Estrogen-related Receptor �� Is a Key Regulator of Muscle Mitochondrial Activity and Oxidative Capacity

Estrogen-related receptor γ (ERRγ) regulates the perinatal switch to oxidative metabolism in the myocardium. We wanted to understand the significance of induction of ERRγ expression in skeletal muscle by exercise. Muscle-specific VP16ERRγ transgenic mice demonstrated an increase in exercise capacity, mitochondrial enzyme activity, and enlarged mitochondria despite lower muscle weights. Furthermore, peak oxidative capacity was higher in the transgenics as compared with control littermates. In contrast, mice lacking one copy of ERRγ exhibited decreased exercise capacity and muscle mitochondrial function. Interestingly, we observed that increased ERRγ in muscle generates a gene expression profile that closely overlays that of red oxidative fiber-type muscle. We further demonstrated that a small molecule agonist of ERRβ/γ can increase mitochondrial function in mouse myotubes. Our data indicate that ERRγ plays an important role in causing a shift toward slow twitch muscle type and, concomitantly, a greater capacity for endurance exercise. Thus, the activation of this nuclear receptor provides a potential node for therapeutic intervention for diseases such as obesity, which is associated with reduced oxidative metabolism and a lower type I fiber content in skeletal muscle.     

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP^C) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP^C extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP^C-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP^C engagement induces an increase in intracellular Ca²⁺ levels. This effect was not detected in PrP^C-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP^C. Using a best candidate approach to test for potential channels involved in Ca²⁺ influx evoked by STI1-PrP^C, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP^C-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP^C and allowed reconstitution of signaling by PrP^C-STI1 interaction. These results indicate that STI1 can interact with the PrP^C·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.     

SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-β Signaling Pathway Targeting the Type II Receptor

Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-β (TGF-β) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-β signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-β signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-β type II receptor (TβRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-β signaling pathway. SPSB1 negatively regulates the TGF-β signaling pathway through its interaction with both endogenous and overexpressed TβRII (and not TβRI) via its Spry domain. As such, TβRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TβRII at a low level by enhancing the ubiquitination levels and degradation rates of TβRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-β signaling and migration and invasion of tumor cells.     

Drosophila GoLoco-Protein Pins Is a Target of Gαo-mediated G Protein–coupled Receptor Signaling

G protein–coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Gα-subunits; the resulting Gα-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein–dependent activities, physically binding Gα-subunits of the Gα_i/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Gα-subunits. Here we demonstrate that the poly-GoLoco–containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Gα_o. We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with Gα_o-GTP. We further pinpoint a lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of Gα_o-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Gα_o acts downstream from Frizzled and redundantly with Gα_i to control the asymmetry of cell divisions.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Zebrafish Dkk3a Protein Regulates the Activity of myf5 Promoter through Interaction with Membrane Receptor Integrin α6b

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the β-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.     

Role of Molecular Chaperones in G Protein ��5/Regulator of G Protein Signaling Dimer Assembly and G Protein �¦� Dimer Specificity

The G protein βγ subunit dimer (Gβγ) and the Gβ5/regulator of G protein signaling (RGS) dimer play fundamental roles in propagating and regulating G protein pathways, respectively. How these complexes form dimers when the individual subunits are unstable is a question that has remained unaddressed for many years. In the case of Gβγ, recent studies have shown that phosducin-like protein 1 (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, it is not known what fraction of the many Gβγ combinations is assembled this way or whether chaperones influence the specificity of Gβγ dimer formation. Moreover, the mechanism of Gβ5-RGS assembly has yet to be assessed experimentally. The current study was undertaken to directly address these issues. The data show that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1–4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results also show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way.Eukaryotic cells utilize receptors coupled to heterotrimeric GTP-binding proteins (G proteins)³ to mediate a vast array of responses ranging from nutrient-induced migration of single-celled organisms to neurotransmitter-regulated neuronal activity in the human brain (1). Ligand binding to a G protein-coupled receptor (GPCR) initiates GTP exchange on the G protein heterotrimer (composed of Gα, Gβ, and Gγ subunits), which in turn causes the release of Gα-GTP from the Gβγ dimer (2–4). Both Gα-GTP and Gβγ propagate and amplify the signal by interacting with effector enzymes and ion channels (1, 5). The duration and amplitude of the signal is dictated by receptor phosphorylation coupled with arrestin binding and internalization (6) and by regulators of G protein signaling (RGS) proteins, which serve as GTPase-activating proteins for the GTP-bound Gα subunit (7, 8). The G protein signaling cycle is reset as the inactive Gα-GDP reassembles with the Gβγ dimer and Gαβγ re-associates with the GPCR (5).To fulfill its essential role in signaling, the G protein heterotrimer must be assembled post-translationally from its nascent polypeptides. Significant progress has been made recently regarding the mechanism by which this process occurs. It has been clear for some time that the Gβγ dimer must assemble first, followed by subsequent association of Gα with Gβγ (9). What has not been clear was how Gβγ assembly would occur given the fact that neither Gβ nor Gγ is structurally stable without the other. An important breakthrough was the finding that phosducin-like protein 1 (PhLP1) functions as a co-chaperone with the chaperonin containing tailless complex polypeptide 1 (CCT) in the folding of nascent Gβ and its association with Gγ (10–15). CCT is an important chaperone that assists in the folding of actin and tubulin and many other cytosolic proteins, including many β propeller proteins like Gβ (16). PhLP1 has been known for some time to interact with Gβγ and was initially believed to inhibit Gβγ function (17). However, several recent studies have demonstrated that PhLP1 and CCT work together in a highly orchestrated manner to form the Gβγ dimer (10–15).Studies on the mechanism of PhLP1-mediated Gβγ assembly have focused on the most common dimer Gβ1γ2 (10, 13, 14), leaving open questions about the role of PhLP1 in the assembly of the other Gβγ combinations. These are important considerations given that humans possess 5 Gβ genes and 12 Gγ genes with some important splice variants (18, 19), resulting in more than 60 possible combinations of Gβγ dimers. Gβ1–4 share between 80 and 90% sequence identity and are broadly expressed (18, 19). Gβ5, the more atypical isoform, shares only ∼53% identity with Gβ1, carries a longer N-terminal domain, and is only expressed in the central nervous system and retina (20). The Gγ protein family is more heterogeneous than the Gβ family. The sequence identity of the 12 Gγ isoforms extends from 10 to 70% (21), and the Gγ family can be separated into 5 subfamilies (21–23). All Gγ proteins carry C-terminal isoprenyl modifications, which contribute to their association with the cell membrane, GPCRs, Gαs, and effectors (9). Subfamily I Gγ isoforms are post-translationally farnesylated, whereas all others are geranylgeranylated (22, 24).There is some inherent selectivity in the assembly of different Gβγ combinations, but in general Gβ1–4 can form dimers with most Gγ subunits (25). The physiological purpose of this large number of Gβγ combinations has intrigued researchers in the field for many years, and a large body of research indicates that GPCRs and effectors couple to a preferred subset of Gβγ combinations based somewhat on specific sequence complementarity, but even more so on cellular expression patterns, subcellular localization, and post-translational modifications (18).In contrast to Gβ1–4, Gβ5 does not interact with Gγ subunits in vivo, but it instead forms irreversible dimers with RGS proteins of the R7 family, which includes RGS proteins 6, 7, 9, and 11 (26). All R7 family proteins contain an N-terminal DEP (disheveled, Egl-10, pleckstrin) domain, a central Gγ-like (GGL) domain, and a C-terminal RGS domain (8, 26). The DEP domain interacts with the membrane anchoring/nuclear shuttling R7-binding protein, and the GGL domain binds to Gβ5 in a manner similar to other Gβγ associations (27, 28). Like Gβγs, Gβ5 and R7 RGS proteins form obligate dimers required for their mutual stability (26). Without their partner, Gβ5 and R7 RGS proteins are rapidly degraded in cells (26, 29). Gβ5-R7 RGS complexes act as important GTPase-accelerating proteins for G_i/oα and G_qα subunits in neuronal cells and some immune cells (26).It has been recently shown that all Gβ isoforms are able to interact with the CCT complex, but to varying degrees (15). Gβ4 and Gβ1 bind CCT better than Gβ2 and Gβ3, whereas Gβ5 binds CCT poorly (15). These results suggest that Gβ1 and Gβ4 might be more dependent on PhLP1 than the other Gβs, given the co-chaperone role of PhLP1 with CCT in Gβ1γ2 assembly. However, another report has indicated that Gγ2 assembly with Gβ1 and Gβ2 is more PhLP1-dependent than with Gβ3 and Gβ4 (30). Thus, it is not clear from current information whether PhLP1 and CCT participate in assembly of all Gβγ combinations or whether they contribute to the specificity of Gβγ dimer formation, nor is it clear whether they or other chaperones are involved in Gβ5-R7 RGS dimer formation. This report was designed to address these issues.     

Regulator of G-Protein Signaling – 5 (RGS5) Is a Novel Repressor of Hedgehog Signaling

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gα_i subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gα_i, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.     

C18 ORF1, a Novel Negative Regulator of Transforming Growth Factor-β Signaling

Transforming growth factor (TGF)-β signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-β signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-β signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-β signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-β type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-β-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-β. Consistently, TGF-β-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-β signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-β signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-β signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-β signaling in a coordinated manner when cells are stimulated with high levels of TGF-β.     

Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) Signaling

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.     

Magnitude of a Conformational Change in the Glycine Receptor ��1-��2 Loop Is Correlated with Agonist Efficacy

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.Glycine receptors (GlyRs)³ are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the γ-aminobutyric acid type-A receptors (GABA_ARs), and serotonin type-3 receptors (5-HT₃Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2–6). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted β-sheets. The inner (vestibule facing) β-sheet comprises seven β-strands, while the outer β-sheet is formed by three β-strands (3). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3).GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine > β-alanine > taurine (7, 8). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9–11). A recent ground-breaking study on an intermediate pre-open state, the so-called “flip” state (12), has provided new insights into the mechanism of partial agonism in Cys-loop receptors (13). This study suggested that agonist efficacy depends on the ability of the agonist to convert the inert agonist-bound receptor to the pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for high and low efficacy agonists. To date there is, however, very little information concerning the structural basis for the lower efficacies of partial agonists. To address this, the present study employed the voltage-clamp fluorometry (VCF) technique (14) to compare the conformational changes induced by glycine, β-alanine, and taurine at various positions in the GlyR LBD.Open in a separate windowFIGURE 1.A, structures of glycine, β-alanine, and taurine. B, model of the LBD, based on carbomylcholine-bound AChBP (PDB code 1uv6). The inner β-sheet is displayed in red, the outer β-sheet in blue. Connecting loops are shown in gray. Colored balls represent approximate locations of selected residues labeled in regions flanking the outer β-sheet (black, G181C in loop F; N203C in loop C; Q219C in the pre-M1 domain) and in the inner β-sheet (yellow, L127C in loop E; Q67C in loop D; A52C in loop 2).VCF involves tethering of an environmentally sensitive fluorophore to a cysteine engineered into a domain of interest. If ligand-binding and/or channel opening leads to a changed dielectric environment surrounding the fluorophore, a change in quantum yield or emission spectrum can be detected. VCF was first employed on voltage-gated potassium channels (15) and has since provided a wealth of information on Cys-loop receptor structure and function (16–23). Here we employ VCF to identify an agonist-specific conformational change that may control or reflect the rate at which the GlyR isomerizes to the flip state.     

Increased Cytokine Production in Interleukin-18 Receptor ��-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

Since interleukin (IL)-18 is a proinflammatory cytokine, mice lacking IL-18 or its ligand-binding receptor (IL-18R) should exhibit decreased cytokine and chemokine production. Indeed, production of IL-1α, IL-6, and MIP-1α was reduced in IL-18 knock-out (ko) mouse embryonic fibroblast (MEF)-like cells. Unexpectedly, we observed a paradoxical 10-fold increase in IL-1β-induced IL-6 production in MEF cells from mice deficient in the IL-18R α-chain (IL-18Rα) compared with wild type MEF. Similar increases were observed for IL-1α, MIP-1α, and prostaglandin E₂. Likewise, coincubation with a specific IL-18Rα-blocking antibody augmented IL-1β-induced cytokines in wild type and IL-18 ko MEF. Stable lines of IL-18Rα-depleted human A549 cells were generated using shRNA, resulting in an increase of IL-1β-induced IL-1α, IL-6, and IL-8 compared to scrambled small hairpin RNA. In addition, we silenced IL-18Rα with small interfering RNA in primary human blood cells and observed up to 4-fold increases in the secretion of lipopolysaccharide- and IL-12/IL-18-induced IL-1β, IL-6, interferon-γ, and CD40L. Mechanistically, despite increases in Stat1 and IL-6, induction of SOCS1 and -3 (suppressor of cytokine signaling 1 and 3) was markedly reduced in the absence of IL-18Rα. Consistent with these observations, activation of the p38α/β and ERK1/2 MAPKs and of protein kinase B/Akt increased in IL-18Rα ko MEF, whereas the negative feedback kinase MSK2 was more active in IL-18 ko cells. These data reveal a role for SOCS1 and -3 in the seemingly paradoxical hyperresponsive state in cells deficient in IL-18Rα, supporting the concept that IL-18Rα participates in both pro- and anti-inflammatory responses and that an endogenous ligand engages IL-18Rα to deliver an inhibitory signal.Often shown to function as a proinflammatory cytokine, structurally related to IL-1β,³ and requiring caspase-1 for processing of its inactive precursor into an active cytokine (1–3), IL-18 is a unique member of the IL-1 family. For example, IL-18 and IL-18 receptor α-chain (IL-18Rα) knock-out (ko) mice unexpectedly overeat and spontaneously become obese, developing insulin resistance and atherosclerosis (4, 5). This phenotype does not occur in mice deficient in other members of the IL-1 family. In the absence of IL-12 and similar co-stimulatory cytokines, IL-18 can act as a typical Th2 cytokine in murine models (6, 7). The affinity of the naturally occurring IL-18-binding protein (IL-18BP) for IL-18 is higher than that of IL-18 for its cognate receptor; thus, low levels of this naturally occurring antagonist effectively neutralize the activity of IL-18 (8–11). In some studies, IL-18 opposes the proinflammatory properties of IL-1β (12). In dextran sodium sulfate-induced colitis, neutralizing antibodies to IL-18 or IL-18BP ameliorate the disease (13, 14), whereas in other studies, mice deficient in IL-18Rα exhibit worsening of the disease (15).IL-18 Induces Several Proinflammatory Cytokines, Such as IL-1β and TNFα, as well as chemokines, nitric oxide, and vascular adhesion molecules (reviewed in Ref. 16). Using mice deficient in IL-18 or neutralization of IL-18, the cytokine appears to play an important role in models of rheumatoid arthritis (17), lupus-like autoimmune disease (18), metastatic melanoma (19), graft versus host disease (20), and myocardial suppression (21, 22). Unlike IL-1, IL-18 also induces Fas ligand and has been proposed as a key mediator of macrophage activation syndrome (23).We have previously reported that whereas deficiency in IL-18 attenuated inflammatory responses to various exogenous stimuli, these responses paradoxically were exaggerated in IL-18Rα ko mice (24). In addition to rejecting insulin-producing islet allografts, splenocytes and peritoneal macrophages from IL-18Rα ko mice produced significantly greater amounts of several proinflammatory cytokines upon stimulation with concanavalin A, TLR2 agonist heat-killed Staphylococcus epidermidis, or anti-CD3 antibodies (24).In the present study, we set out to investigate the fundamental differences in cytokine production between IL-18 ko and IL-18Rα ko mice using mouse embryonic fibroblasts (MEF), which are highly responsive to IL-1 and TNFα stimulation. We also studied the role of IL-18Rα in human cells. IL-18Rα was silenced in human A549 epithelial cells using small hairpin RNA (shRNA) to the IL-18R (shIL-18R) as well as in freshly obtained human peripheral blood mononuclear cells (PBMC). Furthermore, using inhibitors as well as kinase activation studies, real time PCR, and Western blotting, we shed light on the IL-18Rα ko-mediated differences in expression and activation of signaling mediators, such as the suppressors of cytokine signaling (SOCS), MAPKs, protein kinase B/Akt, NF-κB, MSK2/RSKβ, and p70 S6 kinase. The mechanisms underlying the disinhibition of inflammatory responses in IL-18Rα-deficient cells appear to be due to a yet unidentified anti-inflammatory ligand of the IL-18 receptor.     

CFTR Is a Negative Regulator of NFκB Mediated Innate Immune Response

Background

Dysfunctional CFTR in the airways is associated with elevated levels of NFκB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.

Methodology/Principal Findings

We tested the hypothesis that wt-CFTR down-regulates NFκB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFκB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1β induced IL-8, and NFκB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-β-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFκB reporter activities as compared to control cells suggesting a negative regulation of NFκB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFκB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1β in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IκB, an inducer of NFκB mediated signaling in the CFKO mice.

Conclusions/Significance

Our data suggest that CFTR is a negative regulator of NFκB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.