation into functional neurons, and integration into neuronal network. Many intrinsic and extrinsic factors produced from neural stem/progenitor cells and their microenvironment or neurogenic niche take roles in modulating neurogenesis and contribute to the brain repair and functional recoveries in many neurological diseases and psychiatric disorders. In this article, we review current progress about how different growth factors, neurotrophin, neurotransmitters and transcriptional factors work on regulating neurogenic process. In particular, we emphasize the roles of the cellular metabolic factors, such as insulin/IGF signaling, incretins, and lipid metabolic signaling molecules in modulating adult neurogenesis, and discuss their impacts on neurological behaviors. We propose that the metabolic factors could be the new therapeutic targets for adult neurogenesis. Plus, the metabolism-regulating drugs have the potentials for treatment of neurodegenerative diseases and mental disorders.
... APPLs can bind directly to membrane lipids, receptors and signaling molecules to coalesce the players and generate responses at specific membrane domains or cell compartments (8)(9)(10)(11)(12). In the context of the more well-studied isoform APPL1, the N-terminal membrane-curvature BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain bind to the signaling kinases Akt and PI3K, recruiting them for a variety of activated receptors (13,14). In particular, the APPLs are notable for their regulation of signaling for metabolic and inflammatory responses downstream of adiponectin receptors (14)(15)(16). ...
... The depletion of APPL1 had largely dissimilar effects, indicating a major role for APPL1 in supporting or enhancing p38 MAPK signaling and a lesser role in constraining Akt signaling. Despite APPL2 having a major role in influencing Akt signaling in macrophages, intriguingly only APPL1, but not APPL2, can reportedly bind directly to Akt2 (13). APPL2 may thus have its effect combined with APPL1. ...
Macrophages are activated by contact with pathogens to mount innate immune defenses against infection. Toll-like receptor 4 (TLR4) at the macrophage surface recognizes and binds bacterial lipopolysaccharide (LPS), setting off signaling and transcriptional events that lead to the secretion of pro- and anti-inflammatory cytokines; these in turn control inflammatory and antimicrobial responses. Although the complex regulatory pathways downstream of TLR4 have been extensively studied, further molecules critical for modulating the resulting cytokine outputs remain to be characterized. Here we establish potential roles for APPL1 and 2 signaling adaptors as regulators of LPS/TLR4-induced signaling, transcription, and cytokine secretion. APPL1 and 2 are differentially localized to distinct signaling-competent membrane domains on the surface and in endocytic compartments of LPS-activated macrophages. By depleting cells of each adaptor respectively we show separate and opposing functions for APPL1 and 2 in Akt and MAPK signaling. Specifically, APPL2 has a dominant role in nuclear translocation of NF-kB p65 and it serves to constrain the secretion of pro- and anti-inflammatory cytokines. The APPLs, and in particular APPL2, are thus revealed as adaptors with important capacity to modulate inflammatory responses mounted by LPS/TLR4 during infection.
... Similar to APPL1, APPL2 has an N-terminal BAR domain, central PH domain, and C-terminal PTB domain. APPL2 mediates FSH signal transduction by binding to APPL1 via their respective BAR domains, which results in the formation of the FSH receptor -APPL1 -AKT2 complex (Nechamen et al., 2007). Notably, APPL2 does not directly interact with AKT2 (Nechamen et al., 2007;Chial et al., 2008). ...
... APPL2 mediates FSH signal transduction by binding to APPL1 via their respective BAR domains, which results in the formation of the FSH receptor -APPL1 -AKT2 complex (Nechamen et al., 2007). Notably, APPL2 does not directly interact with AKT2 (Nechamen et al., 2007;Chial et al., 2008). Research in our laboratory revealed that APPL2 negatively modulates adiponectin signaling in skeletal muscle cells . ...
Obesity-linked type 2 diabetes is one of the paramount causes of morbidity and mortality worldwide, posing a major threat on human health, productivity, and quality of life. Despite great progress made towards a better understanding of the molecular basis of diabetes, the available clinical counter-measures against insulin resistance, a defect that is central to obesity-linked type 2 diabetes, remain inadequate. Adiponectin, an abundant adipocyte-secreted factor with a wide-range of biological activities, improves insulin sensitivity in major insulin target tissues, modulates inflammatory responses, and plays a crucial role in the regulation of energy metabolism. However, adiponectin as a promising therapeutic approach has not been thoroughly explored in the context of pharmacological intervention, and extensive efforts are being devoted to gain mechanistic understanding of adiponectin signaling and its regulation, and reveal therapeutic targets. Here, we discuss tissue- and cell-specific functions of adiponectin, with an emphasis on the regulation of adiponectin signaling pathways, and the potential crosstalk between the adiponectin and other signaling pathways involved in metabolic regulation. Understanding better just why and how adiponectin and its downstream effector molecules work will be essential, together with empirical trials, to guide us to therapies that target the root cause(s) of type 2 diabetes and insulin resistance.
... In males, on the other hand, both high-quality sperm production and normal testicular volume require normal function of FSH [54]. Accordingly, FSH-inducible transcriptions of SGK1 are also present in spermatogenic cells [55]. ...
Objective
Organisms and cellular viability are of paramount importance to living creatures. Disruption of the balance between cell survival and apoptosis results in compromised viability and even carcinogenesis. One molecule involved in keeping this homeostasis is serum-glucocorticoid regulated kinase (SGK) 1. Emerging evidence points to a significant role of SGK1 in cell growth and survival, cell metabolism, reproduction, and life span, particularly in prenatal programming and reproductive senescence by the same token. Whether the hormone inducible SGK1 kinase is a major driver in the pathophysiological processes of prenatal programming and reproductive senescence?
Method
The PubMed/Medline, Web of Science, Embase/Ovid, and Elsevier Science Direct literature databases were searched for articles in English focusing on SGK1 published up to July 2023
Result
Emerging evidence is accumulating pointing to a pathophysiological role of the ubiquitously expressed SGK1 in the cellular and organismal viability. Under the regulation of specific hormones, extracellular stimuli, and various signals, SGK1 is involved in several biological processes relevant to viability, including cell proliferation and survival, cell migration and differentiation. In line, SGK1 contributes to the development of germ cells, embryos, and fetuses, whereas SGK1 inhibition leads to abnormal gametogenesis, embryo loss, and truncated reproductive lifespan.
Conclution
SGK1 integrates a broad spectrum of effects to maintain the homeostasis of cell survival and apoptosis, conferring viability to multiple cell types as well as both simple and complex organisms, and thus ensuring appropriate prenatal development and reproductive lifespan.
... Other signaling pathways account for Gq protein-mediated phospholipase C (PLC) activation (Lu, Xia, & Zhang, 2001;Su, Xia, Byskov, Fu, & Yang, 1999), resulting in intracellular calcium ion (Ca 2+ ) increase and inositol 1,4,5-trisphosphate (IP3), while other interactors would lead to activation of mammalian target of rapamycin (mTOR) (Chen et al., 2007;Kayampilly & Menon, 2007), β-catenin (Parakh et al., 2006) and protein kinase B (AKT) phosphorylation (Hunzicker-Dunn et al., 2012). A number of other FSHR intracellular interactors were characterized, increasing the complexity of the receptor signaling pattern (Cohen, Nechamen, & Dias, 2004;Dias et al., 2010;Nechamen et al., 2004;Nechamen, Thomas, & Dias, 2007). These events are accompanied by the FSHR intracellular trafficking and cAMP activation, addressing the molecule to recycling to the cell surface or routing it to degradation pathways (Casarini & Simoni, 2021;Sayers & Hanyaloglu, 2018;Sposini et al., 2017;Ulloa-Aguirre, Dias, Bousfield, Huhtaniemi, & Reiter, 2013). ...
... Additional pathways and molecular partners of FSHR activation were described, such as insulin growth factor 1 receptor (IGF-1R), epidermal growth factor receptor (EGFR) (14, 15), 14-3-3t protein (16,17) and forkhead-box transcription factor O (FOXO1a) (18). Moreover, the molecules b-arrestins (19,20), adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL1), are involved in the internalization and recycling of the receptor (21,22). Recently, several studies demonstrated how the predominance of steroidogenic and pro-apoptotic, rather than proliferative signals, could depend on FSHR expression levels on the cell surface (20). ...
An increasing number of pollutants with endocrine disrupting potential are accumulating in the environment, increasing the exposure risk for humans. Several of them are known or suspected to interfere with endocrine signals, impairing reproductive functions. Follicle-stimulating hormone (FSH) is a glycoprotein playing an essential role in supporting antral follicle maturation and may be a target of disrupting chemicals (EDs) likely impacting female fertility. EDs may interfere with FSH-mediated signals at different levels, since they may modulate the mRNA or protein levels of both the hormone and its receptor (FSHR), perturb the functioning of partner membrane molecules, modify intracellular signal transduction pathways and gene expression. In vitro studies and animal models provided results helpful to understand ED modes of action and suggest that they could effectively play a role as molecules interfering with the female reproductive system. However, most of these data are potentially subjected to experimental limitations and need to be confirmed by long-term observations in human.
... The binding of APPL1 with AdipoR1 and AdipoR2 positively mediates adiponectin signaling (11) and facilitates the crosstalk between adiponectin and insulin signal pathways (12). APPL2, an APPL1 isoform displaying 54% identity in protein sequences (13), interacts with multiple receptors and signaling molecules in cells, including AdipoR1 and AdipoR2 (13), Rab5 (14), (14), GIPC1 (15), and FSHR (16). In contrast to APPL1, APPL2 negatively regulates adiponectin signaling and function by blocking the binding of APPL1 to the adiponectin receptors (13). ...
Adiponectin is an adipokine that exerts insulin-sensitizing and anti-inflammatory roles in insulin target tissues including liver. While the insulin-sensitizing function of adiponectin has been extensively investigated, the precise mechanism by which adiponectin alleviates diet-induced hepatic inflammation remains elusive. Here, we report that hepatocyte-specific knockout (KO) of the adaptor protein APPL2 enhanced adiponectin sensitivity and prevented mice from developing high-fat diet-induced inflammation, insulin resistance, and glucose intolerance, although it caused fatty liver. The improved anti-inflammatory and insulin-sensitizing effects in the APPL2 hepatocyte-specific KO mice were largely reversed by knocking out adiponectin. Mechanistically, hepatocyte APPL2 deficiency enhances adiponectin signaling in the liver, which blocks TNF-α-stimulated MCP-1 expression via inhibiting the mTORC1 signaling pathway, leading to reduced macrophage infiltration and thus reduced inflammation in the liver. With results taken together, our study uncovers a mechanism underlying the anti-inflammatory role of adiponectin in the liver and reveals the hepatic APPL2-mTORC1-MCP-1 axis as a potential target for treating overnutrition-induced inflammation in the liver.
... This hypothesis was explored by measuring Gbg-dependent AKT phosphorylation by FSHR-GPER heteromers, in response to FSH (Figure 3H). Acute FSH treatment (15 min) did not induce pAKT activation in HEK293/FSHR cells, which requires chronic FSH stimulation to detect activation (Nechamen et al., 2007). In contrast, pAKT activation was increased in FSH-treated HEK293/FSHR-GPER cells and was inhibited by the selective Gbg inhibitor gallein ( Figure 3I). ...
Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.
... En fonction des voies qu'elles activent, elles sont impliquées dans divers processus cellulaires, tels que le cycle cellulaire ou la réorganisation du cytosquelette. [108]. Il est donc possible que ces interactions induisent des voies de signalisation, d'autant que le complexe APPL1:FSHR induit la production d'IP3 [109]. ...
... (Pink cell) FSH binding to the Fshr G protein-coupled receptor has been shown to activate the Gαs/cAMP/protein kinase A (PKA) signaling pathway [8]. Appl1 interacts with the FshR to trigger the PI3K signaling pathway while Src family members promote a similar activation in an FSHdependant fashion, while triggering a similar response for the ERK cascade [9,10]. Coupling of FshR to Gαh (tissue transglutaminase) leads to PLCδ activation [11]. ...
Objective
Combination of transcriptomic and retrospective clinical data, to assess anti-Mullerian hormone (AMH) functionality at a cumulus cell level and evaluate AMH potential as a suitable marker for IVF outcomes (oocytes retrieved, number of day 3 embryos, gestation outcomes).DesignRaw RNA-sequencing data of cumulus cells sourced from younger (n = 10) patient group (group A) (age 29 (1 year of age), baseline FSH 7.4 (0.5 mIU/ml), AMH 4.67 (1.56 ng/ml)) and older (n = 10) patient group (group B) (age 43 (± 0.55 years of age), baseline FSH 8 (0.8 mIU/ml), AMH 1.07 (0.44 ng/ml)) were employed to derive transcriptomic differences among high vs. low AMH groups. We collected retrospectively patient data from 80 infertile patients selected according to pre-specified inclusion criteria.SettingPublicly available raw RNA-sequencing data were retrieved from the SRA database of NCBI resource GEO Accession (GSM21575/35-44; GEO Accession: GSM21575/45-55). Retrospective data were collected from referrals to the Institute of Reproductive Medicine, Lito Hospital of Athens and the Institute of Life, Iaso Hospital of Athens, between the periods of March 2015 and April 2018.Intervention(s)A fixed human menopausal gonadotropin (hMG) antagonist protocol was used for all patients. All patients had serum AMH levels measured within a 3-month period prior to stimulation and serum levels of FSH and estradiol (day 2 of menstrual cycle; E2) (Clinical Trial code NV24042014).Main outcome measure(s)The primary outcomes were identification of transcriptomic variations among high (group A) vs. low (group B) AMH patients. Retrospective data primary outcomes were number of oocytes retrieved, fertilized successfully (grades A and B, day 2 embryos), and total number of day 3 embryos. Secondary outcome was live birth rate. Finally, we compared primary outcomes with AMH and FSH level as well as their genetic pathways (interacting genes) to demonstrate the predictive accuracy.ResultsEssential players of the AMH signaling cascade, namely, SMAD1, SMAD4, SMAD5, ALK1, and LEF1, were significantly upregulated in group A (n 10) transcriptome. This biological clue was further supported by retrospective clinical data (n 80 participants), where AMH was positively correlated with both oocytes retrieved and fertilized as well as number of day 3 (grades A and B) embryos from patients undergoing IVF, in a statistically significant manner. AMH was further positive trend of association with successful pregnancy outcomes.Conclusion
Overall, this study offers new insight on AMH effects upon cumulus cells and new aspects on how AMH might promote oocyte integrity and embryo viability at a biochemical level as well as add to the current body of evidence supporting AMH clinical potential as a more sensitive marker of IVF outcomes in comparison with FSH, regarding numbers of oocytes received and high-quality day 2 and day 3 embryos.
... This hypothesis was explored by measuring Gβγ-dependent AKT phosphorylation by FSHR-GPER heteromers, in response to FSH (Fig. 4H). Acute FSH treatment (15 min) did not induce pAKT activation in HEK293/FSHR cells, which requires chronic FSH stimulation to detect activation 43 . In contrast, pAKT activation was increased in FSH-treated HEK293/FSHR-GPER cells and was inhibited by the selective Gβγ inhibitor gallein. ...
Classically, follicle stimulating hormone receptor (FSHR) driven cAMP-mediated signaling boosts human ovarian follicle growth and would be essential for oocyte maturation. However, contradicting in vitro suggest a different view on physiological and clinical significance of FSHR-mediated cAMP signaling. We found that the G protein coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are effectively delivered upon equal expression levels of both receptors, while they are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with FSH responsiveness of patients undergoing controlled ovarian stimulation. Consistent with high FSHR expression levels during follicular selection, cell viability is dramatically reduced in FSHR overexpressing cells due to preferential coupling to the Gαs protein/cAMP pathway. In contrast, FSHR/GPER heteromer formation resulted in FSH-triggered anti-apoptotic/proliferative signaling delivered via the Gβγ dimer while heteromer impairment or GPER-associated Gαs inhibitory protein complexes resulted in cell death. GPER-depleted granulosa cells have an amplified FSH-dependent decrease in cell viability and steroidogenesis, consistent with the requirement of estrogen signaling for successful oocyte growth. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.
One Sentence Summary
FSHR/GPER heteromers block cAMP-dependent selection of ovarian follicles and target tumor growth and poor FSH-response in women.
... AdipoR1 activate AMPK. The mechanism latent have insulin-sensitizing action when induce adiponectin, so when found in skeletal muscle and liver the fAdiponectin stimulated AMPK phosphorylation, whereas gAdiponectin done stimulation only in skeletal muscle (Nechamen et al., 2007). AMPK activation is blocked by using the dominant-negative mutant and they inhibit the effects of both fAdiponectin or gAdiponectin, this activity indicate that through activation of AMPK stimulation of glucose for utilization and fatty acid breakdown is proceeding. ...
... A recent study indicated that APPL2 forms dimers with APPL1 via the BAR domain. APPL2 initiates follicle stimulation hormone signaling by forming a complex with the FSH receptor, APPL1, and Akt 2 [36]. Another study showed that APPL2 functions as a negative regulator in adiponectin signaling by competitively binding to the intracellular domain of AdipoR1 against APPL1 and, thereby, blocking adiponectin signaling in muscle cells [37]. ...
Adiponectin is the richest adipokine in human plasma, and it is mainly secreted from white adipose tissue. Adiponectin circulates in blood as high-molecular, middle-molecular, and low-molecular weight isoforms. Numerous studies have demonstrated its insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects. Additionally, decreased serum levels of adiponectin is associated with chronic inflammation of metabolic disorders including Type 2 diabetes, obesity, and atherosclerosis. However, recent studies showed that adiponectin could have pro-inflammatory roles in patients with autoimmune diseases. In particular, its high serum level was positively associated with inflammation severity and pathological progression in rheumatoid arthritis, chronic kidney disease, and inflammatory bowel disease. Thus, adiponectin seems to have both pro-inflammatory and anti-inflammatory effects. This indirectly indicates that adiponectin has different physiological roles according to an isoform and effector tissue. Knowledge on the specific functions of isoforms would help develop potential anti-inflammatory therapeutics to target specific adiponectin isoforms against metabolic disorders and autoimmune diseases. This review summarizes the current roles of adiponectin in metabolic disorders and autoimmune diseases.
... This conclusion is reflected in the follicular-like protein network shown in Figure 5A. If this transcriptional pattern is preserved at a protein level, we hypothesize that a direct interaction of Foxo1 with membrane-localized Fshr to form an atypical signaling complex would preclude the nuclear translocation of Foxo1 in aged multiparous ovarian cells, as observed in HEK 293 cells [54]. ...
In middle-aged women, the decline of ovarian follicle reserve below a critical threshold marks menopause, leading to hormonal, inflammatory, and metabolic changes linked to disease. The highest incidence and mortality of sporadic ovarian cancer (OC) occur at post-menopause, while OC risk is reduced by full-term pregnancies during former fertile life. Herein, we investigate how parity history modulates the ovarian transcriptome related to such declining follicle pool and systemic inflammation in reproductively-aged mice. Female C57BL/6 mice were housed under multiparous and virgin (nulliparous) breeding regimens from adulthood until estropause. The ovaries were then subjected to follicle count and transcriptional profiling, while a cytokine panel was determined in the sera. As expected, the follicle number was markedly decreased just by aging. Importantly, a significantly higher count of primordial and total follicles was observed in aged multiparous relative to aged virgin ovaries. Consistently, among the 65 genes of higher expression in aged multiparous ovaries, 27 showed a follicle count-like pattern, 21 had traceable evidence of roles in follicular/oocyte homeostasis, and 7 were transforming-growth factor beta (TGF-β)/bone morphogenetic protein (BMP) superfamily members. The remaining genes were enriched in cell chemotaxis and innate-immunity, and resembled the profiles of circulating CXCL1, CXCL2, CXCL5, CSF3, and CCL3, chemokines detected at higher levels in aged multiparous mice. We conclude that multiparity during reproductive life promotes the retention of follicle remnants while improving local (ovarian) and systemic immune-innate surveillance in aged female mice. These findings could underlie the mechanisms by which pregnancy promotes the long-term reduced OC risk observed at post-menopause.
... These phospho-proteins are key players modulating steroidogenesis, proliferation and survival/apoptosis (8,11), all molecular events underlying reproductive functions (12). Upon ligand binding, FSHR recruits other heterotrimeric Gα proteins, including Gαq and Gαi (13)(14)(15)(16), as well as other interactors (17), linking FSH action to multiple intracellular signaling pathways, such as the rapidly-activated, phospholipase C-dependent (18), cytosolic calcium cation (Ca 2+ ) release (19). FSHR internalization and recycling is mediated by βarrestin 1 and 2, which triggers G protein-independent ERK1/2 signaling (20,21). ...
Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10−3-1 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC50 range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 μg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.
... It is worth noting that the FSHR does not display a known PDZ ligand in its C-tail and the exact mechanism on how GIPC controls FSHR fate remains to be determined. However, APPL1, a known FSHR binding partner, localizes to a subset of VEE and displays a PDZ motif previously shown to interact with GIPC (64,65,77). A possible scenario would be that FSHR, via its interaction with APPL1, connects with GIPC and is targeted to VEE where it activates ERK (78). ...
Knowledge on G protein-coupled receptor (GPCRs) structure and mechanism of activation has profoundly evolved over the past years. The way drugs targeting this family of receptors are discovered and used has also changed. Ligands appear to bind a growing number of GPCRs in a competitive or allosteric manner to elicit balanced signaling or biased signaling (i.e., differential efficacy in activating or inhibiting selective signaling pathway(s) compared to the reference ligand). These novel concepts and developments transform our understanding of the follicle-stimulating hormone (FSH) receptor (FSHR) biology and the way it could be pharmacologically modulated in the future. The FSHR is expressed in somatic cells of the gonads and plays a major role in reproduction. When compared to classical GPCRs, the FSHR exhibits intrinsic peculiarities, such as a very large NH2-terminal extracellular domain that binds a naturally heterogeneous, large heterodimeric glycoprotein, namely FSH. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα subunits. G protein-coupled receptor kinases and β-arrestins are also recruited to this receptor and account for its desensitization, trafficking, and intracellular signaling. Different classes of pharmacological tools capable of biasing FSHR signaling have been reported and open promising prospects both in basic research and for therapeutic applications. Here we provide an updated review of the most salient peculiarities of FSHR signaling and its selective modulation.
... It is widely accepted that following FSHR activation, multiple signaling pathways are stimulated in parallel or in a sequential manner. The interaction of activated and phosphorylated FSHR with several receptor-associated proteins, including b-arrestins (101-104) and adaptor proteins containing PH domain, PTB domain, and leucine zipper motif (38,(105)(106)(107), is crucial for regulation of their signaling and functional outcomes. These associated proteins regulate several signaling pathways (38,108), including Erk, inositol triphosphate, and Ca 2+ . ...
Follicle stimulating hormone (FSHR) belongs to the leucine rich repeat family of GPCR (LGR) which includes the glycoprotein hormone receptors, LHR, TSHR and other LGRs 4-7. FSH is the key regulator of folliculogenesis in female and spermatogenesis in male. FSH elicits its physiological response through its cognate receptor on the cell surface. Binding of the hormone, FSH to its receptor, FSHR brings about conformational changes in the receptor which are transduced through the transmembrane domain to the intracellular region where the downstream effector interaction takes place leading to activation of the downstream signaling cascade. Identification of small molecules that could activate or antagonize FSHR provided interesting tools to study the signal transduction mechanism of the receptor. However, because of the nature of the ligand-receptor interaction of FSH-FSHR that contains multiple sites in the extracellular binding domain, most of the small molecule modulators of FSHR are unable to bind to the orthosteric site of the receptors, rather they modulate receptor activation through allosteric sites in the transmembrane region. This review will discuss allosteric modulation of FSHR primarily through the discovery of small molecule modulators focusing on current data on the status of development and the utility of these as tools to better understand signaling mechanisms.
... APPL1 implicates in its signal transduction to regulate Akt and/or MAPK pathways and exerts its effects on regulating cell growth in addition to the modulating glucose and lipid metabolism [27][28][29]. APPL2, the other APPL isoform sharing 54% identity sequences with APPL1, was documented to regulate glucose uptake in skeletal muscle through PI3K/Akt pathway [30][31][32][33]. However, whether APPLs could impact the central GR signaling and consequently affect adult neurogenesis as well as animal behaviors remain unexplored. ...
Adaptor proteins containing the pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPLs) are multifunctional adaptor proteins involved in regulating many biological activities and processes. The newly identified metabolic factor APPL2 showed the potentials to modulate cell growth, but whether APPL2 could affect adult neurogenesis and animal mood behaviors remains unknown. In the present study, APPL2 transgenic (Tg) mice and wild-type littermates were used for testing our hypothesis that APPL2 could affect glucocorticoid receptor (GR) signaling and modulate hippocampal neurogenesis, which contributes to depressive and anxiety behaviors. Compared with WT littermates, APPL2 Tg mice had enhanced GR phosphorylation under basic condition but had no different plasma corticosterone (CORT) level and GR phosphorylation under stress stimulation. APPL2 Tg mice had decreased hippocampal neurogenesis that was reversed by GR antagonist RU486. APPL2 Tg mice also showed the impaired hippocampal neurogenesis and presented the depressive and anxiety behaviors. In conclusion, APPL2 could be an important regulator for adult neurogenesis. APPL2 overexpression could blunt the activation of glucocorticoid receptor when undergoing environmental stress. Our study suggests that APPL2 might be a new therapeutic target for mental disorders.
... Similar to APPL1, APPL2 has an N-terminal BAR domain, central PH domain, and C-terminal PTB domain. APPL2 involves follicle-stimulating hormone signal transduction pathway by binding to APPL1 via their respective BAR domains [44]. ...
Adiponectin is the most abundant peptide secreted by adipocytes, whose reduction plays a central role in obesity-related diseases, including insulin resistance/type 2 diabetes and cardiovascular disease. In addition to adipocytes, other cell types, such as skeletal and cardiac myocytes and endothelial cells, can also produce this adipocytokine. Adiponectin effects are mediated by adiponectin receptors, which occur as two isoforms (AdipoR1 and AdipoR2). Adiponectin has direct actions in liver, skeletal muscle, and the vasculature.Adiponectin exists in the circulation as varying molecular weight forms, produced by multimerization. Several endoplasmic reticulum ER-associated proteins, including ER oxidoreductase 1-α (Ero1-α), ER resident protein 44 (ERp44), disulfide-bond A oxidoreductase-like protein (DsbA-L), and glucose-regulated protein 94 (GPR94), have recently been found to be involved in the assembly and secretion of higher-order adiponectin complexes. Recent data indicate that the high-molecular weight (HMW) complexes have the predominant action in metabolic tissues. Studies have shown that adiponectin administration in humans and rodents has insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects, and, in certain settings, also decreases body weight. Therefore, adiponectin replacement therapy in humans may suggest potential versatile therapeutic targets in the treatment of obesity, insulin resistance/type 2 diabetes, and atherosclerosis. The current knowledge on regulation and function of adiponectin in obesity, insulin resistance, and cardiovascular disease is summarized in this review.
... TrkC has a role in regulating growth and survival of many human cancer tissues and has been found to be a potential tumor suppressor in colon cancer that is commonly inactivated by both epigenetic and genetic mechanisms [41][42][43] and activates three downstream signaling pathways [44]. APPL1 which is involved in the regulation of cell proliferation [45], encodes a protein that interacts with p110α (encoded by PIK3CA gene) [46], Akt2 [47], p85 regulatory subunit of PI3K [48], and deleted in colorectal cancer (DCC) protein [49]. ...
PI3K/AKT signaling is involved in cell survival, proliferation, and migration. In this pathway, PI3Kα enzyme is composed of a regulatory protein encoded by p85 gene and a catalytic protein encoded by PIK3CA gene. Human PIK3CA locus is amplified in several cancers including lung and colorectal cancer (CRC). Therefore, microRNAs (miRNAs) that are encoded within the PIK3CA gene might have a role in cancer development. Here, we report a novel microRNA named PIK3CA-miR1 (EBI accession no. LN626315), which is located within PIK3CA gene. A DNA segment corresponding to PIK3CA-premir1 sequence was transfected in human cell lines that resulted in generation of mature exogenous PIK3CA-miR1. Following the overexpression of PIK3CA-miR1, its predicted target genes (APPL1 and TrkC) were significantly downregulated in the CRC-originated HCT116 and SW480 cell lines, detected by qRT-PCR. Then, dual luciferase assay supported the interaction of PIK3CA-miR1 with APPL1 and TrkC transcripts. Endogenous PIK3CA-miR1 expression was also detected in several cell lines (highly in HCT116 and SW480) and highly in CRC specimens. Consistently, overexpression of PIK3CA-premir1 in HCT116 and SW480 cells resulted in significant reduction of the sub-G1 cell distribution and apoptotic cell rate, as detected by flowcytometry, and resulted in increased cell proliferation, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PIK3CA-miR1 overexpression also resulted in Wnt signaling upregulation detected by Top/Fop assay. Overall, accumulative evidences indicated the presence of a bona fide novel onco-miRNA encoded within the PIK3CA oncogene, which is highly expressed in colorectal cancer and has a survival effect in CRC-originated cells.
... APPL1 also binds to signaling proteins such as PI3K and growth factor receptors (Lin et al., 2006;Nechamen et al., 2007;Tan et al., 2010;Wang et al., 2012). Interestingly, another form of LTD, induced by metabotropic glutamate receptors (mGluR-LTD), does involve activation of PI3K signaling, in addition to AMPAR internalization (Snyder et al., 2001;Hou and Klann, 2004). ...
Hippocampal synaptic plasticity involves both membrane trafficking events and intracellular signaling, but how these are coordinated is far from clear. The endosomal transport of glutamate receptors in and out of the postsynaptic membrane responds to multiple signaling cascades triggered by synaptic activity. In this work, we have identified APPL1 (Adaptor protein containing PH domain, PTB domain and leucine zipper motif 1) as a critical element linking trafficking and signaling during synaptic plasticity. We show that APPL1 knock-down specifically impairs PI3K-dependent forms of synaptic plasticity, such as long-term potentiation (LTP) and metabotropic glutamate receptor-dependent long-term depression (mGluR LTD). Indeed, we demonstrate that APPL1 is required for the activation of the PIP3 pathway in response to LTP induction. This requirement can be bypassed by membrane localization of PI3K and is related to phosphoinositide binding. Interestingly, inhibitors of PDK1 and Akt have no effect on LTP expression. Therefore, we conclude that APPL1 gates PI3K activation at the plasma membrane upon LTP induction, which is then relayed by downstream PIP3 effectors different from PDK1 and Akt.
... Endocytic adaptor proteins have also been observed in nuclear signaling. Nuclear localization signals have been detected in the adaptors HIP1, DAB1 and APPL2 (Honda and Nakajima, 2006;Mills et al., 2005;Nechamen et al., 2007). EPS15, Epsin1, CALM and β-arrestin have also been detected in the nucleus, though no nuclear localization signals have been identified in these proteins (Archangelo et al., 2006;Hyman et al., 2000;Poupon et al., 2002;Wang et al., 2003). ...
The receptor tyrosine kinase (RTK) signaling pathway is strongly activated in most forms of cancer. As a result, manipulation of the RTK pathway is a common means of promoting oncogenesis. Recently, proteins in the RTK endocytosis pathway have been added to the list of oncoproteins that affect RTK signaling. Among these proteins is Huntingtin Interacting Protein 1 (HIP1), an endocytic adaptor protein that interacts with clathrin, actin, adaptor protein 2 (AP2) and cargo proteins, including RTKs. HIP1 is expressed at high levels in many forms of cancer in a manner that also correlates with RTK overexpression. Additionally, HIP1 overexpression transforms fibroblasts by increasing RTK expression to enable anchorage-independent growth. Though HIP1 has been found to interact with and stabilize multiple RTKs following stimulation, the precise role(s) of HIP1 during the endocytic process remain unclear.
This thesis examines the interaction between HIP1 and RTKs. First, high HIP1 levels were found in a rare, deadly, form of cancer, Merkel cell carcinoma (MCC), which also expresses the RTK c-Kit at high levels. Patients with metastatic MCC also demonstrated high levels of circulating antibody produced against HIP1. Additionally, HIP1 was found to interact with c-Kit and also stabilize this receptor following stem cell factor (SCF) ligand stimulation. These data collectively indicate that targeting HIP1 in MCC may improve patient prognosis. To further investigate a means of targeting HIP1 interaction with RTKs, we found that the EGFR kinase domain was necessary for HIP1 association with this receptor. HIP1 was also found to be tyrosine phosphorylated when HIP1 and EGFR are co-expressed. This phosphorylation is maintained for 15 to 30 minutes after stimulation, at which time HIP1 and EGFR localize to Rab5 sorting endosomes. Furthermore, a novel form of EGFR was identified that strongly interacts with HIP1 and structurally resembles the oncoprotein EGFRvIII. These findings all provide insight into possible mechanisms of how HIP1-mediates RTK stabilization in cancer.
... However, opposite results describing negative cooperativity between FSHR and LHCGR were previously found (Feng et al., 2013), revealing decreased G a s protein-dependent signaling in cotransfected HEK293 cells under FSH or hCG treatment. It suggests that cell-dependent, peculiar FSHR:LHCGR ratio and intracellular milieu may result in partial receptor homo-oligomers and different coupling to receptor-specific intracellular interactors (Kanamarlapudi et al., 2012;Nechamen et al., 2007), thus modulating the downstream signaling. Therefore, further studies are needed to fully elucidate the mechanisms underlying gonadotropin receptor heterodimers. ...
Luteinizing hormone (LH) and choriogonadotropin (hCG) are glycoprotein hormones regulating ovarian function and pregnancy, respectively. Since these molecules act on the same receptor (LHCGR), they were traditionally assumed as equivalent in assisted reproduction techniques (ART), although differences between LH and hCG were demonstrated at molecular and physiological level. In this study, we demonstrated for the first time that co-treatment with a follicle-stimulating hormone (FSH) dose in the ART therapeutic range potentiates different LH- and hCG-dependent responses in vitro, measured in terms of cAMP, phospho-CREB, -ERK1/2 and -AKT activation, gene expression, progesterone and estradiol production in human granulosa-lutein cells (hGLC). We show that in the presence of FSH, hCG biopotency is about 5-fold increased, in the presence of FSH, in terms of cAMP activation. Accordingly, CREB phosphorylation and steroid production is increased under hCG and FSH co-treatment. LH effects, evaluated as steroidogenic cAMP/PKA pathway activation, do not change in the presence of FSH, which, however, increases LH-dependent ERK1/2 and AKT, but not CREB phosphorylation, resulting in anti-apoptotic effects. The different modulatory activity of FSH on LH and hCG action in vitro corresponds to their different physiological functions, reflecting proliferative effects exerted by LH during the follicular phase and before trophoblast development, and the high steroidogenic potential of hCG requested to sustain pregnancy from the luteal phase onwards.
... Although, activation of GPHRs induces the coupling of different G proteins (60), most of physiological activities are mediated through a Gαs protein induced adenyl cyclase catalyzing the conversion of ATP into cAMP (1). However, as suggested by studies on TSHR deletions and others focusing on GPHRs signaling and trafficking, "biased agonism, " also referred to as "ligand directed signaling" is likely caused by a spectrum of different ligand-receptor complex conformations in combination with other cell-specific factors (1,(61)(62)(63). Recent studies indicated that different GPH glycoforms may have distinct effects on signaling and result in a biased agonism (64). ...
Last two decades of structure-function studies performed in numerous laboratories provided substantial progress in understanding basic science, physiological, pathophysiological, pharmacological, and comparative aspects of glycoprotein hormones (GPHs) and their cognate receptors. Multiple concepts and models developed based on experimental data in the past stood the test of time and have been, at least in part, confirmed and/or remained compatible with the new structures resolved at the atomic level. Major advances in understanding of the ligand-receptor relationships are heralding the dawn of a new era for GPHs and their receptors, although many basic questions still remain unanswered. This article examines retrospectively several basic science aspects of GPH super-agonists and related "biosuperiors" in a broader context of the advances in the ligand-receptor structure-function relationships and new mechanistic models generated based on the structure elucidation. Due to selective focus of my comments and perspectives in certain parts, the reader is directed to the most relevant publications and reviews in the field for more comprehensive analyses.
... PTX3 is an important component of the cumulus expansion process (Salustri et al., 2004), NR3C1 has been shown to play a role in suppression of apoptosis in bovine corpus luteum (Komiyama et al., 2008) and APPL2 has been identified as a binding partner of FSHR in complex with akt2 and FOXO1a (Nechamen et al., 2007) and has a possible role in FSH signaling. Taken together, these results suggest that CDKN2A plays a major role in the arrest of proliferation and maturation of human CC during final folliculogenesis. ...
Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mam-malian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment.
... Another report showed that both APPL proteins have opposing effects in adiponectin-and insulin-mediated signaling, where APPL2 competitively inhibits APPL1 binding to the receptors and downregulates signaling (Wang et al., 2009). Our (Miaczynska et al., 2004;Nechamen et al., 2007;Rashid et al., 2009;Wang et al., 2009Wang et al., , 2012Zoncu et al., 2009;Bohdanowicz et al., 2012). ...
Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to active innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking and here we show in macrophages that Rab31-GTP is additionally specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P2 to PI(3,4,5)P3 and it persists on PI(3)P-enriched phagosomes. During early phagocytosis we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P3 and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Interestingly, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex that is also involved in phagocytosis and signaling.
© 2015 by The American Society for Cell Biology.
... Several Gαs-independent mechanisms capable of functionally coupling the FSHR with intracellular signaling cascades have been reported to physically and/or functionally interact with the FSHR and to trigger downstream signaling events (Zeleznik et al., 2003;Escamilla-Hernandez et al., 2008;Gloaguen et al., 2011). These mechanisms involve Gαi (Arey et al., 1997;Crépieux et al., 2001), β-arrestins (Kara et al., 2006;Wehbi et al., 2010a,b;Tranchant et al., 2011), Epithelial Growth Factor Receptor (EGFR) (Cottom et al., 2003;Andric and Ascoli, 2006;Yang and Roy, 2006;Wayne et al., 2007) and Adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) (Nechamen et al., 2004(Nechamen et al., , 2007Thomas et al., 2011). Although the coupling mechanisms that promote Phosphoinositide 3-kinase (PI3K) (Zeleznik et al., 2003) and Phosphatase and Tensin homolog deleted on chromosome 10 (PTEN) (Dupont et al., 2010) activation upon FSH exposure are as yet unidentified, APPL1 is a likely candidate because it has been reported to trigger downstream signaling through PI3K (Nechamen et al., 2004). ...
Follicle-stimulating hormone (FSH) is a master endocrine regulator of mammalian reproductive functions. Hence, it is used to stimulate folliculogenesis in assisted reproductive technologies (ART), both in women and in breeding animals. However, the side effects that hormone administration induces in some instances jeopardize the success of ART. Similarly, the luteinizing hormone (LH) is also of paramount importance in the reproductive function because it regulates steroidogenesis and the LH surge is a pre-requisite to ovulation. Gaining knowledge as extensive as possible on gonadotropin-induced biological responses could certainly lead to precise selection of their effects in vivo by the use of selective agonists at the hormone receptors. Hence, over the years, numerous groups have contributed to decipher the cellular events induced by FSH and LH in their gonadal target cells. Although little is known on the effect of gonadotropins on microRNA expression so far, recent data have highlighted that a microRNA regulatory network is likely to superimpose on the signaling protein network. No doubt that this will dramatically alter our current understanding of the gonadotropin-induced signaling networks. This is the topic of this review to present this additional level of complexity within the gonadotropin signaling network, in the context of recent findings on the microRNA machinery in the gonad.
... On the other hand, the present study shows that APPL2 suppresses insulin-dependent glucose uptake at a step downstream of Akt. Unlike APPL1, APPL2 does not interact with Akt or the regulatory subunit p85 and catalytic subunit p110 of PI3K (7,29,30). Indeed, overexpression of APPL2 results in only a modest decrease in insulin-elicited Akt phosphorylation in myotubes, whereas targeted deletion of APPL2 in skeletal muscle has no obvious effect on Akt phosphorylation. Such a modest effect of APPL2 on Akt activity in cultured cells is perhaps a result of its heterodimerization with APPL1, which in turn prevents the binding of APPL1 to Akt (16). ...
Insulin stimulates glucose uptake by promoting the trafficking of GLUT4 to the plasma membrane in muscle cells, and impairment of this insulin action contributes to hyperglycemia in type 2 diabetes. The adaptor protein APPL1 potentiates insulin-stimulated Akt activation and downstream actions. However, the physiological functions of APPL2, a close homologue of APPL1, in regulating glucose metabolism remain elusive. Here we showed that insulin-evoked plasma membrane recruitment of GLUT4 and glucose uptake were impaired by APPL2 overexpression, but enhanced by APPL2 knockdown. Likewise, conditional deletion of APPL2 in skeletal muscles enhanced insulin sensitivity, leading to an improvement of glucose tolerance. We identified the Rab-GTPase-activating protein TBC1D1 as an interacting partner of APPL2. Insulin stimulated TBC1D1 phosphorylation on serine-235, leading to enhanced interaction with the BAR domain of APPL2, which in turn suppressed insulin-evoked TBC1D1 phosphorylation on threonine-596 in cultured myotubes and skeletal muscle. Substitution of serine-235 with alanine diminished APPL2-mediated inhibition on insulin-dependent TBC1D1 phosphorylation on threonine-596 and the suppressive effects of TBC1D1 on insulin-induced glucose uptake and GLUT4 translocation to the plasma membrane in cultured myotubes. Therefore, the APPL2-TBC1D1 interaction serves as a key step to fine-tune insulin-stimulated glucose uptake by regulating the membrane recruitment of GLUT4 in skeletal muscle.
Gonadotropins are glycoprotein sex hormones regulating development and reproduction and bind to specific G protein-coupled receptors expressed in the gonads. Their effects on multiple signaling cascades and intracellular events have recently been characterized using novel technological and scientific tools. The impact of allosteric modulators on gonadotropin signaling, the role of sugars linked to the hormone backbone, the detection of endosomal compartments supporting signaling modules, and the dissection of different effects mediated by these molecules are areas that have advanced significantly in the last decade. The classic view providing the exclusive activation of the cAMP/protein kinase A (PKA) and the steroidogenic pathway by these hormones has been expanded with the addition of novel signaling cascades as determined by high-resolution imaging techniques. These new findings provided new potential therapeutic applications. Despite these improvements, unanswered issues of gonadotropin physiology, such as the intrinsic pro-apoptotic potential to these hormones, the existence of receptors assembled as heteromers, and their expression in extragonadal tissues, remain to be studied. Elucidating these issues is a challenge for future research.
G-protein-coupled receptors (GPCRs) form the largest family of receptors in humans, with over 800 members. These receptors mediate the effects of many hormones and convey numerous endocrine functions including growth, appetite regulation, bone development, glucose homeostasis and reproduction. Consequently, mutations in these receptors cause a spectrum of disorders and studies of these diseases has provided new insights into GPCR functions. This has led to an enhanced appreciation of the complexity of GPCR signaling achieved, in part, by the ability of receptors to couple to multiple G-protein pathways, utilization of sophisticated spatiotemporal signaling from plasma membrane and intracellular compartments, oligomerization of receptors and activation of G-protein-independent pathways. Pharmacological strategies for targeting GPCRs have become increasingly nuanced with the development of chimeric ligands targeting multiple receptors and the emergence of compounds that enable biased signaling. Future therapeutic approaches are likely to further enhance tissue and functional specificity. This article focusses on GPCRs involved in energy metabolism and growth (melanocortin, ghrelin, somatostatin, GLP-1 and GIP receptors); calcium homeostasis and skeletal development (calcium-sensing and parathyroid hormone receptors); thyroid function (TSH receptor); and reproduction (GnRHR, LHR, FSHR, KISS1R). Mutations in these GPCRs contribute to diverse human disorders including severe obesity, hyper/hypocalcaemia, skeletal dysplasia, hyper/hypothyroidism, precocious puberty, and delayed sexual development, and many are targets for currently approved drugs. For each receptor an understanding of the biological function, signal transduction mechanisms and pathophysiological consequences of human mutations will be explained, and current and future therapeutic strategies for targeting each receptor described.
Follicle-stimulating hormone receptor (FSHR) is a class A G protein-coupled receptor that belongs to the subfamily of glycoprotein hormone receptors (GPHRs). The interaction of FSH with FSHR triggers downstream signaling pathways that play a central role in mammalian reproduction, such as folliculogenesis in females and the maintenance of spermatogenesis in males. This warrants a detailed investigation into FSHR, from its genesis, to the post-translational modifications that enable it to become functionally competent, followed by its trafficking to the cell membrane. Subsequently, FSH-stimulated Gs uncoupling and transduction of G protein-mediated signaling pathways takes place, after which the receptor undergoes β-arrestin-mediated internalization and may trigger other noncanonical signaling pathways. The majority of the FSH-FSHR complexes are recycled back to the cell surface and only a small proportion are routed to lysosomal degradation pathways, thus completing the lifecycle of the FSH receptor. Information about important epitopes and aspects of FSH receptor function has been gleaned from a number of sources, including structure-function studies on both naturally occurring and induced mutations, single nucleotide polymorphisms, peptides and antipeptide antibodies corresponding to predicted functional residues, X-ray crystallography analysis and high resolution imaging studies, in addition to the information available for the other GPHRs. In this review, we have traversed through the life cycle of the FSH receptor and discuss the reproductive pathophysiologies that could result from an impairment in receptor function, as may arise from defects during its journey from its birth to its degradation. Moreover, the unresolved questions and challenges that require exploration have been highlighted.
Adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) plays a central role as the main contributing factor in the adiponectin and insulin signaling. This review aims to discuss previous and recent findings concerning the role of APPL1 in the polycystic ovary syndrome (PCOS) patients with conclusions regarding more efficient therapeutic approaches. A literature review was performed in PubMed, Web of Science, ScienceDirect, Scopus and, Google Scholar from Aug 1999 to May 2020. This study reveals APPL1 has a key role in adiponectin, insulin, and follicle‐stimulating hormone (FSH) signaling pathways occurring within the ovaries. Recent studies in mouse model systems have indicated that APPL1 can prevent diabetes, endothelial disorders, and insulin resistance. In contrast, APPL1 deficiency can lead to the metabolic and vascular disorders. APPL1 due to its potential roles in different signaling pathways might be suggested as a novel diagnostic and therapeutic option for prediction of ovarian dysfunctions, and treatment of reproductive disorders especially PCOS.
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Background
Adiponectin is a protein hormone secreted by adipocytes.
Aim
The aim of present review is to enlist them all, and mention their significance in obesity, which helps researchers in drug discovery and future medicine.
Material and methods
The last 20 years data has been searched and studied to mention their current role in obesity treatment and ailments of disease.
Results and conclusion
Adiponectin exert its action through receptors AdipoR1, AdipoR2, T-cadherin. In muscle AdipoR1 is exhibit, while in liver AdipoR2 is mainly exhibit. AMPK and PPARα pathway in liver and skeletal muscle are magnified by adiponectin. Adiponectin show many function like expand fatty acids oxidation, control glucose level and manage receptor activity. Circulating level of adiponectin is lower in case of obesity, insulin resistance and other metabolic disorder. In this review we will focus on role of adiponectin and its receptors in metabolic disorder obesity.
Models of G protein-coupled receptor (GPCR) signaling have dramatically altered over the past two decades. Indeed, GPCRs such as the follicle-stimulating hormone receptor (FSHR) have contributed to these new emerging models. We now understand that receptor signaling is highly organized at a spatial level, whereby signaling not only occurs from the plasma membrane but distinct intracellular compartments. Recent studies in the role of membrane trafficking and spatial organization of GPCR signaling in regulating gonadotropin hormone receptor activity has identified novel intracellular compartments, which are tightly linked with receptor signaling and reciprocally regulated by the cellular trafficking machinery. Understanding the impact of these cell biological mechanisms to physiology and pathophysiology is emerging for certain GPCRs. However, for FSHR, the potential impact in both health and disease and the therapeutic possibilities of these newly identified systems is currently unknown, but offers the potential to reassess prior strategies, or unveil novel opportunities, in targeting this receptor.
Our understanding of G protein-coupled receptor (GPCR) signalling has significantly evolved over the past decade, whereby signalling not only occurs from the plasma membrane but continues, or is reactivated, following internalisation in to endosomal compartments. The spatial organisation of GPCRs is thus essential to decode dynamic and complex signals, and to activate specific downstream pathways that elicit the appropriate cellular response. For the gonadotrophin hormone receptors, membrane trafficking has been demonstrated to play a significant role in regulating its signal activity that in turn would impact at physiological and even pathophysiological level. Here we will describe the developments in our understanding of the role of ‘location’ in gonadotrophin hormone receptor signalling, and how these receptors have unveiled fundamental mechanisms of signal regulation likely to be pertinent for other GPCRs. We will also discuss the potential impact of spatially-controlled gonadotrophin hormone receptor signalling in both health and disease, and the therapeutic possibilities this new understanding of these receptors, so key in reproduction, offers.
The risk of osteoporosis and cardiovascular disease increases significantly in postmenopausal women. Until recently, the underlying mechanisms have been primarily attributed to estrogen decline following menopause. However, follicle-stimulating hormone (FSH) levels rise sharply during menopausal transition and are maintained at elevated levels for many years. FSH receptor has been detected in various extragonadal sites, including osteoclasts and endothelial cells. Recent advances suggest FSH may contribute to postmenopausal osteoporosis and cardiovascular disease. Here, we review the key actions through which FSH contributes to the risk of osteoporosis and cardiovascular disease in women as they transition through menopause. Advancing our understanding of the precise mechanisms through which FSH promotes osteoporosis and cardiovascular disease may provide new opportunities for improving health-span for postmenopausal women.
FSH is synthesized in the pituitary by gonadotrope cells. By binding to and interacting with its cognate receptor (FSHR) in the gonads, this gonadotropin plays a key role in the control of gonadal function and reproduction. Upon activation, the FSHR undergoes conformational changes leading to transduction of intracellular signals, including dissociation of G protein complexes into components and activation of several associated interacting partners, which concertedly regulate downstream effectors. The canonical Gs/cAMP/protein kinase A pathway, considered for a long time as the sole effector of FSHR-mediated signaling, is now viewed as one of several mechanisms employed by this receptor to transduce intracellular signals in response to the FSH stimulus. This complex network of signaling pathways allows for a fine-tuning regulation of the gonadotropic stimulus, where activation/inhibition of its multiple components vary depending on the cell context, cell developmental stage, and concentration of associated receptors and corresponding ligands. Activation of these multiple signaling modules eventually converge to the hormone-integrated biological response, including survival, proliferation and differentiation of target cells, synthesis and secretion of paracrine/autocrine regulators and, at the molecular level, functional selectivity and differential gene expression.
In this minireview, we briefly discuss the complexity of FSHR-mediated intracellular signals activated in response to ligand stimulation. A better understanding of the signaling pathways involved in FSH action might potentially influence the development of new therapeutic strategies for reproductive disorders.
Signaling activated by binding of the C-X-C motif chemokine ligand CXCL12 to its cognate G protein-coupled receptor (GPCR), chemokine C-X-C motif receptor 4 (CXCR4), is linked to metastatic disease. Yet the mechanisms governing CXCR4 signaling remain poorly understood. Here we show that endocytosis and early endosome antigen 1 (EEA1), which is part of the endosome fusion machinery, are required for CXCL12-mediated AKT Ser/Thr kinase (Akt) signaling selective for certain Akt substrates. Pharmacological inhibition of endocytosis partially attenuated CXCL12-induced phosphorylation of Akt, but not phosphorylation of ERK-1/2. Similarly, phosphorylation of Akt, but not ERK-1/2, stimulated by CXCL13, the cognate ligand for the chemokine receptor CXCR5, was also attenuated by inhibited endocytosis. Further, siRNA-mediated depletion of the Rab5-effector EEA1, but not of adaptor protein, phosphotyrosine-interacting with PH domain and leucine zipper 1 (APPL1), partially attenuated Akt, but not ERK-1/2, phosphorylation promoted by CXCR4. Attenuation of Akt phosphorylation through inhibition of endocytosis or EEA1 depletion was associated with reduced signaling to Akt substrates forkhead box O1/3a, but not the Akt substrates TSC complex subunit 2 or glycogen synthase kinase 3β. This suggested that endocytosis and endosomes govern discrete aspects of CXCR4- or CXCR5-mediated Akt signaling. Consistent with this hypothesis, depletion of EEA1 reduced the ability of CXCL12 to attenuate apoptosis in suspended, but not adherent, HeLa cells. Our results suggest a mechanism whereby compartmentalized chemokine-mediated Akt signaling from endosomes suppresses the cancer related process known as anoikis. Targeting this signaling pathway may help inhibit metastatic cancer involving receptors such as CXCR4.
The gonads (ovary, testis) produce gametes (oocytes and spermatozoa). The pituitary glycoprotein hormones luteinizing hormone (LH, lutropin) and follicle-stimulating hormone (FSH, follitropin) are gonadotropins because their targets are the gonads. Another hormone produced by the syncytiotrophoblast of the human placenta is human chorionic gonadotropin (hCG, choriogonadotropin), and its activity is like LH. This review describes the protein structure, gene structure, and regulation of biosynthesis of both the gonadotropins and their membrane bound receptors. The functions of both hormones and receptors are covered extensively, as well as naturally occurring mutations that have been identified in disease states and therapies derived from their study.
Endosomal adaptor proteins are important regulators of signaling pathways underlying many biological processes. These adaptors can integrate signals from multiple pathways via localization to specific endosomal compartments, as well as through multiple protein- protein interactions. One such adaptor protein that has been implicated in regulating signaling pathways is the adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1). APPL1 localizes to a subset of Rab5-positive endosomes through its Bin-Amphiphysin-Rvs and PH domains, and it coordinates signaling pathways through its interaction with many signaling receptors and proteins through its PTB domain. This review discusses our current understanding of the role of APPL1 in signaling and trafficking, as well as highlights recent work into the function of APPL1 in cell migration and adhesion.
AIMS
Previously, we verified that overtrained mice upregulated the TRB3 levels, its association with Akt, and the hepatic concentrations of glycogen. It is known that APPL1 can limit the interaction between TRB3 and Akt, playing an important role in the glucose homeostasis. Thus, we verified the effects of three overtraining protocols on the hepatic levels of APPL1 and APPL2.
METHODS
Rodents were divided into control (CT), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR). The hepatic contents of APPL1 and APPl2 were measured by the immunoblotting technique.
RESULTS
Significant elevation of APPL1 observed in the OTR/down and OTR/up groups, as well as the tendency of increase (p=0.071) observed in the OTR group.
CONCLUSION
These results indicate that this particular protein is likely to participate in the glucose homeostasis previously observed in response to these OT protocols.
Epidermal growth factor receptor (EGFR) mediates multiple signaling pathways that regulate cell migration, proliferation, and differentiation. Adaptor protein APPL1 has been reported to function as a downstream effector of EGFR signaling pathway. However, molecular mechanisms underlying the role of APPL1 downstream of EGFR signaling remains elusive. Here, we identified APPL1 as a critical molecule that interacts with EGFR. Suppression of APPL1 by siRNA inhibited EGF-stimulated Akt phosphorylation. Functionally, EGF stimulation of cells caused phosphorylation of APPL1 at Ser636, which subsequently promoted the interaction between APPL1 and EGFR, indicating that APPL1 sensitizes EGF stimulation by acting at a site downstream of the EGFR signaling. Importantly, non-phosphorylatable mutant of APPL1 reduced cell migration compared with wild-type APPL1 in an Akt-dependent manner. Our study reveals a novel function of APPL1 in EGF signaling and defines a novel molecular mechanism by which phosphorylation of APPL1 upon EGF stimulation regulates cell migration underlying EGF-stimulated Akt pathway.
Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the β2-adrenergic receptor (β2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the β2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the β2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates β2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the β2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of β-arrestin, β-arrestin-(319–418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the β2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the β2AR is in part due to trafficking of the β2AR via the clathrin-coated pit endosomal pathway to lysosomes.
The FKHR gene was first identified from its disruption by the t(2;13) chromosomal translocation seen in the pediatric tumor alveolar rhabdomyosarcoma. It encodes for a member of the forkhead family of transcription factors. Recently, a homolog of FKHR in the nematode Caenorhabditis elegans was identified called DAF-16, which is a downstream target of two Akt homologs in an insulin-related signaling pathway. We have examined the possible role of Akt in the regulation of FKHR. We find that FKHR can bind in vitro to the insulin-responsive sequence (IRS) in the insulin-like growth factor-binding protein 1 promoter and can activate transcription from a reporter plasmid containing multiple copies of the IRS. Expression of active but not inactive Akt can suppress FKHR-mediated transcriptional activation. Akt can phosphorylate FKHR in vitro on three phosphoacceptor sites, at least a subset of which can also be phosphorylated by Akt in vivo. Importantly, mutation of these three sites to alanine residues enhances the transcriptional activity of FKHR and renders it resistant to inhibition by Akt. Expression of an Akt-resistant mutant of FKHR causes apoptosis in 293T cells in a manner dependent on DNA binding. These results suggest that FKHR may be a direct nuclear regulatory target for Akt in both metabolic and cell survival pathways.
AKT2 is a serine/threonine kinase implicated in human ovarian and pancreatic cancers. AKT2 is activated by a variety of growth factors and insulin via phosphatidylinositol 3-kinase (PI3K). However, its normal cellular role is not well understood. To gain insight into the function of AKT2, we performed yeast two-hybrid system to screen for interacting proteins. Using this technique, we identified a novel interactor, designated APPL, which contains a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a leucine zipper, classes of motifs defined in signaling molecules as functional interaction domains with specific targets. The PH domain of APPL shows similarity to those found in GTPase-activating proteins such as oligophrenin-1 and Graf, whereas its PTB domain exhibits homology with CED-6, an adaptor protein that promotes engulfment of apoptotic cells, and IB1, a transactivator of the GLUT2 gene. APPL is highly expressed in skeletal muscle, heart, ovary and pancreas, tissues in which AKT2 mRNA is abundant. APPL interacts with the inactive form of AKT2; moreover, APPL binds to the PI3K catalytic subunit, p110alpha. These data suggest that APPL is an adaptor that may tether inactive AKT2 to p110alpha in the cytoplasm and thereby may expedite recruitment of AKT2 and p110alpha to the cell membrane upon mitogenic stimulation. Furthermore, the APPL gene was mapped to human chromosome 3p14.3-p21.1, where deletions and other rearrangements have often been reported in a variety of tumor types. The identification of APPL may facilitate further analysis of the physiological and oncogenic activities of AKT2.
Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide
phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for
a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this
sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin
2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes
with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of
these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable
dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.
Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.
DCC (deleted in colorectal cancer) is a candidate tumor suppressor gene. However the function ofDCC remains elusive. Previously, we demonstrated that forced expression of DCC induces apoptosis or cell cycle arrest (Chen, Y. Q., Hsieh, J. T., Yao, F., Fang, B., Pong, R. C., Cipriano, S. C. & Krepulat,
F. (1999) Oncogene 18, 2747–2754). To delineate the DCC-induced apoptotic pathway, we have identified a protein, DIP13α, which
interacts with DCC. The DIP13α protein has a pleckstrin homology domain and a phosphotyrosine binding domain. It interacts
with a region on the DCC cytoplasmic domain that is required for the induction of apoptosis. Although ectopic expression of
DIP13α alone causes only a slight increase in apoptosis, co-expression of DCC and DIP13αresults in an ∼5-fold increase in apoptosis. Removal of the DCC-interacting domain on DIP13α abolishes its ability to enhance
DCC-induced apoptosis. Inhibition of endogenous DIP13αexpression by small interfering RNA blocks DCC-induced apoptosis. Our data suggest that DIP13α is a mediator of the DCC apoptotic
pathway.
In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote
activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the
ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic
stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates
with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation.
FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition
and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with
this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide
Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by
a 100-kDa phosphotyrosine phosphatase.
APPL may function as an adapter protein to modulate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Although we have previously proven that the PI3K/Akt pathway can suppress androgen receptor (AR) transactivation, the potential linkage from APPL to the AR remains unclear. Here we demonstrated that APPL could suppress AR-mediated transactivation in a dose-dependent manner in LNCaP and PC-3 cells. This suppressive effect could be blocked by either dominant-negative Akt or dominant-negative PI3K or LY294002, suggesting that the APPL-mediated suppression of AR transactivation is dependent on the PI3K/Akt pathway. We also observed that APPL could further enhance the Akt-mediated suppression of AR transactivation and AR target gene using the reporter gene and Northern blot assay. APPL was able to enhance insulin-like growth factor (IGF-1)-mediated Akt activation. The abrogation of IGF-1-mediated Akt activation by the dominant-negative PI3K or LY294002 or antisense APPL suggests that APPL may function as an important adapter protein in controlling the IGF-1 --> Akt signal pathway. Co-immunoprecipitation and glutathione S-transferase pull-down assays suggest that APPL, Akt, and AR may exist in a complex and Akt may serve as an important bridge factor for the association of APPL with AR. Together, our data indicate that APPL may suppress AR transactivation via potentiating Akt activity.
The forkhead family of transcription factors is conserved in evolution and known to play critical roles in the regulation of cellular differentiation and proliferation in many systems. The current studies demonstrate for the first time that forkhead homolog in rhabdomyosarcoma (FKHR) (FoxO1a) is expressed in porcine granulosa cells, and FSH stimulates FKHR phosphorylation and regulates its subcellular localization in this system. RT-PCR and Western blot studies demonstrated that FKHR is expressed and showed no change in FKHR message or protein levels in response to FSH (0-6 h). However, [32p]-orthophosphate labeling of cultured granulosa cells revealed robust phosphorylation after FSH treatment for 30 min. In addition, FSH caused nuclear exclusion of FKHR in these cells, apparently through the phosphatidylinositol 3-kinase signal transduction pathway. The cytosolic accumulation of FKHR protein that was observed in FSH-treated cells both by Western blot and immunohistochemistry was blocked when the cells were preincubated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our data also demonstrate that Akt/protein kinase B, an established kinase for FKHR, is phosphorylated in response to FSH treatment. Interestingly, although FKHR was phosphorylated by 30 min after FSH treatment, the time course for Akt phosphorylation was relatively delayed and sustained. Although these studies do not preclude Akt involvement in FSH-stimulated FKHR phosphorylation, they do suggest that other kinases may contribute to rapid signaling to FKHR. Because FKHR has been shown to activate genes involved in apoptosis and growth inhibition, FSH may promote growth and survival by initiating the phosphorylation of FKHR, causing its nuclear exclusion, and reducing its effect as a cell cycle arrest or death-promoting transcription factor.
The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in
endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes.
With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo.
The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that
BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal
BAR domain is a dimerization, membrane-binding, and curvature-sensing module.
We sought to elucidate the role of AKT in follicle-stimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Our results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol (PI) 3-kinase/AKT activity leads to Rheb (Ras homolog enriched in brain) and subsequent mTOR (mammalian target of rapamycin) activation. mTOR then stimulates translation by phosphorylating p70 S6 kinase and, consequently, the 40 S ribosomal protein S6. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-alpha, microtubule-associated protein 2D, and the PKA type IIbeta regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSH-stimulated HIF-1 activity is inhibited by the PI 3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277 (farnesyltransferase inhibitor-277), and the mTOR inhibitor rapamycin. Finally, we find that the FSH-mediated up-regulation of reporter activities for LHR, inhibin-alpha, and vascular endothelial growth factor is dependent upon HIF-1 activity, because a dominant negative form of HIF-1alpha interferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI 3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers.
A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype.
RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.
BAR-domains recently took centre stage in science through a report on the crystal structure of this domain in Drosophila Amphiphysin. Though only weakly conserved at the sequence level, the structure of the BAR domain shows striking similarity to the GTPase-binding domain of Arfaptin 2, an effector of Rho- and Arf- GTPases. On the basis of this sequence and structural similarity, these two proteins have been classified as belonging to the same family, the BAR-domain family, and they probably also have similar functional characteristics. Presented here are the results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2. This search identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis. For example, the BAR-domain family includes Endophilins, GTPase-activating proteins of the Centaurinbeta family and Oligophrenins, the adaptor proteins APPL1 and APPL2 that were recently shown to interact with the small GTPase Rab5, as well as members of the Sorting nexin family. On the basis of the structures of Amphiphysin and Arfaptin 2 and the cellular role of Amphiphysins in the early steps of endocytosis, the functions of the BAR domain have been defined as a dimerization motif and as sensing and inducing membrane curvature. However, data on Arfaptin 2 and now also on the Adaptor proteins APPL1 and 2 suggest that another function of the BAR domain is to bind to small GTPases.
This study investigates the possibility that FSH activates the p38 mitogen-activated protein kinase (MAPK) pathway in immature granulosa cells (GC). FSH induced the phosphorylation (activation) of p38 MAPK as evaluated by immunoprecipitation and by phosphorylation-specific immunoblotting. FSH-induced phosphorylation of p38 MAPK was blocked by pretreatment with the protein kinase A (PKA) inhibitor H89 and mimicked by the cAMP generating agonist forskolin, indicating that FSH-induced cAMP productin and PKA activation are necessary and sufficient for the activation of p38 MAPK in GC. The small heat shock protein HSP-27 comprises a downstream phosphorylation target for the p38 MAPK pathway. FSH-induced phosphorylation of HSP-27 was blocked by pretreatment with the p38 MAPK inhibitor SB 203580, indicating that p38 MAPK activation is necessary for FSH-induced HSP-27 phosphorylation. FSH-induced GC rounding/aggregation was blocked by pretreatment with SB 203580 indicating that p38 MAPK activation is necessary for FSH-induced GC cell shape change. The results of these experiments show that the p38 MAPK pathway is activated in GC in response to FSH in a cAMP/PKA-dependent manner, and that p38 MAPK activity is required for FSH-induced HSP-27 phosphorylation as well as rounding/aggregation in GC.
The AKT2 oncogene encodes a protein-serine/threonine kinase that was recently shown to be activated by a variety of growth factors. In addition, we previously showed that AKT2 is abundant in brown fat and skeletal muscle, tissues that are highly insulin responsive and that play a role in glucose metabolism. In this study, we demonstrate that AKT2 is activated in response to stimulation by insulin in a dose- and time-dependent manner in human ovarian carcinoma cells and that activation of AKT2 is abolished in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). Activation of AKT2 is manifested by changes in its phosphorylation state. Immunofluorescence experiments demonstrate that AKT2 is translocated to the plasma membrane after insulin stimulation, and this translocation is abolished by wortmannin. Both wild-type AKT2 activated by insulin and constitutively active AKT2, which has been targeted to the membrane by the addition of a myristoylation signal, were found to inactivate glycogen synthase kinase-3 (GSK-3) in vitro. GSK-3 was not inactivated by a catalytically inactive AKT2 mutant. Collectively, these data indicate that activation of AKT2 by insulin is mediated by PI 3-kinase and that GSK-3 is a downstream target of AKT2, suggesting a potentially important role of AKT2 in glycogen synthesis and other GSK-3 signaling pathways. J. Cell. Biochem. 70:433–441, 1998. © 1998 Wiley-Liss, Inc.
Excited states of 142Ce, populated in deep inelastic reactions of 82Se projectiles bombarding 139La target, have been studied up to medium spins using in-beam γ spectroscopy techniques. Three new levels have been identified
at 2625, 2995, 3834 keV, and assigned as 8+, 9(-) and 11(-), respectively. These new yrast states follow closely the level systematics of the even mass N = 84 isotones. Their strctures have been discussed with the help of empirical shell model calculations.
Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
This study investigates the possibility that FSH activates the p38 mitogen-activated protein kinase (MAPK) pathway in immature granulosa cells (GC). FSH induced the phosphorylation (activation) of p38 MAPK as evaluated by immunoprecipitation and by phosphorylation-specific immunoblotting. FSH-induced phosphorylation of p38 MAPK was blocked by pretreatment with the protein kinase A (PKA) inhibitor H89 and mimicked by the cAMP generating agonist forskolin, indicating that FSH-induced cAMP production and PKA activation are necessary and sufficient for the activation of p38 MAPK in GC. The small heat shock protein HSP-27 comprises a downstream phosphorylation target for the p38 MAPK pathway. FSH-induced phosphorylation of HSP-27 was blocked by pretreatment with the p38 MAPK inhibitor SB 203580, indicating that p38 MAPK activation is necessary for FSH-induced HSP-27 phosphorylation. FSH-induced GC rounding/aggregation was blocked by pretreatment with SB 203580 indicating that p38 MAPK activation is necessary for FSH-induced GC cell shape change. The results of these experiments show that the p38 MAPK pathway is activated in GC in response to FSH in a cAMP/PKA-dependent manner, and that p38 MAPK activity is required for FSH-induced HSP-27 phosphorylation as well as rounding/aggregation in GC.
The AKT2 oncogene encodes a protein-serine/threonine kinase that was recently shown to be activated by a variety of growth factors. In addition, we previously showed that AKT2 is abundant in brown fat and skeletal muscle, tissues that are highly insulin responsive and that play a role in glucose metabolism. In this study, we demonstrate that AKT2 is activated in response to stimulation by insulin in a dose- and time-dependent manner in human ovarian carcinoma cells and that activation of AKT2 is abolished in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). Activation of AKT2 is manifested by changes in its phosphorylation state. Immunofluorescence experiments demonstrate that AKT2 is translocated to the plasma membrane after insulin stimulation, and this translocation is abolished by wortmannin. Both wild-type AKT2 activated by insulin and constitutively active AKT2, which has been targeted to the membrane by the addition of a myristoylation signal, were found to inactivate glycogen synthase kinase-3 (GSK-3) in vitro. GSK-3 was not inactivated by a catalytically inactive AKT2 mutant. Collectively, these data indicate that activation of AKT2 by insulin is mediated by PI 3-kinase and that GSK-3 is a downstream target of AKT2, suggesting a potentially important role of AKT2 in glycogen synthesis and other GSK-3 signaling pathways.
FSH stimulates in ovarian granulosa cells diverse, differentiation-dependent responses that implicate activation of specific cellular signaling cascades. In these studies three kinases were investigated to determine their relationship to FSH, cAMP, and A kinase signaling: protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase (Sgk), and p38 mitogen-activated protein kinase (p38MAPK). The phosphorylation (activation) of these kinases was analyzed by using selective agonists/inhibitors: forskolin/H89 for cAMP-dependent protein kinase (A kinase), insulin-like growth factor I (IGF-I)/LY294002 and wortmannin for phosphatidylinositol-dependent kinase (PI3-K), and phorbol myristate (PMA)/GF109203X for diacylglycerol and Ca++-dependent kinases (C kinases). An inhibitor (PD98059) of MEK1, which regulates extracellular regulated kinases (ERKs), and SB203580, which inhibits p38MAPK, were also used. In addition, we analyzed the expression of the recently described, cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFI and GEFII) that impact Ras-related GTPases and Raf kinases, known regulators of various protein kinase cascades. We provide evidence that FSH, forskolin, and 8-bromo-cAMP stimulate phosphorylation of PKB by mechanisms involving PI3-K (LY294002/wortmannin sensitive) not A kinase (H89 insensitive), a pattern of response mimicking that of IGF-I. In contrast, FSH induction and phosphorylation of Sgk protein requires A kinase (H89 sensitive) but also involves PI3-K (LY294002 sensitive) as well as p38MAPK (SB203580 sensitive) pathways. PMA (C kinase) abolished FSH-mediated (but not IGF-I-mediated) phosphorylation of PKB at a step(s) upstream of PI3-K and independent of A kinase. Lastly, FSH-mediated phosphorylation of p38MAPK is negatively affected by A kinase and PI3-K, suggesting that it may be downstream of specific members of the cAMP-GEF/Rap/Raf pathway. We propose that cAMP activation of A kinase is obligatory for transcription of Sgk in granulosa cells whereas cAMP (IGF-I-like)-mediated phosphorylation (activation) of PKB and Sgk (via PI3-K), as well as p38MAPK, involves other cellular events. These results provide new and exciting evidence that cAMP acts in granulosa cells by A kinase-dependent and -independent mechanisms, each of which controls specific kinase cascades.
Investigations of the human Follitropin receptor (hFSHR) have failed to identify the tertiary structure that forms the active hormone-receptor interaction site which is essential to develop an immunocontraceptive based upon the receptor. To identify such a domain of hFSHR, an immunoneutralizing monoclonal antibody (mAb) 106-105 (IgG2b) was generated. Flow cytometry tested whether mAb 106-105 recognized native hFSHR. The epitope of mAb 106-105 was mapped by Western blot and by peptide ELISA. Inhibition of hFSH binding and bioactivity was determined by radioreceptor assay and by cAMP production, respectively. MAb 106-105 bound native hFSHR through an epitope including residues 300-315. MAb 106-105 completely blocked hormone binding to receptor and cAMP production by Y1-R cells expressing hFSHR. These effects were completely reversible by increasing the concentration of hFSH. Coincubation of this antibody with peptide D300-F315 blocked antibody activity. These data demonstrate that a discrete linear hFSHR epitope is a target for interference with hormone activity. These results further demonstrate that antibody binding to the extracellular domain (ECD) of hFSHR and subsequent bioactivation can be modulated through a domain specific hindrance, offering a reversible immunoneutralizing target.
Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.
Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.
Follitropin and the follitropin receptor are essential for normal gamete development in males and females. This review discusses the molecular genetics and structural and cellular biology of the follitropin/follitropin receptor system. Emphasis is placed on the human molecules when possible. The structure and regulation of the genes for the follitropin beta subunit and the follitropin receptor is discussed. Control of systemic and cellular protein levels is explained. The structural biology of each protein is described, including protein structure, motifs, and activity relationships. Finally, the follitropin/follitropin receptor signal transduction system is discussed.
Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27(Kip1) and p19(INK4) proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.
Selection of a dominant follicle that will ovulate likely occurs by activation of cell survival pathways and suppression of death-promoting pathways in a mechanism involving FSH and its cognate receptor (FSHR). A yeast two-hybrid screen of an ovarian cDNA library was employed to identify potential interacting partners with human FSHR intracellular loops 1 and 2. Among eight cDNA clones identified in the screen, APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif; also known as APPL or DIP13alpha) was chosen for further analysis. APPL1 appears to coimmunoprecipitate with FSHR in HEK 293 cells stably expressing FSHR (293/FSHR cells), confirming APPL1 as a potential FSHR-interacting partner. The phosphorylation status of members of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway was also examined because of the proposed role of APPL1 in the antiapoptotic PI3K/Akt pathway. FOXO1a, also referred to as forkhead homologue in rhabdomyosarcoma, is a downstream effector in the pathway and tightly linked to expression of proapoptotic genes. FOXO1a, but not the upstream kinase Akt, is rapidly phosphorylated, and FOXO1a is thereby inactivated when 293/FSHR cells are treated with FSH. In addition, FSHR coimmunoprecipitates with Akt. The identification of APPL1 as a potential interactor with FSHR and the finding that FOXO1a is phosphorylated in response to FSH provide a possible link between FSH and PI3K/Akt signaling, which may help to delineate a survival mechanism whereby FSH selects the dominant follicle to survive.
The human follitropin (follicle stimulating hormone, FSH) receptor (FSHR) is a G protein-coupled receptor (GPCR). To identify cytoplasmic proteins that may regulate FSHR function, a yeast-based interaction trap was performed. A linked construct of the first and second intracellular loops (iL1-iL2 bait) of FSHR was used as bait and a human ovarian cDNA library was used as prey. Among the proteins identified that interacted with the bait was 14-3-3tau, a member of a family of homodimeric cytoplasmic adapter proteins. Human granulosa cells, the site of FSHR expression in the ovary, were found to contain 14-3-3tau. Importantly, 14-3-3tau co-immunoprecipitated with FSHR stably expressed in HEK 293 cells. Its association with FSHR was follitropin-dependent. Over-expression of 14-3-3tau resulted in a modest decrease of follitropin-induced cAMP accumulation. Collectively, these data support a role for 14-3-3tau in follitropin action. The finding that 14-3-3tau interacts with FSHR is novel and should lead to new insights into the regulation of GPCR in general and FSHR specifically.
Intracellular signaling involves assembly and regulation of multiprotein complexes. These complexes are functional units of signal transduction and are a means by which protein networks carry out tasks within the cell. One mechanism to influence the components, the subcellular localization, and the activity of these complexes, involves scaffold proteins. Scaffold proteins facilitate signal transduction by tethering molecules together and serving as molecular backbones for signaling complex assembly. Recent studies, particularly in the field of signaling kinases, have considerably advanced our understanding of the role that scaffold proteins play within multiprotein complexes in cardiac and other cell types.
Multiprotein signalling networks create focal points of enzyme activity that disseminate the intracellular action of many hormones and neurotransmitters. Accordingly, the spatio-temporal activation of protein kinases and phosphatases is an important factor in controlling where and when phosphorylation events occur. Anchoring proteins provide a molecular framework that orients these enzymes towards selected substrates. A-kinase anchoring proteins (AKAPs) are signal-organizing molecules that compartmentalize various enzymes that are regulated by second messengers.
Accumulating evidence suggests that the human follitropin receptor is unusually sensitive to mutation. Previous results (Mol. Cell. Endo. 166 (2000) 101) determined that scanning mutations in a.a. 12-14 and 22-30 neither bound follitropin nor were present on the cell surface, suggesting that these regions are involved in either hormone binding or trafficking. To distinguish between these hypotheses, single alanine substitutions in a.a. 12-14 and 22-30 were generated, all of which appeared to bind 125I-follitropin with an affinity constant similar to wild type (wt) follitropin receptor. However, the level of receptor on the cell surface varied widely, in some cases 100-fold lower than wt. Expression on the cell surface corresponded to expression of the mature 80 kD follitropin receptor. An accumulation of the ER-resident 62 kD band of follitropin receptor was observed in mutants that had low surface expression of receptor, suggesting that misfolded protein was trapped in the ER by a quality control mechanism.
Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.
Follicle-stimulating hormone activation of Dias Molecular, structural, and cellular biology of follitropin and follitropin receptor
- H Alam
- E T Maizels
- Y Park
- S Ghaey
- Z J Feiger
- N S Chandel
- M Hunzicker-Dunn
Alam, H., Maizels, E.T., Park, Y., Ghaey, S., Feiger, Z.J., Chandel, N.S., Hunzicker-Dunn, M., 2004. Follicle-stimulating hormone activation of Dias, J.A., Cohen, B.D., Lindau-Shepard, B., Nechamen, C.A., Peterson, A.J., Schmidt, A., 2002. Molecular, structural, and cellular biology of follitropin and follitropin receptor. In: Litwack, G. (Ed.), Vitamins and Hormones, first ed. Academic Press, New York, NY, pp. 249–322.
The BAR-domain family of proteins: a case of bend-ing and binding? The membrane bending and GTPase-binding functions of proteins from the BAR-domain family
- B Habermann
Habermann, B., 2004. The BAR-domain family of proteins: a case of bend-ing and binding? The membrane bending and GTPase-binding functions of proteins from the BAR-domain family. EMBO Rep. 5, 250–255.
- Oj Bjerrum
Bjerrum, OJ.; Schafer-Nielsen, C. Analytical Electrophoresis. Dunn, MJ., editor. Verlag Chemie;
Weinheim: 1986. p. 315
- Ja Dias
- Bd Cohen
- B Lindau-Shepard
- Ca Nechamen
- Aj Peterson
- A Schmidt
Dias, JA.; Cohen, BD.; Lindau-Shepard, B.; Nechamen, CA.; Peterson, AJ.; Schmidt, A. Molecular,
Structural, and Cellular Biology of Follitropin and Follitropin Receptor. In: Litwack, G., editor.
Vitamins and Hormones. 1 Ed.. Academic Press; New York, NY: 2002. p. 249-322.