Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na⁺ channel (ENaC) is modulated by Na⁺ self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na⁺. A His residue within the extracellular domain of γENaC (γHis²³⁹) was found to have a critical role in Na⁺ self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na⁺ self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na⁺ self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na⁺ self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the γH239F mutant showed that, in the absence of Na⁺ self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that γHis²³⁹ functions within an extracellular allosteric regulatory subdomain of the γ subunit that has an important role in conferring the response of the channel to external Na⁺.     

Dystroglycan Binding to α-Neurexin Competes with Neurexophilin-1 and Neuroligin in the Brain

α-Neurexins (α-Nrxn) are mostly presynaptic cell surface molecules essential for neurotransmission that are linked to neuro-developmental disorders as autism or schizophrenia. Several interaction partners of α-Nrxn are identified that depend on alternative splicing, including neuroligins (Nlgn) and dystroglycan (αDAG). The trans-synaptic complex with Nlgn1 was extensively characterized and shown to partially mediate α-Nrxn function. However, the interactions of α-Nrxn with αDAG, neurexophilins (Nxph1) and Nlgn2, ligands that occur specifically at inhibitory synapses, are incompletely understood. Using site-directed mutagenesis, we demonstrate the exact binding epitopes of αDAG and Nxph1 on Nrxn1α and show that their binding is mutually exclusive. Identification of an unusual cysteine bridge pattern and complex type glycans in Nxph1 ensure binding to the second laminin/neurexin/sex hormone binding (LNS2) domain of Nrxn1α, but this association does not interfere with Nlgn binding at LNS6. αDAG, in contrast, interacts with both LNS2 and LNS6 domains without inserts in splice sites SS#2 or SS#4 mostly via LARGE (like-acetylglucosaminyltransferase)-dependent glycans attached to the mucin region. Unexpectedly, binding of αDAG at LNS2 prevents interaction of Nlgn at LNS6 with or without splice insert in SS#4, presumably by sterically hindering each other in the u-form conformation of α-Nrxn. Thus, expression of αDAG and Nxph1 together with alternative splicing in Nrxn1α may prevent or facilitate formation of distinct trans-synaptic Nrxn·Nlgn complexes, revealing an unanticipated way to contribute to the identity of synaptic subpopulations.     

Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

Among the three extracellular domains of the tetrameric voltage-gated K⁺ (Kv) channels consisting of six membrane-spanning helical segments named S1–S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2–4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.     

Measuring the Influence of the BKCa β1 Subunit on Ca2+ Binding to the BKCa Channel

The large-conductance Ca²⁺-activated potassium (BK_Ca) channel of smooth muscle is unusually sensitive to Ca²⁺ as compared with the BK_Ca channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BK_Ca auxiliary subunit β1, whose presence dramatically increases both the potency and efficacy of Ca²⁺ in promoting channel opening. β1 contains no Ca²⁺ binding sites of its own, and thus the mechanism by which it increases the BK_Ca channel''s Ca²⁺ sensitivity has been of some interest. Previously, we demonstrated that β1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with β1 present. This decreases the work that Ca²⁺ must do to open the channel and thereby increases the channel''s apparent Ca²⁺ affinity without altering the real affinities of the channel''s Ca²⁺ binding sites. To explain the full effect of β1 on the channel''s Ca²⁺ sensitivity, however, we also proposed that there must be effects of β1 on Ca²⁺ binding. Here, to test this hypothesis, we have used high-resolution Ca²⁺ dose–response curves together with binding site–specific mutations to measure the effects of β1 on Ca²⁺ binding. We find that coexpression of β1 alters Ca²⁺ binding at both of the BK_Ca channel''s two types of high-affinity Ca²⁺ binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca²⁺ bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca²⁺ binding at either site has a larger effect on channel opening when β1 is present.     

Improved Binding of Raf to Ras��GDP Is Correlated with Biological Activity

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras·Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras·GTP versus Ras·GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.     

Binding of Extracellular Maspin to ��1 Integrins Inhibits Vascular Smooth Muscle Cell Migration

Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells (VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the β1 integrin subunit, and subsequently both α3β1 and α5β1, but not αvβ3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to α5β1 was confirmed using a recombinant α5β1-Fc fusion protein. Using conformation-dependent anti-β1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of α5β1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human α5 integrin, but not those lacking α5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment.Maspin is a member of the serpin family of serine protease inhibitors (SERPINB5).² It was originally identified as a gene down-regulated in invasive breast cancer and proposed as a class II tumor suppressor (1), and has since been shown to have many effects on cellular behavior that are consistent with this activity. It has been shown to decrease the proliferation, migration, and metastasis of tumor cells in vivo (1, 2) and their invasion in vitro (3, 4), and to increase apoptosis of endothelial cells (5) and inhibit angiogenesis (6). However, the cellular effects of maspin are not restricted to tumor cells, and we have demonstrated that maspin can inhibit the migration of vascular smooth muscle cells (7).VSMC migration is a key event in the development of atherosclerosis (8), and contributes significantly to restenosis after angioplasty (9) and transplant arteriosclerosis (10). VSMC are not terminally differentiated and acquire migratory capacity as part of a phenotypic switch from a contractile, quiescent state to a dedifferentiated phenotype, characterized by proliferation and increased extracellular matrix synthesis, in addition to motility (11). This allows VSMC to respond to environmental cues following vascular injury. The phenotypic plasticity of VSMC is regulated by an array of signals, among which integrin-mediated association with surrounding extracellular matrix and changes in the expression of matrix-degrading proteases are prominent (12–14).How maspin mediates its various cellular effects is unclear. Maspin has been reported to be an inhibitor of plasminogen activation (3, 15, 16), but we have shown that maspin is unable to inhibit either uPA- or tPA-catalyzed plasminogen activation under conditions in which the serpin PAI-1 was completely inhibitory (7). The anti-proteolytic inhibitory mechanism of serpins is dependent on characteristics of the reactive center loop (RCL) allowing it to adopt the necessary canonical conformation and rearrangements subsequent to protease binding (17). The RCL of maspin does not have the required characteristics (7, 18), and the conclusion that maspin is a non-inhibitory serpin is fully supported by its crystal structure (19, 20).Another confounding factor in understanding the mechanisms underlying the cellular effects of maspin is that, in common with the serpin PAI-2, it lacks an authentic secretion signal sequence. Nevertheless it has been shown to enter secretory vesicles (21) and is found extracellularly, in the cytoplasm and also in the nucleus (21, 22). Cytoplasmic and nuclear binding proteins for maspin have been identified (23–25), and may be responsible for its effects on proliferation and apoptosis. How secreted, extracellular maspin exerts its effects is unclear, but a function as a cell signaling ligand has been proposed (26–28). However, the characteristics of the maspin inhibitory effect on VSMC migration point to a more direct effect of maspin.To determine the mechanism of the maspin effect on VSMC migration, we have now attempted to identify maspin-binding proteins on the surface of these cells. In this report we provide biochemical, cellular, and functional evidence that the effect of maspin on cell migration is mediated by specific binding to cell adhesion receptors of the integrin family. We find that maspin binds specifically to β1 integrins on the surface of dedifferentiated VSMC, which leads to a reduction in the activation status of the integrin, and that the binding of maspin to α5β1 is sufficient for its inhibitory effects on cell migration and may represent a more general mechanism underlying its diverse biological effects.     

Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Voltage-gated Na⁺ channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na⁺ channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na⁺ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.     

Structural Basis for Calcium and Magnesium Regulation of a Large Conductance Calcium-activated Potassium Channel with β1 Subunits

Large conductance Ca²⁺- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary β subunits, play important roles in diverse physiological activities. The β1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca²⁺ sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and β1 remains elusive. Using macroscopic ionic current recordings in various Ca²⁺ and Mg²⁺ concentrations, we identified two binding sites on the cytosolic N terminus of β1, namely the electrostatic enhancing site (mSlo1(K392,R393)-β1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-β1(L5,V6,M7)), passing the physical force from the Ca²⁺ bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg²⁺ sensitivity. A comprehensive structural model of the BK(mSlo1 α-β1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.     

Binding of histamine to the H1 receptor—a molecular dynamics study

Binding of histamine to the G-protein coupled histamine H₁ receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp107^3.32, Tyr108^3.33, Thr194^5.43, Asn198^5.46, Trp428^6.48, Tyr431^6.51, Phe432^6.52, and Phe435^6.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 μs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp107^3.32 for histamine binding.     

The Direct Binding of Insulin-like Growth Factor-1 (IGF-1) to Integrin ��v��3 Is Involved in IGF-1 Signaling

It has been proposed that ligand occupancy of integrin αvβ3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of αvβ3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that αvβ3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of αvβ3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin αvβ3 plays a role in IGF-1 signaling through ternary complex formation (αvβ3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.Integrins are a family of cell adhesion receptors that mediate cell-extracellular matrix (ECM)³ interaction and cell-cell interaction (1). It has been proposed that signaling from inside the cells regulates the ligand binding affinity of integrins (inside-out signaling) (2). Each integrin is a heterodimer containing α and β subunits. At present 18 α and 8 β subunits have been identified that combine to form 24 integrins (3).It has been reported that integrin αvβ3 plays a role in cancer proliferation and invasiveness. High levels of integrin αvβ3 correlate with growth and/or progression of melanoma (4, 5), neuroblastoma (6), breast cancer (7, 8), colon cancer (9), ovarian cancer (10), and cervical cancer (11). Moreover, individuals homozygous for the β3L33P polymorphism that enhances the ligand binding affinity of β3 integrins have an increased risk to develop breast cancer, ovarian cancer, and melanoma (12). However, it remains unclear whether and how increased levels of αvβ3 on tumor cells contribute to cancer development.Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone (75 kDa) that has a high degree of structural similarity to human proinsulin. IGF-1 acts through binding to the type I IGF receptor (IGF1R), a receptor tyrosine kinase. The IGF1R is a heterotetramer that consists of two α-subunits that contain the ligand-binding domains and two β-subunits that contain the tyrosine kinase activity. After ligand binding, the receptor undergoes a conformational change resulting in the activation of the tyrosine kinase, which results in transphosphorylation of the opposite β-subunit on specific tyrosine residues. These phosphotyrosines then bind to adapter molecules such as Shc and IRS-1. Phosphorylation of these proteins leads to activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) signaling pathways (reviewed in Ref. 13).IGF-1 has been implicated in cancer progression (14). One of the major actions of IGF-1 is to inhibit apoptosis. IGF-1 confers resistance to chemotherapy and radiation therapy. IGF-1 expression levels are increased in breast, lung, prostate, and many other cancers. Several strategies to target IGF-1 signaling have been extensively studied, including small interfering RNA and monoclonal antibodies for IGF1R and kinase inhibitors to inhibit the enzymatic activity of the receptor. The IGF-1 system is a therapeutic target for cancer, and elucidation of the IGF-1 signaling pathway should have a major impact in designing new therapeutic strategies.It has been proposed that ligand occupancy of αvβ3 with ECM ligands such as vitronectin plays a critical role in enhancing IGF-1 signaling (14). It has been reported that inhibiting αvβ3-ECM interaction (“ligand occupancy”) of αvβ3 inhibited IGF-1 actions selectively in cell types that express αvβ3 (14). Inhibiting ligand occupancy of αvβ3 blocked IGF-1-induced cell migration (15), DNA synthesis, IRS-1 phosphorylation, and IGF1R-linked downstream signaling events, such as activation of phosphatidylinositol 3-kinase and ERK1/2 (16).In the present study, we demonstrated that expression of αvβ3 enhanced proliferation of ovarian cancer cells in the presence of fetal bovine serum (FBS) and in serum-free conditions if IGF-1 was present. This suggests that IGF-1 is involved in enhanced proliferation of αvβ3-expressing cells. We demonstrated that αvβ3 bound to IGF-1 in several different binding assays. We found that two Arg residues at positions 36 and 37 in the C-domain of IGF-1 are critical for integrin binding by docking simulation and mutagenesis. Mutation of these Arg residues to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, the R36E/R37E mutant was defective in inducing cell proliferation and IGF-1 intracellular signaling, although it still bound to IGF1R. We demonstrated that wild type IGF-1 mediated co-precipitation of αvβ3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 plays a role in IGF-1 signaling.