Concept: Cell signaling
N-methyl-D-aspartate (NMDA) receptors are glutamate- and glycine-gated channels composed of two GluN1 and two GluN2 or/and GluN3 subunits. GluN3A expression is developmentally regulated, and changes in this normal pattern of expression, which occur in several brain disorders, alter synaptic maturation and function by unknown mechanisms. Uniquely within the NMDA receptor family, GluN1/GluN3 receptors produce glycine-gated deeply desensitising currents that are insensitive to glutamate and NMDA; these currents remain poorly characterised and their cellular functions are unknown. Here, we show that extracellular acidification strongly potentiated glycine-gated currents from recombinant GluN1/GluN3A receptors, with half-maximal effect in the physiologic pH range. This was largely due to slower current desensitisation and faster current recovery from desensitisation, and was mediated by residues facing the heterodimer interface of the ligand-binding domain. Consistent with the observed changes in desensitisation kinetics, acidic shifts increased the GluN1/GluN3A equilibrium current and depolarized the membrane in a glycine concentration-dependent manner. These results reveal novel modulatory mechanisms for GluN1/GluN3A receptors that further differentiate them from the canonical glutamatergic GluN1/GluN2 receptors and provide a new and potent pharmacologic tool to assist the detection, identification, and the further study of GluN1/GluN3A currents in native preparations.
Integrin clustering plays a pivotal role in a host of cell functions. Hetero-dimeric integrin adhesion receptors regulate cell migration, survival, and differentiation by communicating signals bidirectionally across the plasma membrane. Thus far, crystallographic structures of integrin components are solved only separately, and for some integrin types. Also, the sequence of interactions that leads to signal transduction remains ambiguous. Particularly, it remains controversial whether the homo-dimerization of integrin transmembrane domains occurs following the integrin activation (i.e. when integrin ectodomain is stretched out) or if it regulates integrin clustering. This study employs molecular dynamics modeling approaches to address these questions in molecular details and sheds light on the crucial effect of the plasma membrane. Conducting a normal mode analysis of the intact αllbβ3 integrin, it is demonstrated that the ectodomain and transmembrane-cytoplasmic domains are connected via a membrane-proximal hinge region, thus merely transmembrane-cytoplasmic domains are modeled. By measuring the free energy change and force required to form integrin homo-oligomers, this study suggests that the β-subunit homo-oligomerization potentially regulates integrin clustering, as opposed to α-subunit, which appears to be a poor regulator for the clustering process. If α-subunits are to regulate the clustering they should overcome a high-energy barrier formed by a stable lipid pack around them. Finally, an outside-in activation-clustering scenario is speculated, explaining how further loading the already-active integrin affects its homo-oligomerization so that focal adhesions grow in size.
In response to toxic stimuli, BCL2L11 (also known as BIM), a BH3-only protein, is released from its interaction with dynein light chain 1 (DYNLL1 also known as LC8) and can induce apoptosis by inactivating anti-apoptotic BCL2 proteins and by activating BAX-BAK1. Recently, we discovered that BCL2L11 interacts with BECN1 (Beclin 1), and that this interaction is facilitated by DYNLL1. BCL2L11 recruits BECN1 to microtubules by bridging BECN1 and DYNLL1, thereby inhibiting autophagy. In starvation conditions, BCL2L11 is phosphorylated by MAPK8/JNK and this phosphorylation abolishes the BCL2L11-DYNLL1 interaction, allowing dissociation of BCL2L11 and BECN1, thereby ameliorating autophagy inhibition. This finding demonstrates a novel function of BIM beyond its roles in apoptosis, highlighting the crosstalk between autophagy and apoptosis, and suggests that BCL2L11’s dual effects in inhibiting autophagy and promoting apoptosis may have important roles in disease pathogenesis.
BACKGROUND: Cells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory proteins that, all together, form focal adhesions (FAs). The molecular basis of how these sensing units are regulated, how they are implicated in transducing mechanical stimuli, and how this leads to a spatiotemporal coordination of FAs is unclear. RESULTS: Here we show that vinculin, through its links to the talin-integrin complex and F-actin, regulates the transmission of mechanical signals from the extracellular matrix to the actomyosin machinery. We demonstrate that the vinculin interaction with the talin-integrin complex drives the recruitment and release of core FA components. The activation state of vinculin is itself regulated by force, as underscored by our observation that vinculin localization to FAs is dependent on actomyosin contraction. Using a variety of vinculin mutants, we establish which components of the cell-matrix adhesion network are coordinated through direct and indirect associations with vinculin. Moreover, using cyclic stretching, we demonstrate that vinculin plays a key role in the transmission of extracellular mechanical stimuli leading to the reorganization of cell polarity. Of particular importance is the actin-binding tail region of vinculin, without which the cell’s ability to repolarize in response to cyclic stretching is perturbed. CONCLUSIONS: Overall our data promote a model whereby vinculin controls the transmission of intracellular and extracellular mechanical cues that are important for the spatiotemporal assembly, disassembly, and reorganization of FAs to coordinate polarized cell motility.
BACKGROUND: Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterialphosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents asignaling unit that acts as a sensory element and measures the activity of the central metabolism.Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr),which communicate with each other by phosphate exchange. Recent experimental results showed a cross talkbetween the two branches. However, the functional role of the crosstalk remains open. RESULTS: A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one ofthe PTS proteins, for different environmental conditions and different strain variants. Additionally, data fromflux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based onthe calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructosecould be determined. CONCLUSION: Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major partof the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a newrole of the second branch of the PTS and will serve as a basis for further experimental studies.
Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5) isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.
BACKGROUND: Honokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. RESULTS: Honokiol had a potent (EC50 [almost equal to] 5 mumol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding. CONCLUSIONS: It is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.
Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4+CD3+CD4+) and show that this group of cells is responsive to sustained nicotine exposure. In circulation, spleen, and thymus we find that nicotine promotes an increase in CD3+CD4+ cells via its activation of the α4 nAChR and regulation of Gαo, Gprin1, and CDC42 signaling within T-cells. In particular, nicotine is found to promote a Th2, adaptive, immunological response within T-cells, which was absent in α4-/- mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T-cells, possibly accounting for the effect of smoking on the immune system.
Gap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells.
The frequent alterations of the PI3K/Akt/mTOR-growth signaling pathway are proposed mechanisms for resistance to endocrine therapy in breast cancer, partly through regulation of estrogen receptor α (ER) activity. Reliable biomarkers for treatment prediction are required for improved individualized treatment. We performed a retrospective immunohistochemical analysis of primary tumors from 912 postmenopausal patients with node-negative breast cancer, randomized to either tamoxifen or no adjuvant treatment. Phosphorylated (p) Akt-serine (s) 473, p-mTOR-s2448, and ER phosphorylations-s167 and -s305 were evaluated as potential biomarkers of prognosis and tamoxifen treatment efficacy. High expression of p-mTOR indicated a reduced response to tamoxifen, most pronounced in the ER+/progesterone receptor (PgR) + subgroup (tamoxifen vs. no tamoxifen: hazard ratio (HR), 0.86; 95 % confidence interval (CI), 0.31-2.38; P = 0.78), whereas low p-mTOR expression predicted tamoxifen benefit (HR, 0.29; 95 % CI, 0.18-0.49; P = 0.000002). In addition, nuclear p-Akt-s473 as well as p-ER at -s167 and/or -s305 showed interaction with tamoxifen efficacy with borderline statistical significance. A combination score of positive pathway markers including p-Akt, p-mTOR, and p-ER showed significant association with tamoxifen benefit (test for interaction; P = 0.029). Cross-talk between growth signaling pathways and ER-signaling has been proposed to affect tamoxifen response in hormone receptor-positive breast cancer. The results support this hypothesis, as an overactive pathway was significantly associated with reduced response to tamoxifen. A clinical pre-treatment test for cross-talk markers would be a step toward individualized adjuvant endocrine treatment with or without the addition of PI3K/Akt/mTOR pathway inhibitors.