ADAM17 co-precipitates with α5β1 integrin and dissociates from the integrin during GPCR stimulation.

<p>(A) Control (−) and 1 µM 5-HT stimulated (+) mesangial cell lysates were immunoprecipitated (IP) with either ADAM17 antibody or Ig control (Ig C), resolved on 3–8% Tris-acetate gel and probed for the presence of β1 integrin and α5 integrin by Western blotting. (B) ADAM17 blot shows that equal amount of ADAM17 were precipitated from each sample. Arrows point to specific and non-specific (NS) bands. One representative example out of four experiments is shown.</p

Cell free assays show ADAM17 binding to α5β1 integrin and changes in ADAM17 activity.

<p>(A) Recombinant ADAM17 binds purified α5β1 integrin and recombinant β1 integrin in a cell free binding assay. Plates pre-coated with antibodies against α5β1 integrin or β1 integrin were incubated with purified α5β1 integrin or with β1 recombinant integrin, individually. Recombinant ADAM17 was then added at the indicated concentrations and ADAM17 binding was measured using a colorimetric assay at 450 nm as described in Methods. Data are expressed as mean±S.D. *p<0.05, **p<0.01 <i>vs</i> control; data from 4 experiments with 3 parallels/each condition are shown. (B) Purified α5β1 integrin and recombinant β1 integrin decrease ADAM17 enzymatic activity. Recombinant ADAM17 (10 ng/ml) was incubated alone or together with either purified α5β1 integrin (25 ng/ml or 250 ng/ml) or with β1 recombinant integrin (12 ng/ml or 120 ng/ml), in OG buffer in the presence of a quenched fluorogenic ADAM17 substrate. Enzyme activity was expressed as the rate of change of relative fluorescence units (ΔRFUs⁻¹); *p<0.05, **p<0.01 <i>vs</i> control; data from four experiments with eight parallels/each condition are shown.</p

Integrin expression regulates ADAM17 sheddase activity.

<p>(A) Time-dependent release of alkaline phosphatase (AP)-tagged HB-EGF by unstimulated (C) and 5-HT stimulated cells. Cells were transfected with AP-HB-EGF expressing plasmid and 2 days after transfection they were stimulated with 5-HT for the indicated time. AP activity of cell supernatants was determined using Attophos substrate. Data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units; *p<0.05, **p<0.01 <i>vs</i> control at same time point, n = eight experiments, three parallels/each condition. (B) β1 integrin silencing promotes 5-HT-induced AP-HB-EGF shedding. Cells were transfected with AP-HB-EGF expression plasmid together with β1 integrin siRNA (β1-siRNA) or a non-targeting (nt−) siRNA. Successful silencing of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. (C) β1 integrin overexpression inhibits 5-HT induced AP-HB-EGF release in mesangial cells. Cells were transfected with AP-HB-EGF and with β1 integrin expressing plasmid (β1-plasmid) or control DNA (Co-DNA). Successful overexpression of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. Activity data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units (RFU); *p<0.05, **p<0.01 <i>vs</i> unstimulated control nt-siRNA or control DNA-transfected cells; ^# p<0.05 and ^## p<0.01 <i>vs</i> 5-HT stimulated nt-siRNA or DNA-transfected cells; n = five experiments, three parallels/each condition.</p

Co-localization of ADAM17 and α5β1 integrin in rat mesangial cells.

<p>Control (C) and 1 µM 5-HT -stimulated mesangial cell were fixed, permeabilized, and (A) co-immunostained using ADAM17 antibody (green) and β1 integrin antibody (red) as indicated in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033350#s4" target="_blank">Material and Methods</a>”. Arrows indicate co-localization of ADAM17 and α5β1 integrin immunopositive areas (yellow). For the negative controls we omitted the primary antibodies and used PBS followed by secondary antibodies. (B) Parallel samples were incubated with oligonucleotide-labeled PLA probes after incubation with primary antibodies. PLA signals as fluorescence dots were imaged and quantified. As negative control we used either ADAM17 or α5β1 integrin antibody alone followed by the oligonucleotide-labeled PLA probes. Cartoon explains binding of the fluorescence detection reagent only to antibodies in close proximity; **p<0.01. Representative examples out of three experiments are shown.</p

Glutamate-dependent ectodomain shedding of neuregulin-1 type II precursors in rat forebrain neurons

<div><p>The neurotrophic factor neuregulin 1 (NRG1) regulates neuronal development, glial differentiation, and excitatory synapse maturation. NRG1 is synthesized as a membrane-anchored precursor and is then liberated by proteolytic processing or exocytosis. Mature NRG1 then binds to its receptors expressed by neighboring neurons or glial cells. However, the molecular mechanisms that govern this process in the nervous system are not defined in detail. Here we prepared neuron-enriched and glia-enriched cultures from embryonic rat neocortex to investigate the role of neurotransmitters that regulate the liberation/release of NRG1 from the membrane of neurons or glial cells. Using a two-site enzyme immunoassay to detect soluble NRG1, we show that, of various neurotransmitters, glutamate was the most potent inducer of NRG1 release in neuron-enriched cultures. NRG1 release in glia-enriched cultures was relatively limited. Furthermore, among glutamate receptor agonists, N-Methyl-D-Aspartate (NMDA) and kainate (KA), but not AMPA or tACPD, mimicked the effects of glutamate. Similar findings were acquired from analysis of the hippocampus of rats with KA-induced seizures. To evaluate the contribution of members of a disintegrin and metalloproteinase (ADAM) families to NRG1 release, we transfected primary cultures of neurons with cDNA vectors encoding NRG1 types I, II, or III precursors, each tagged with the alkaline phosphatase reporter. Analysis of alkaline phosphatase activity revealed that the NRG1 type II precursor was subjected to tumor necrosis factor-α-converting enzyme (TACE) / a Disintegrin And Metalloproteinase 17 (ADAM17) -dependent ectodomain shedding in a protein kinase C-dependent manner. These results suggest that glutamatergic neurotransmission positively regulates the ectodomain shedding of NRG1 type II precursors and liberates the active NRG1 domain in an activity-dependent manner.</p></div

Manganase²⁺ stimulation decreases association of ADAM17 to β1 integrin and leads to increased ADAM17 activity.

<p>(A) Control and manganese stimulated (1 mM MnCl₂ in PIPES buffer for 2 min) mesangial cell lysates were immunoprecipitated with ADAM17 antibody. Samples were resolved on a 3–8% Tris-acetate gel and probed for β1 integrin and α5 integrin by Western blotting. Immunoblotting for ADAM17 served as loading control. Arrows point to specific and non-specific (NS) bands. One representative blot out of three is shown. (B) Mesangial cells were transfected with AP-HB-EGF construct and stimulated with 1 mM MnCl₂ in PIPES buffer. HB-EGF shedding was expressed as mean±S.D. of fold increase in the rate of change of relative fluorescence units (RFU); **p<0.01 <i>vs</i> control; n = three experiments, 6 parallels/each condition.</p