Expression of interferon receptor subunits,IFNAR1 and IFNAR2, in the ovine uterus

Interferon-tau (IFN-tau) is the antiluteolytic factor released by concepti of ruminant ungulate species prior to implantation. All type I interferons, including IFN-tau, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c, but the distribution of the two polypeptides in uterine endometrium has not been examined. In situ hybridization and immunohistochemistry on sections from pregnant and nonpregnant ovine uteri at Days 14 and 15 after estrus and mating showed that both IFNAR1 and IFNAR2 mRNA and protein were strongly expressed in endometrial luminal epithelium (LE), superficial glandular epithelium (GE), and stromal cells, within but not outside caruncles. Similar staining patterns were noted in pregnant and nonpregnant uteri for both subunits. Western blot analysis of membrane fractions from cell lines derived from endometrial LE, GE, and stromal cells, and affinity cross-linking experiments with radioactively labeled IFN-tau performed on crude endometrial membranes indicated the presence of both high ( approximately 110 kDa) and low (75-80 kDa) molecular mass forms of the two receptor subunits. To localize where IFN-tau binds when it is introduced into the uterine lumen, immunohistochemistry with an antiserum against IFN-tau was performed on sections of uteri from Day 14 nonpregnant ewes whose uteri had previously been infused with IFN-tau. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues. These studies demonstrate the heavy concentration of IFNAR1 and IFNAR2 in cells of the LE and superficial GE, which appear to be the main targets for IFN-tau.     

Phosphorylation and specific ubiquitin acceptor sites are required for ubiquitination and degradation of the IFNAR1 subunit of type I interferon receptor

Ubiquitination, endocytosis, and lysosomal degradation of the IFNAR1 (interferon alpha receptor 1) subunit of the type I interferon (IFN) receptor is mediated by the SCFbeta-Trcp (Skp1-Cullin1-F-box protein beta transducin repeat-containing protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. In addition, stability of IFNAR1 is regulated by its binding to Tyk2 kinase. Here we characterize the determinants of IFNAR1 ubiquitination and degradation. We found that the integrity of two Ser residues at positions 535 and 539 within the specific destruction motif present in the cytoplasmic tail of IFNAR1 is essential for the ability of IFNAR1 to recruit beta-Trcp as well as to undergo efficient ubiquitination and degradation. Using an antibody that specifically recognizes IFNAR1 phosphorylated on Ser535 we found that IFNAR1 is phosphorylated on this residue in cells. This phosphorylation is promoted by treatment of cells with IFNalpha. Although the cytoplasmic tail of IFNAR1 contains seven Lys residues that could function as potential ubiquitin acceptor sites, we found that only three (Lys501, Lys525, and Lys526), all located proximal to the destruction motif, are essential for ubiquitination and degradation of IFNAR1. Expression of Tyk2 stabilized IFNAR1 in a manner that was dependent neither on its binding to beta-Trcp nor IFNAR1 ubiquitination. We discuss the complexities and specifics of the ubiquitination and degradation of IFNAR1, which is a beta-Trcp substrate that undergoes degradation via a lysosomal pathway.     

Ammonium chloride causes reversible inhibition of low density lipoprotein receptor recycling and accelerates receptor degradation

The effects of the acidotropic agent, NH4Cl, on the recycling and turnover of low density lipoprotein (LDL) receptors were analyzed in human skin fibroblasts using ligand binding assays, [35S]methionine pulse-chase experiments, and electron microscopy. NH4Cl did not prevent receptor internalization but caused a marked redistribution of LDL receptors to intracellular sites (endosomes) that was completely dependent on the presence of apolipoprotein-B- or -E-containing ligands. Maximal inhibition of recycling was observed at LDL concentrations that only partially saturated receptors, suggesting that the receptors function as oligomers. In contrast, full receptor occupancy by the multivalent, apolipoprotein-E-containing beta-very low density lipoprotein was required for the same effect. The intracellular accumulation was reversible and the majority of receptors returned to the cell surface when NH4Cl was removed after short treatments. The rate of degradation of LDL receptors was greatly accelerated in the presence of NH4Cl and ligand, with a t1/2 of about 2 h (approximately 6 times faster than receptor degradation in the absence of NH4Cl). Neither the redistribution nor the accelerated loss of immunoprecipitable LDL receptors was observed in an LDL receptor internalization-defective mutant cell line. We conclude that NH4Cl inhibited the recycling specifically of occupied receptors, thereby accelerating their degradation, probably in endosomes.     

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.     

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Agonists stimulate cannabinoid 1 receptor (CB₁R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization – likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB₁) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB₁R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB₁Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB₁R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of β-arrestin-2 recruitment to mutant CB₁R suggests that putative carboxy-terminal phosphorylation sites mediate β-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB₁R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB₁Rs.     

Pathogen recognition by the cell surface receptor CD46 induces autophagy

Autophagy is a degradative mechanism involved in cell protection against invading pathogens. Although the autophagic process is well characterized, the molecular pathways leading to its activation upon pathogen binding remain poorly understood. Our recent work demonstrates that the cell surface pathogen receptor CD46 induces autophagy upon pathogen recognition. The molecular pathway linking CD46 to the autophagosome machinery relies on the scaffold protein GOPC and on the autophagosome formation complex Beclin 1/VPS34. The CD46-dependent autophagy is critical to an early control of infection.     

Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling

Eph receptors and their ephrin ligands are involved in various aspects of cell-cell communication during development, including axonal pathfinding in the nervous system and cell-cell interactions of the vascular endothelial cells. Recent structural studies revealed unique molecular features, not previously seen in any other receptor-ligand families, and explained many of the biochemical and signaling properties of Ephs and ephrins. However, unresolved questions remain regarding the potential oligomerization and clustering of these important signaling molecules. In this study, the biophysical properties and receptor-binding preferences of the extracellular domain of ephrin-B1 were investigated and its crystal structure was determined at 2.65 A resolution. Ephrin-B1 is a monomer both in solution and in the crystals, while it was previously shown that the closely related ephrin-B2 forms homodimers. The main structural difference between ephrin-B1 and ephrin-B2 is the conformation of the receptor-binding G-H loop and the partially disordered N-terminal tetramerization region of ephrin-B1. The G-H loop is structurally rigid in ephrin-B2 and adopts the same conformation in both the receptor-bound and unbound ligand, where it mediates receptor-independent homodimerization. In the ephrin-B1 structure, on the other hand, the G-H loop is not involved in any homotypic interactions and adopts a new, distinct conformation. The implications of the ephrin-B1 structure, in context of available ephrin-B1 mutagenesis data, for the mechanism of Eph-ephrin recognition and signaling initiation are discussed.     

Disruption of tumor necrosis factor alpha receptor 1 signaling accelerates NAFLD progression in mice upon a high-fat diet

Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1β release and impairment of insulin signaling are still unknown, so we determined whether IL-1β affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1β plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1β-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.     

Phage SPP1 reversible adsorption to Bacillus subtilis cell wall teichoic acids accelerates virus recognition of membrane receptor YueB

Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect Bacillus subtilis. Interestingly, SPP1 still binds to the surface of yueB mutants, although in a completely reversible way. We evaluated here the relevance of a reversible step in SPP1 adsorption and identified the receptor(s) involved. We show that reversible adsorption is impaired in B. subtilis mutants defective in the glucosylation pathway of teichoic acids or displaying a modified chemical composition of these polymers. The results indicate that glucosylated poly(glycerolphosphate) cell wall teichoic acid is the major target for SPP1 reversible binding. Interaction with this polymer is characterized by a fast adsorption rate showing low-temperature dependence, followed by a rapid establishment of an equilibrium state between adsorbed and free phages. This equilibrium is basically determined by the rate of phage dissociation, which exhibits a strong dependence on temperature compatible with an Arrhenius law. This allowed us to determine an activation energy of 22.6 kcal/mol for phage release. Finally, we show that SPP1 reversible interaction strongly accelerates irreversible binding to YueB. Our results support a model in which fast SPP1 adsorption to and desorption from teichoic acids allows SPP1 to scan the bacterial surface for rapid YueB recognition.     

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.     

A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.     

Polyadenylation accelerates degradation of chloroplast mRNA.

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.     

Odor recognition and second messenger signaling in olfactory receptor neurons

The detection of volatile odorants is supposed to begin with their interaction with soluble binding proteins which shuttle the hydrophobic ligands through the aqueous mucus layer towards specific odorant receptors in the ciliary membrane of olfactory neurons. A large family of receptors for odorants has been identified recently; individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, ultimately leading to an electrical response of the receptor neuron. Olfactory signaling is terminated by phosphorylation of receptors via a negative feedback reaction catalyzed by two types of kinases.     

Cbl-b-dependent coordinated degradation of the epidermal growth factor receptor signaling complex

Cbl proteins function as ubiquitin protein ligases for the activated epidermal growth factor receptor and, thus, negatively regulate its activity. Here we show that Cbl-b is ubiquitinated and degraded upon activation of the receptor. Epidermal growth factor (EGF)-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and requires binding of the Cbl-b protein to the activated EGF receptor (EGFR). Degradation of both the EGFR and the Cbl-b protein is blocked by lysosomal and proteasomal inhibitors. Other components of the EGFR-signaling complex (i.e. Grb2 and Shc) are also degraded in an EGF-induced Cbl-b-dependent fashion. Our results suggest that the ubiquitin protein ligase function of Cbl-b is regulated by coordinated degradation of the Cbl-b protein along with its substrate. Furthermore, the data demonstrate that Cbl-b mediates degradation of multiple proteins in the EGFR-signaling complex.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

AH23848 accelerates inducible nitric oxide synthase degradation through attenuation of cAMP signaling in glomerular mesangial cells.

Excessive release of nitric oxide (NO) by mesangial cells contributes to the pathogenesis of glomerulonephritis. Prostaglandin E(2) (PGE(2)) produced at inflammatory sites regulates the release of NO through its downstream signaling. In glomerular mesangial cells (MES-13 cells), PGE(2) modulated NO production mainly through EP4 receptor in a cAMP-dependent manner. Lipopolysaccharide and interferon-gamma (LPS+IFNgamma)-induced NO production, inducible nitric oxide synthase (iNOS) gene and protein expression were greatly inhibited by AH23848, an EP4 antagonist. Further investigation indicated that AH23848 attenuated endogenous cAMP accumulation in MES-13 cells and modulated NO production through declination of iNOS gene expression and acceleration of iNOS protein degradation. AH23848 downregulated the iNOS protein in MES-13 cells through protein kinase A (PKA) since KT5720, a PKA-specific inhibitor, reduced iNOS protein stability. A short exposure of activated MES-13 cells to okadaic acid augmented iNOS activity. AH23848 and KT5720 attenuated serine/threonine phosphorylation of iNOS protein in LPS + IFNgamma-stimulated MES-13 cells. The results of this study led us to speculate that cAMP might regulate iNOS-stimulated NO synthesis through posttranslational mechanisms. Attenuation of cAMP signaling and the phosphorylation status of the iNOS protein may account for the effect of AH23848 in accelerating iNOS protein degradation in MES-13 cells.