Does plasma membrane H+-ATPase activation by fusicoccin involve protein kinase?

Sugar beet ( Beta vulgaris L.) root suspension-cultured cells were converted to protoplasts which responded to fusicoccin (FC) by a rise in cytoplasmic pH (pH_cyt) averaging 0.25 units in the fluorimetric assay. This effect was blocked by erythrosin B, a specific inhibitor of the plasma membrane H₊-ATPase. A protein kinase inhibitor, staurosporine also caused cytosolic alkalinization that was sensitive to H⁺-ATPase inhibitors. Most strikingly, the effect of staurosporine was suppressed by fusicoccin and vice versa. Addition of okadaic acid, entailing overall protein phosphorylation, also led to H⁺-ATPase activation, whereupon fusicoccin lost its effect on proton transport. In parallel, kinetic and inhibitor analyses demonstrated that FC binding to the protoplast plasma membrane involved two sites with dissociation constants of 1 n M and 0.2 μ M and was indifferent to phosphorylation and dephosphorylation inhibitors. Thus, it could be concluded that (1) the effect of FC on cytoplasmic pH probably depends on the phosphorylation state of plasma membrane proteins and may have either sign; (2) the activation of H⁺-ATPase by FC most likely proceeds directly through conformational receptor-enzyme interaction.     

How does the plasma membrane participate in cellular signaling by receptors for immunoglobulin E?

Accumulating evidence strongly supports the view that the plasma membrane participates in transmembrane signaling by IgE-receptors (IgE-Fc epsilon RI) through the formation of lipid-based domains, also known as rafts. Ongoing biochemical and biophysical experiments investigate the composition, structure, and dynamics of the corresponding membrane components and how these are related to functional coupling between Fc epsilon RI and Lyn tyrosine kinase to initiate signaling in mast cells.     

Ankyrin-G palmitoylation and βII-spectrin binding to phosphoinositide lipids drive lateral membrane assembly

Ankyrin-G and βII-spectrin colocalize at sites of cell–cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and βII-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that βII-spectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and βII-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G–βII-spectrin interaction or βII-spectrin–phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with βII-spectrin, and βII-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells.     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.     

Does zaltoprofen antagonize the bradykinin receptors?

Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B₂ receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B₂ receptor antagonists (contractility, human isolated umbilical vein) or B₁ receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg⁹-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg⁹-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kinin-stimulated phospholipase A₂ activity in HEK 293 cells expressing recombinant forms of the rabbit B₁ or B₂ receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems.     

Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?

The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.     

Does amiodarone inhibit T3 binding to solubilized nuclear receptors in vitro?

One of the proposed mechanisms of action of amiodarone is by interfering with T3-receptor interactions. However, reports in the literature on this matter are contradictory and, in an attempt to resolve these contradictions, the effects of amiodarone on T3 binding to solubilized nuclear receptors from rat liver were investigated. A drug concentration-related competitive inhibition of T3 binding occurred in the presence of considerable amiodarone precipitation. Measurement of the proportion of soluble amiodarone over the range where significant inhibition of T3 binding was observed, revealed that the soluble amiodarone decreased over this range. Since the amiodarone preparation was free of any contaminant, these findings suggested that a product generated from amiodarone during incubation was responsible for the inhibition. HPLC analysis of the soluble amiodarone fraction indicated that, during incubation, two substances were generated from the parent compound. However, only the concentration of one of the substances continually increased whereas the other decreased with increasing concentrations of amiodarone added to the samples. These findings suggest that the inhibition of T3 binding to solubilized nuclear receptors in the presence of amiodarone results from the generation of a specific product from the amiodarone under certain conditions during incubation.     

Does estrogen contribute to the hepatic regeneration following portal branch ligation in rats?

The aim of this study was to determine whether estrogen plays any role in the hepatic regeneration of nonligated lobe following portal branch ligation (PBL). Male rats were subjected to PBL on the left and middle lobes. Two and 7 days after PBL, the rats were killed and blood and liver samples were analyzed. Sham animals underwent only laparotomy. The serum estradiol levels were significantly elevated on day 2 following PBL and returned to normal levels on day 7. The expression of estrogen receptors (ER) in the liver evaluated by Western blotting did not show any change in the nonligated lobe compared with shams. Immunohistochemical study for ER showed a predominant ER expression in the hepatocyte nucleus in periportal area (zone 1), although there was no apparent difference in the amount and expression pattern between sham and PBL. However, chronic inhibition of ER by an ER antagonist (ICI 182,780) showed a significantly lower regeneration rate of the nonligated lobe compared with vehicle treatment. Liver regeneration-associated genes also were less activated in the ICI group. Moreover, portal venous flow, determined by fluorescent microsphere injection, was significantly lower in the ICI group compared with vehicle group. These changes correlated with the attenuated expression of endothelial nitric oxide synthase mRNA in both superior mesenteric arteries and veins. In conclusion, these results indicate that the estrogen's contribution on hepatic regeneration following PBL is at least partly mediated through maintaining mesenteric blood flow by mesenteric endothelial nitric oxide synthase upregulation rather than directly activating liver regeneration in the liver.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Proteomics and the dynamic plasma membrane: Quo Vadis?

Quo Vadis: where are you going? Advances in MS-based proteomics have enabled research to move from obtaining the basic protein inventory of cells and organelles to the ability of monitoring their dynamics, including changes in abundance, location and various PTMs. In this respect, the cellular plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma membrane proteome is crucial for understanding fundamental biological processes, disease mechanisms and for finding drug targets. Protein identification, characterization of dynamic PTMs and protein-ligand interactions, and determination of transient changes in protein expression and composition are among the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required for detailed functional and comparative analysis of the dynamic plasma membrane proteome.     

TGF-β, to target or not to target; to prevent thyroid cancer progression?

Thyroid cancer (TC) is a common endocrine cancer with a rising incidence. Current treatment fails to eliminate aggressive thyroid tumours, prompting an investigation into the processes that cause disease progression. In this review, we provide insight into TGF-β driven epithelial to mesenchymal transition (EMT), summarizing the current literature surrounding thyroid carcinogenesis, and discuss the potential for therapeutic strategies targeting the TGF-β signalling pathway. Understanding the underlying mechanisms that regulate cancer stem cell (CSC) growth and TGF-β signalling may provide novel therapeutic approaches for highly resistant TCs.     

Does inbreeding affect N-glycosylation of human plasma proteins?

Inbreeding depression and heterosis are the two ends of phenotypic changes defined by the genome-wide homozygosity. The aim of this study was to investigate the association of genetic marker-based homozygosity estimates with 46 N-glycan features measured in human plasma. The study was based on a total of 2,341 subjects, originating from three isolated island communities in Croatia (Vis and Korcula islands) and Scotland (Orkney Islands). Inbreeding estimates were associated with an increase in tetrantennary and tetrasialylated glycans, and a decrease in digalactosylated glycans (P?相似文献   

The Neurospora crassa exocyst complex tethers Spitzenk?rper vesicles to the apical plasma membrane during polarized growth

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.     

Peptide ligands that use a novel binding site to target both TGF-β receptors

The transforming growth factor beta (TGF-β) signaling pathway plays myriad roles in development and disease. TGF-β isoforms initiate signaling by organizing their cell surface receptors TβRI and TβRII. Exploration and exploitation of the versatility of TGF-β signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of TβR1 (TβRI-ED) as a target for effector screening. The observation that TβRI-ED can bind to a TGF-β coreceptor (endoglin) suggests that the TβRI-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TβRI-ED (K(d)≈ 10(-5) M). Although our screen focused on TβRI-ED, the hit peptides interact with the TβRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TβRI and TβRII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-β binding. These results indicate that both TβRI and TβRII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-β. To convert these compounds into high affinity probes, we exploited the observation that TβRI and TβRII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-β function.