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951.
Razvan L. Cornea Florentin R. Nitu Montserrat Samsó David D. Thomas Bradley R. Fruen 《The Journal of biological chemistry》2010,285(25):19219-19226
The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca2+ release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [3H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model. 相似文献
952.
Wataru Masuda Matthew J. Betzenhauser David I. Yule 《The Journal of biological chemistry》2010,285(48):37927-37938
Ca2+ release through inositol 1,4,5-trisphosphate receptors (InsP3R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca2+ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca2+ release via InsP3R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP3R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP3R-1 subtype resulted in enhanced Ca2+ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP3R subtype, and phosphorylation of IRAG by PKG attenuated Ca2+ release through all InsP3R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca2+ release through InsP3R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP3R-2. Phosphorylation of IRAG resulted in reduced Ca2+ release through all InsP3R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways. 相似文献
953.
Eva Sammels Benoit Devogelaere Djalila Mekahli Geert Bultynck Ludwig Missiaen Jan B. Parys Yiqiang Cai Stefan Somlo Humbert De Smedt 《The Journal of biological chemistry》2010,285(24):18794-18805
Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca2+ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release mechanism, which only occurred in the presence of the TRPP2-IP3R interaction, and not via altered IP3R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca2+ microdomains necessary for initiating Ca2+-induced Ca2+ release. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease. 相似文献
954.
Lars Zimmermann Wolfgang Paster Julian Weghuber Paul Eckerstorfer Hannes Stockinger Gerhard J. Sch��tz 《The Journal of biological chemistry》2010,285(9):6063-6070
Palmitoylation represents a common motif for anchorage of cytosolic proteins to the plasma membrane. Being reversible, it allows for controlled exchange between cytosolic and plasma membrane-bound subpopulations. In this study, we present a live cell single molecule approach for quantifying the exchange kinetics of plasma membrane and cytosolic populations of fluorescently labeled Lck, the key Src family kinase involved in early T cell signaling. Total internal reflection (TIR) fluorescence microscopy was employed for confining the analysis to membrane-proximal molecules. Upon photobleaching Lck-YFP in TIR configuration, fluorescence recovery proceeds first via the cytosol outside of the evanescent field, so that in the early phase fluorescence signal arises predominantly from membrane-proximal cytosolic Lck. The diffusion constant of each molecule allowed us to distinguish whether the molecule has already associated with the plasma membrane or was still freely diffusing in the cytosol. From the number of molecules that inserted during the recovery time we quantified the insertion kinetics: on average, membrane-proximal molecules within the evanescent field needed ∼400 ms to be inserted. The average lifetime of Lck in the plasma membrane was estimated at 50 s; together with the mobility of 0.26 μm2/s this provides sufficient time to explore the surface of the whole T cell before dissociation into the cytosol. Experiments on palmitoylation-deficient Lck mutants yielded similar on-rates, but substantially increased off-rates. We discuss our findings based on a model for the plasma membrane association and dissociation kinetics of Lck, which accounts for reversible palmitoylation on cysteine 3 and 5. 相似文献
955.
Juliati Rahajeng Sai S. P. Giridharan Naava Naslavsky Steve Caplan 《The Journal of biological chemistry》2010,285(42):31918-31922
Endocytosis is a conserved cellular process in which nutrients, lipids, and receptors are internalized and transported to early endosomes, where they are sorted and either channeled to degradative pathways or recycled to the plasma membrane. MICAL-L1 and EHD1 are important regulatory proteins that control key endocytic transport steps. However, the precise mechanisms by which they mediate transport, and particularly the mode by which they connect to motor proteins, have remained enigmatic. Here we have identified the collapsin response mediator protein-2 (Crmp2) as an interaction partner of MICAL-L1 in non-neuronal cells. Crmp2 interacts with tubulin dimers and kinesin and negatively regulates dynein-based transport in neuronal cells, but its expression and function in non-neuronal cells have remained poorly characterized. Upon Crmp2 depletion, we observed dramatic relocalization of internalized transferrin (Tf) from peripheral vesicles to the endocytic recycling compartment (ERC), similar to the effect of depleting either MICAL-L1 or EHD1. Moreover, Tf relocalization to the ERC could be inhibited by interfering with microtubule polymerization, consistent with a role for uncoupled motor protein-based transport upon depletion of Crmp2, MICAL-L1, or EHD1. Finally, transfection of dynamitin, a component of the dynactin complex whose overexpression inhibits dynein activity, prevented the relocalization of internalized Tf to the ERC upon depletion of Crmp2, MICAL-L1, or EHD1. These data provide the first trafficking regulatory role for Crmp2 in non-neuronal cells and support a model in which Crmp2 is an important endocytic regulatory protein that links MICAL-L1·EHD1-based vesicular transport to dynein motors. 相似文献
956.
The two‐way communication between the ECM (extracellular matrix) and the cytoplasm via the integrins has many functions in cancer cells, including the suppression of apoptosis. As cells in a 3D (three‐dimensional) architecture resemble the in vivo situation more closely than do cells in more conventional 2D cultures, we have employed a substratum that prevents cell adhesion and induces cell aggregation to determine why highly metastatic B16F10 melanoma cells resist anoikis. We compared the behaviour of B16F10 cells in 2D [on tPS (tissue culture polystyrene)] and 3D culture {on polyHEMA [poly(2‐hydroxyethylmethacrylate)]} configurations. For this, we analysed cell morphology, proliferation, apoptosis and the activation status of several proteins involved in cell proliferation and survival [RhoA, FAK (focal adhesion kinase), Akt, ERK1/2 (extracellular‐signal‐regulated kinase 1/2)]. B16F10 cells in 3D architecture were able to proliferate as cell aggregates for 3 days, after which the number of cells decreased. The normal Swiss 3T3 cells used as an anoikis‐sensitive control did not proliferate on the anti‐adhesive substratum. Rho A was activated in B16F10 aggregates throughout their time in culture, whereas it was not in Swiss 3T3 aggregates. An absence of apoptotic activity was correlated with the proliferation of B16F10 cells in aggregates: caspase 3 was significantly activated only after 3 days in culture on polyHEMA. FAK and Akt were transiently activated, and their inactivation was correlated with the induction of apoptosis. ERK1/2 were activated throughout the 3D culture. No survival protein was activated in Swiss 3T3 aggregates. Data obtained from cells in 3D culture suggest that B16F10 cells are resistant to anoikis through the activation of the FAK and Akt signalling pathways. 相似文献
957.
Clegg JS 《Cell biology international》2010,34(11):1105-1107
The 'microtrabecular lattice' (MTL) that Keith Porter described in the 1970s and 1980s is reconsidered as a proposed fundamental cytoplasmic structure of eukaryotic cells. Although considered to be an artefact by most cell biologists of his time (and probably ours), the case is made that something like the MTL may well exist, but in a much more dynamic form than images from electron microscopy imply and convey. 相似文献
958.
Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for
its successful replication within the host. A number of evidence suggest that changes in parasite Ca2+ levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC.
However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca2+ increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca2+ levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an
increase of cytosolic Ca2+ in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise
in [Ca2+]c. Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites,
suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in
this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca2+]c in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells. 相似文献
959.
Mary G. O’Keeffe Peter R. Thorne Gary D. Housley Simon C. Robson Srdjan M. Vlajkovic 《Purinergic signalling》2010,6(2):249-261
Membrane-bound ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) in the inner ear regulate complex extracellular
purinergic type-2 (P2) receptor signalling pathways through hydrolysis of extracellular nucleoside 5′-triphosphates and diphosphates.
This study investigated the distribution of NTPDase5 and NTPDase6, two intracellular members of the E-NTPDase family, and
linked this to regulation of P2 receptor signalling in the adult rat cochlea. These extracellular ectonucleotidases preferentially
hydrolyse nucleoside 5′-diphosphates such as UDP and GDP. Expression of both enzymes at mRNA and protein level was detected
in cochlear tissues and there was in vivo release of soluble NTPDase5 and 6 into cochlear fluids. Strong NTPDase5 immunostaining
was found in the spiral ganglion neurones and supporting Deiters’ cells of the organ of Corti, while NTPDase6 was confined
to the inner hair cells. Upregulation of NTPDase5 after exposure to loud sound indicates a dynamic role for NTPDase5 in cochlear
response to stress, whereas NTPDase6 may have more limited extracellular roles. Noise-induced upregulation of co-localised
UDP-preferring P2Y6 receptors in the spiral ganglion neurons further supports the involvement of NTPDase5 in regulation of P2Y receptor signalling.
Noise stress also induced P2Y14 (UDP- and UDP-glucose preferring) receptor expression in the root processes of the outer sulcus cells, but this was not associated
with localization of the E-NTPDases. 相似文献
960.
Like animals, plants have evolved into complex organisms. Developmental cohesion between tissues and cells is possible due to signaling molecules (messengers) like hormones. The first hormone discovered in plants was auxin. This phytohormone was first noticed because of its involvement in the response to directional light. Nowadays, auxin has been established as a central key player in the regulation of plant growth and development and in responses to environmental changes. At the cellular level, auxin controls division, elongation, and differentiation as well as the polarity of the cell. Auxin, to integrate so many different signals, needs to be regulated at many different levels. A tight regulation of auxin synthesis, activity, degradation as well as transport has been demonstrated. Another possibility to modulate auxin signaling is to modify the capacity of response of the cells by expressing differentially the signaling components. In this review, we provide an overview of the present knowledge in auxin biology, with emphasis on root development. 相似文献