C-myc Epitope Research Articles

Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein.

P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

Functional role of surface layer proteins of Lactobacillus acidophilus L-92 in stress tolerance and binding to host cell proteins.

Lactobacillus acidophilus surface layer proteins (SLPs) self-assemble into a monolayer that is non-covalently bound to the outer surface of the cells. There they are in direct contact with the environment, environmental stressors and gut components of the host in which the organism resides. The role of L. acidophilus SLPs is not entirely understood, although SLPs seem to be essential for bacterial growth. We constructed three L. acidophilus L-92 strains, each expressing a mutant of the most abundant SLP, SlpA. Each carried a 12-amino acid c-myc epitope substitution at a different position in the protein. A strain was also obtained that expressed the SlpA paralog SlpB from an originally silent slpB gene. All four strains behaved differently with respect to growth under various stress conditions, such as the presence of salt, ox gall or ethanol, suggesting that SlpA affects stress tolerance in L. acidophilus L-92. Also, the four mutants showed differential in vitro binding ability to human host cell proteins such as uromodulin or dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). Furthermore, co-culture of murine immature DCs with a mutant strain expressing one of the recombinant SlpA proteins changed the concentrations of the cytokines IL-10 and IL-12. Our data suggest that SlpA and SlpB of L. acidophilus participate in bacterial stress tolerance and binding to uromodulin or DC-SIGN, possibly leading to effective immune-modification.

Enhancement of cell proliferation and motility of mammalian cells grown in co-culture with Pichia pastoris expressing recombinant human FGF-2

Many studies examining the biological function of recombinant proteins and their effects on the physiology of mammalian cells stipulate that the proteins be purified before being used as therapeutic agents. In this study, we explored the possibility of using unpurified recombinant proteins to treat mammalian cells. The recombinant protein was used directly from the expression source and the biological function was compared to purified commercially available, equivalent protein. The model for this purpose was recombinant FGF-2, expressed by Pichia pastoris, which was used to treat the murine fibroblast cell line, NIH/3T3. We generated a P. pastoris strain (yHL11) that constitutively secreted a biologically active recombinant FGF-2 protein containing an N-terminal c-myc epitope (Myc-FGF-2). Myc-FGF-2 was then used without purification either a) in the form of conditioned mammalian cell culture medium or b) during co-cultures of yHL11 with NIH/3T3 to induce higher proliferation and motility of NIH/3T3 cells. The effects of Myc-FGF-2 on cell physiology were comparable to commercially available FGF-2. To our knowledge, this is the first time the physiology of cultured mammalian cells had been successfully altered with a recombinant protein secreted by P. pastoris while the two species shared the same medium and culture conditions. Our data demonstrated the biological activity of unpurified recombinant FGF-2 on NIH/3T3 cells and provided a foundation for directly using unpurified recombinant proteins expressed by P. pastoris with mammalian cells, potentially as wound-healing therapeutics.

Quantification of interacting cognate odorants with olfactory receptors in nanovesicles

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors.

Biological activity and dimerization state of modified phytochrome A proteins.

To assess potential physical interactions of type I phyA with the type II phyB-phyE phytochromes in vivo, transgenes expressing fusion gene forms of phyA were introduced into the Arabidopsis phyA mutant background. When a single c-Myc (myc) epitope is added to either the N- or C-terminus of phyA, the constructs completely complement phyA mutant phenotypes. However, addition of larger tags, such as six consecutive myc epitopes or the yellow fluorescent protein sequence, result in fusion proteins that show reduced activity. All the tagged phyA proteins migrate as dimers on native gels and co-immunoprecipitation reveals no binding interaction of phyA to any of the type II phys in the dark or under continuous far-red light. Dimers of the phyA 1–615 amino acid N-terminal photosensory domain (NphyA), generated in vivo with a yeast GAL4 dimerization domain and attached to a constitutive nuclear localization sequence, are expressed at a low level and, although they cause a cop phenotype in darkness and mediate a very low fluence response to pulses of FR, have no activity under continuous FR. It is concluded that type I phyA in its Pr form is present in plants predominantly or exclusively as a homodimer and does not stably interact with type II phys in a dimer-to-dimer manner. In addition, its activity in mediating response to continuous FR is sensitive to modification of its N- or C-terminus.

Heterologous expression of β-γ-type cyclodextrin glycosyltransferase of newly isolated alkaliphilicBacillussp. SD5 inPichia pastoris

Cyclodextrin glycosyltransferase (CGTase) enzyme is used industrially to obtain cyclodextrins. In this study, a newly isolated alkaliphilic CGTase producing Bacillus sp. SD5 was used as the source organism for the gene encoding for CGTase. The gene was amplified and sequenced in preparation for cloning into Pichia pastoris for methanol-induced expression and extracellular secretion. Three strategies were employed in designing the amplification primers for obtaining the fragments to be used in transformation. Hence, three P. pastoris X33 clones were obtained: N2 (with natural gene sequence), S3 (also encoding the c-myc epitope and polyhistidine tail), and M5 (encoding the polyhistidine tail). Extracellular CGTase activity was monitored throughout fermentation for each clone. The molecular weights of the enzymes were in the range of 85–90 kDa and all three clones showed maximum CGTase activity upon 24 h of induced fermentation. The CGTase enzyme obtained from the M5 clone had Km and Vmax values 13.59 mg/mL and 1153 µmol/(mg min), respectively. The enzyme was purified and displayed optimum activity at 50°C and pH 6 and pH 9. HPLC analysis of reaction products following 10 min of incubation in a reaction solution containing 4% starch showed 852.5 mg/L β-cyclodextrin and 89.6 mg/L γ-cyclodextrin being produced.

Heterologous expression of β-γ-type cyclodextrin glycosyltransferase of newly isolated alkaliphilic Bacillus sp. SD5 in Pichia pastoris

Cyclodextrin glycosyltransferase (CGTase) enzyme is used industrially to obtain cyclodextrins. In this study, a newly isolated alkaliphilic CGTase producing Bacillus sp. SD5 was used as the source organism for the gene encoding for CGTase. The gene was amplified and sequenced in preparation for cloning into P. pastoris for methanol induced expression and extracellular secretion. Three strategies were employed in designing the amplification primers for obtaining the fragments to be used in transformation. Hence three Pichia pastoris X33 clones were obtained; N2 (with natural gene sequence), S3 (also encoding the c-myc epitope and polyhistidine tail) and M5 (encoding the polyhistidine tail). Extracellular CGTase activity was monitored throughout fermentation for each clone. The molecular weights of the enzymes were in the range of 85-90 kDa and all three clones showed maximum CGTase activity upon 24 hours of induced fermentation. The CGTase enzyme obtained from the M5 clone had Km and Vmax values 13.59 mg/mL and 1153 μmol/(mg.min), respectively. The enzyme was purified and displayed optimum activity at 50 °C and pH 6 and pH 9. HPLC analysis of reaction products following 10 minutes of incubation in a reaction solution containing 4% starch showed 852.5 mg/L β-cyclodextrin and 89.6 mg/L γ-cyclodextrin being produced.

PRODUCTION OF ANTIBODY IgY ANTI-c-Myc IN CHICKEN EGGS

<p>Bioinformatics analysis has been performed on c-Myc peptide sequences to predict its epitopes in order to design antigen for IgY anti-c-Myc production in chickens. Peptide sequences for human c-Myc protein obtained from the NCBI database were analyzed with ProPred-1 softwares. Manual screening has been conducted on the prediction results.One candidate of c-Myc epitope, EQKLISEEDL, have been selected and prepared as an antigen, by adding one molecule of Cystein to the peptide, and conjugated it with Keyhole limpet hemocyanin (KLH). Twelve hens, aged 22-24 weeks, were immunized a week after their first eggs were produced. Antigen diluted in aquabidest and emulsified with Freund’s Complete Adjuvant. The suspension was injected into the area of the chicken breast, subcutaneously or intramuscularly, with a dose of 0.1 mg for the first immunization. Booster injection performed on days 10, 20, and 30 with the same volume of emulsion, but using Incomplete Freund’s Adjuvant and the amount of antigen half of the first immunization. Extraction of IgY from egg has been carried out using kits from Gallus Immunotech. IgY levels quantified using a spectrophotometer, absorbance measured at 280 nm. The results showed that chicken antibody production gave rise to a sigmoid curve pattern, just like in mammals. The IgY content of the egg was stable at high levels (> 5 mg / ml yolk) after the third immunization. In the intramuscular group, the immune response seems to work earlier than in the subcutaneous group, but eventually the subcutaneous group reaches the same level, and even higher. In general, from an egg produced after the second booster it can be extracted at least 50 mg of IgY. Higher yields could be obtained when IgY was extracted from egg 2 to 3 days after the third booster. </p><p>Keywords: epitope prediction, c-Myc, IgY antibody, ProPred-1</p>

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity.

Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >10(3) times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated saporin L3 (amino acids 22-280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94–101, 93–101, and 93–102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93–102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93–102 residues to 8 aa residues at positions 94–101 in 3A protein.

One-for-all: a monoclonal antibody specific to different recombinant proteins in transgenic citrus plants

The easy and rapid identification of a recombinant protein in transgenic plants is becoming increasingly relevant as more transgenic plants are used for research and commercial applications. Tagging recombinant proteins with a small peptide (epitope) can perform such a task using a variety of immunological methods. Epitope tags are short, hydrophilic peptide sequences recognized by specific antibodies. Compared with larger protein fusions, the small size of epitope tags makes them less likely to interfere with protein folding and function.We describe herein the detection of the c-myc epitope using different immunological methods in citrus transgenic plants. A c-myc tag sequence (N-EQKLISEEDL-C, corresponding to the C-terminal amino acids (410-419) of human c-myc protein) was added to the DNA sequence by PCR and the resulting proteins are being tested at the CREC. Our experiments with a genetically altered endogenous citrus gene modified to produce a protein with the c-myc tag demonstrate the utility of this technique for detection of trans-proteins in Citrus. Since this tag can be incorporated in the C terminal end of any protein, this technology simplifies different assays that require recognition by protein specific antibodies. We could detect different trans-proteins using the same antibody against the Myc epitope by ELISA or Western blotting. Moreover, expression of recombinant proteins bearing epitope tags can also eliminate the need of isolating proteins and producing antibodies for each new recombinant protein to be studied, which requires more cost and time, and can be problematic as a result of low antigenicity or high background cross-reaction with other proteins.

Improved expression of single-chain antibodies in Ustilago maydis

To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

Expression of Medicago truncatula ecto-apyrase MtAPY1;1 in leaves of Nicotiana benthamiana restricts necrotic lesions induced by a virulent fungus

Ecto-apyrase(s) participates in cell-wall-associated defense through ATP hydrolysis. Here we analyzed Medicago truncatula genes through cDNA screening and in silico analyses against known databases. This study revealed seven genes, five of which (MtAPY1;1 to MtAPY1;5) are members of a legume-specific family, whereas two genes (MtAPY2;1 and MtAPY2;2) are close to those in other plants. Agrobacterium-based transient expression in Nicotiana benthamiana, combined with a c-myc epitope tag technology, confirmed that the MtAPY1;1 is a secreted protein. Transient expression of MtAPY1;1 in leaves of N. benthamiana restricted disease development by a virulent fungus, suggesting a role in disease resistance.

Purification of the goldfish membrane progestin receptor α (mPRα) expressedin yeast Pichia pastoris

Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestins on plasma membranes. We established a procedure for the expression and purification of recombinant goldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, the recombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an active form as the receptor for maturation-inducing steroids of fish. Expressed proteins were bound reversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. After solubilization of mPRα with n-dodecyl-β-D-maltoside (DDM) from yeast membranes, the recombinant protein was purified using three different columns: first it was affinity-purified over nickel-nitrilotriaceticacid (Ni-NTA), then bound to a cellulose resin with free amino groups and finally to a column with affinity for the c-Myc epitope. The identity of the purified protein was verified by MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purification of mPRα protein in its functional form will enable the screening of ligands and the determination of its three dimensional structure.

Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified. [BMB Reports 2013; 46(10): 495-500]

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome

Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3′ region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor.

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Spx activates transcription initiation in Bacillus subtilis by directly interacting with the C-terminal domain of the RNA polymerase (RNAP) holoenzyme α subunit, which generates a complex that recognizes the promoter regions of genes within the Spx regulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneously contact RNAP. The composition of Spx/RNAP was examined in vitro using an Spx variant (SpxΔCHA) bearing a 12-amino-acid deletion of the C terminus (SpxΔC) and a hemagglutinin (HA) epitope tag and Spxc-Myc, a full-length Spx with a C-terminal myelocytomatosis oncoprotein (c-Myc) epitope tag. All Spx/RNAP complexes bearing deletion or C-terminal-tagged variants were transcriptionally active in vivo and in vitro. Reaction mixtures containing SpxΔCHA and Spxc-Myc combined with RNAP were applied to either anti-HA or anti-c-Myc affinity columns. Eluted fractions contained RNAP with only one of the epitope-tagged Spx derivatives. The resin-bound RNAP complex bearing a single epitope-tagged Spx derivative was transcriptionally active. In vivo production of SpxΔC and SpxΔCHA followed by anti-HA affinity column chromatography of a cleared lysate resulted in retrieval of Spx/RNAP with only the SpxΔCHA derivative. Binding reactions that combined active Spxc-Myc, inactive Spx(R60E)ΔCHA, and RNAP, when applied to the anti-HA affinity column, yielded only inactive Spx(R60E)ΔCHA/RNAP complexes. The results strongly argue for a model in which a single Spx monomer engages RNAP to generate an active transcriptional complex.

Translocation of calcium-permeable TRPV2 channel to the podosome: Its role in the regulation of podosome assembly

The present study was conducted to investigate localization and function of TRPV2 channel in a mouse macrophage cell line, TtT/M87. We infected an adenovirus vector encoding TRPV2 tagged with c-Myc in the extracellular domain. Immunoreactivity of c-Myc epitope exposed to the cell surface formed a ring structure, which was colocalized with markers of the podosome, namely β-integrin, paxillin and Pyk2. The ring structure was also observed in TRPV2-GFP-expressing cells using total internal reflection fluorescent microscopy. Addition of formyl-Met-Leu-Phe (fMLP) increased the number of podosome and increased the intensity of the TRPV2 signal associated with the podosome. Measurement of subplasmalenmal free calcium concentration ([Ca2+]pm) revealed that [Ca2+]pm was elevated around the podosome. fMLP further increased [Ca2+]pm in this region, which was abolished by a TRPV2 inhibitor ruthenium red. Phosphorylated Pyk2 was detected in fMLP-treated cells, and knockdown of TRPV2 reduced the expression of phospho-Pyk2. Introduction of dominant-negative Pyk2 or knockdown of TRPV2 increased the number of podosome. Conversely, elevation of [Ca2+]pm by the addition of ionomycin reduced the number of podosome. These results indicate that TRPV2 is localized abundantly in the podosome and increases [Ca2+]pm by the podosome. The elevation of [Ca2+]pm is critical to regulate assembly of the podosome.

S125 Potential interactions of CRACM ion channels with the calcium activated potassium channel KCa3.1 in human lung mast cells

Introduction and ObjectivesThe influx of extracellular Ca2+ is essential for the IgE-dependent release of both preformed mediators and newly-generated autacoids and cytokines from human lung mast cells (HLMC). Members of...