Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Distribution of Nedd4-2 and TRPV6 in rat intestinal tract. A, RT-PCR results showing expression of TRPV6, Nedd4, and Nedd4-2 in rat intestine at the mRNA level. All PCR experiments were performed for 25 cycles. B, anti-Nedd4-2 antiserum specifically recognized Nedd4-2 but not Nedd4 exogenously expressed in X. laevis oocytes by Western blot analysis. A band with the same size as Nedd4-2 and two bands of lower molecular weight were recognized by this antiserum in rat colon lysate. C, immunofluorescent staining of oocytes injected with cRNAs for TRPV6, Nedd4-2, Nedd4, or water (as controls) with antisera against TRPV6 and Nedd4-2, respectively. TRPV6 and Nedd4-2 antisera recognized TRPV6 and Nedd4-2 expressed in the plasma membrane and cytoplasma of oocytes without specific staining in water or Nedd4 cRNA-injected oocytes. Scale bar, 50 μm. D, immunofluorescent staining of rat intestine tissues with TRPV6 and Nedd4-2 antisera in adjacent sections. TRPV6 antiserum labeled strongly in apical membrane in all segments of rat intestine tested. Nedd4-2 antiserum exhibited weak intracellular staining in duodenum and jejunum and strong staining in ileum, cecum, and colon. Preimmune serum (for control of TRPV6 staining) and secondary antibody alone (for control of Nedd4-2 staining) exhibited no specific staining in colon, respectively. Magnification was ×250 and ×800 for regular and enlarged sections, respectively.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Molecular Weight, Staining, Injection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 and Nedd4 decreased Ca2+ uptake, Na+ current, and protein abundance of TRPV6 and TRPV5 in X. laevis oocytes. A, Ca2+ uptake values of control oocytes or oocytes expressing Nedd4-2, Nedd4, TRPV6, or TRPV6 together with Nedd4-2 or Nedd4. Data are presented as means ± S.E. (error bars) of six independent experiments. *, p < 0.01 versus TRPV6 alone group. Representative Western blot analyses for proteins extracted from different groups using antisera against TRPV6 and β-actin (as loading control) are shown (bottom). B, current-voltage (I-V) plots of Na+-evoked currents in oocytes expressing TRPV6 alone or together with Nedd4-2 or Nedd4. Oocytes expressing Nedd4-2 or Nedd4 exhibited negligible currents similar to water-injected oocytes (not shown). Data are expressed as means ± S.E. of 14 oocytes in each group from four independent experiments. C, Ca2+ uptake in control oocytes (water-injected) or oocytes injected with TRPV5 cRNA alone or together with Nedd4-2 and Nedd4, respectively. Data are presented as means ± S.E. of six independent experiments. *, p < 0.01 versus TRPV5 alone group. TRPV5 protein level was decreased by Nedd4-2 or Nedd4 (bottom). D, current-voltage plots of Na+-evoked currents in oocytes expressing TRPV5 alone or together with Nedd4-2 or Nedd4. Data are expressed as means ± S.E. of 18 oocytes in each group from three independent experiments. All experiments were performed at 2 days after injection of cRNAs.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Expressing, Western Blot, Injection

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 dose-dependently decreased TRPV6 protein abundance (A) and TRPV6-mediated Ca2+ uptake (B). Groups of X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA with 0, 3.1, 6.3, 12.5, or 25 ng of Nedd4-2 cRNA, and Ca2+ uptake experiments were performed 2 days later. Data are presented as a percentage of Ca2+ uptake of the group injected with TRPV6 cRNA alone. Representative Western blot analyses show that TRPV6 protein level decreased as Nedd4-2 protein level increased, and β-actin (loading control) was at a similar level in all of the groups (A). Error bars, S.E.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 decreased TRPV6 stability. X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA alone or together with 12.5 ng of Nedd4-2 cRNA. Two days after injection, the stability of TRPV6 protein was examined by treating the oocytes with 100 μg/ml cycloheximide, an inhibitor of protein synthesis, for 0, 3, 6, or 9 h. A, representative Western blot analyses show the level of TRPV6 proteins at each time point in the presence and absence of Nedd4-2. The level of β-actin determined by Western blotting was used as a control for equal loading. B, assessment of the linear range of TRPV6 band intensity. Oocyte lysates containing TRPV6 protein were loaded at varying levels in different lanes and were run together with samples in A, and the linear range of TRPV6 protein level and band intensity were determined. Shown are a representative Western blot for TRPV6 (top) and the derived relationship of band intensity and TRPV6 protein level (bottom). TRPV6 band intensity is expressed as a percentage of that at 100% input. C, rate of TRPV6 protein degradation in the presence or absence of Nedd4-2. Protein level is expressed as a percentage of band intensity at time 0. Data from seven blots are shown as means ± S.E. (error bars). *, p < 0.05 versus the TRPV6 alone group.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Injection, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: The ubiquitin E3 ligase activity of Nedd4-2 was essential to its inhibitory effect on TRPV6. A, ubiquitination of TRPV6 was enhanced by Nedd4-2. Oocytes were injected with cRNA for TRPV6, Nedd4-2, and HA-tagged ubiquitin (HA-Ub) alone or in combination. Twenty-four hours after injection, oocytes were treated with 25 μm MG132 for 5 h before harvesting. TRPV6 was immunoprecipitated (IP) with a TRPV6 antiserum and immunoblotted (IB) with anti-HA to detect ubiquitinated TRPV6. A Western blot with TRPV6 antiserum is shown at the bottom. B, ligase activity of Nedd4-2 was necessary for the down-regulation of TRPV6. TRPV6 was expressed alone or together with WT Nedd4-2 or Nedd4-2 C922S mutant in oocytes. Data are presented as means ± S.E. (error bars) of three independent experiments. *, p < 0.01 versus TRPV6 alone.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Activity Assay, Injection, Immunoprecipitation, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 did not increase the endocytosis TRPV6 but facilitated the proteasomal degradation of TRPV6. A, surface TRPV6 abundance was decreased by Nedd4-2. Oocytes expressing TRPV6 alone or with Nedd4-2 and control oocytes were probed with an antiserum that recognizes the first extracellular loop of TRPV6. The oocytes were then fixed for 1 h at 4 °C and then incubated with an HRP-coupled secondary antibody. Chemiluminescence signals of individual oocytes developed with HRP substrate were detected using a luminometer. The values are expressed as percentages of the chemiluminescence signal level of the TRPV6 alone group. Data from 40–57 oocytes in each group from two batches of oocytes are presented as mean ± S.E. (error bars). *, p < 0.01 versus TRPV6 alone group. B, Nedd4-2 did not alter the endocytosis of surface TRPV6. Oocytes were treated with the same maneuver as in A except that they were incubated for 0, 15, and 30 min for endocytosis at room temperature before incubation with secondary antibody. The values are expressed as percentages of the chemiluminescence signal levels at time 0 of the TRPV6 group or the TRPV6 plus Nedd4-2 group. Background values of water-injected control groups were subtracted. Data from 18–61 oocytes in each group from two batches of oocytes are presented as mean ± S.E. C, effects of proteasome inhibitor MG132 and lysosome inhibitor chloroquine on the regulation of TRPV6 by Nedd4-2. Oocytes expressing TRPV6 alone or with Nedd4-2 were incubated with 25 μm MG132 or 100 μm chloroquine for 5 h before Ca2+ uptake and Western blot experiments were performed. Data are presented as means ± S.E. of three independent experiments. *, p < 0.01 versus the TRPV6 plus Nedd4-2 group; NS, not statistically significant (p > 0.01).

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Expressing, Incubation, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Assessment of Nedd4-2 domains on TRPV6. A and B, WT Nedd4-2 and Nedd4-2 constructs lacking C2 (ΔC2, without amino acids 1–166), WW1-2 (ΔWW1-2, without amino acids 194–398), WW3-4 (ΔWW3-4, without amino acids 478–561), or HECT (ΔHECT, without amino acids 618–955) were co-injected with TRPV6, and the Ca2+ uptake and TRPV6 protein level were determined 2 days later. A, all cRNAs were injected at 12.5 ng/oocyte; B, WT and ΔWW1-2 were injected at 1.6 ng/oocyte because ΔWW1-2 construct expression made oocytes unhealthy. *, p < 0.01 versus WT plus TRPV6 group. β-Actin was detected by Western blot analysis as a loading control. Only the ΔHECT construct was unable to inhibit TRPV6. C, HECT domain was necessary for the association between Nedd4-2 and TRPV6. Top, immunoblotting (IB) with the anti-HA antibody showed the expression of HA tagged wild-type and ΔHECT Nedd4-2 constructs. Bottom, HA-tagged Nedd4-2 proteins co-immunoprecipitated (IP) with TRPV6 proteins were detected by anti-HA antibody. HECT domain deletion abolished the association between Nedd4-2 and TRPV6. Error bars, S.E.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Construct, Injection, Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: TRPV6 directly interacted with Nedd4-2. A, autoradiography of GST fusion proteins with the Nedd4-2 C2 region (amino acids 1–166, including the C2 domain (amino acids 21–124)), WW1-2 region (amino acids 167–438), WW3-4 region (amino acids 439–590), or HECT region (amino acids 591–955, including the HECT domain 618–955) probed with 35S-labeled TRPV6 N-terminal (top) or C-terminal region (middle). Purified GST fusion proteins stained with Coomassie Blue are shown at the bottom. Note that the bands with lower molecular weights were present in lanes loaded with WW1-2 and HECT fusion proteins (top bands), respectively. The nature of these bands was unknown, but they may represent degraded products. B, autoradiography of individual GST fusion Nedd4-2 WW domains probed with 35S-labeled TRPV6 N-terminal (top) or C-terminal (middle) region. Purified GST fusion WW domains stained with Coomassie Blue are shown at the bottom. C, autoradiography of in vitro synthesized 35S-labeled TRPV6 N-terminal region (1–327 amino acids) and C-terminal region (578–725 amino acids) that were used as probes for A and B. D, autoradiography of GST fusion C-terminal region of TRPV6 (amino acids 597–725; the fusion protein was close to the 43 kDa marker) (top) probed with 35S-labeled Nedd4-2 full-length, ΔC2, ΔWW1-2, ΔWW3-4, and ΔHECT constructs (bottom) described in the legend to Fig. 7. The amounts of different probes were adjusted to the same level for each experiment in A, B, and D based on the band intensity.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Autoradiography, Labeling, Purification, Staining, In Vitro, Synthesized, Marker, Construct

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Asp204 and Asp376 of Nedd4-2 were critical to the interaction between TRPV6 N-terminal region and WW1 and WW2 domains, respectively. A, sequence alignment of four WW domains of Nedd4-2. *, amino acid residues in WW1 domain that are conserved in WW2 but not in WW3 and WW4 domains. B–D, Far Western analyses of key amino acid residues of WW1 in its interaction with TRPV6 terminal regions. GST fusion WW1, WW2, and WW4 peptides, including the WT and constructs with the indicated mutations, were probed with the 35S-labeled TRPV6 N-terminal region (amino acids 1–327; top) and C-terminal region (amino acids 578–725; middle), respectively. Purified GST fusion WW peptides stained with Coomassie Blue are shown at the bottom. B, D204H mutation in WW1 significantly attenuated the interaction between the TRPV6 N-terminal and WW1 domain. C, D204A mutation in WW1 also attenuated the interaction between the TRPV6 N-terminal region and the WW1 domain. The H539D mutation in the WW4 domain increased the interaction between the TRPV6 N-terminal region and WW4 domain. His539 in WW4 is the counterpart of Asp204 in WW1. D, D376H mutation in the WW2 domain also attenuated the interaction between the TRPV6 N-terminal region and WW2 domain. Asp376 in WW2 is the counterpart of Asp204 in WW1.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Sequencing, Western Blot, Construct, Labeling, Purification, Staining, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: D204H mutation in Nedd4-2 enhanced its ability to inhibit TRPV6. Top, TRPV6-mediated Ca2+ uptake in oocytes expressing TRPV6 alone or together with WT Nedd4-2, D204H, D376H, or D204H/D376H mutant. Oocytes injected with water (Water) were used as control. Data are presented as means ± S.E. (error bars) of three independent experiments. *, p < 0.01 versus TRPV6 alone group; #, p < 0.01 versus TRPV6 plus wild-type Nedd4-2 group. Representative Western blot analyses using antibodies against TRPV6, Nedd4-2, and β-actin are shown at the bottom.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Mutagenesis, Expressing, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 with D204H/D376H double mutations increased TRPV6 ubiquitination without compromising its association with TRPV6. A, association of TRPV6 with HA-Nedd4-2 and HA-Nedd4-2 D204H/D376H in oocytes. Top, immunoblotting (IB) with the anti-HA antibody indicates the equal level of wild-type HA-Nedd4-2 and HA-Nedd4-2 containing the D204H/D376H mutation. HA-Nedd4-2 proteins (wild type or mutant) co-immunoprecipitated with TRPV6 proteins were detected by anti-HA antibody. Bottom, intensity of the co-immunoprecipitated Nedd4-2 band was significantly decreased by the D204H/D376H mutation. Data from four experiments were normalized against band intensity of wild-type or mutant Nedd4-2. *, p < 0.01. AU, arbitrary unit. B, Nedd4-2 D204H/D376H mutant increased the ubiquitination of TRPV6 compared with the wild-type Nedd4-2. Details of the experiments were similar to those described in the legend to Fig. 5A. Error bars, S.E.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation