The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels

Abstract

TRPV4 is a widely expressed member of the transient receptor potential (TRP) family that facilitates Ca(2+) entry into nonexcitable cells. TRPV4 is activated by several stimuli, but it is largely unknown how the activity of this channel is terminated. Here, we show that ubiquitination represents an important mechanism to control the presence of TRPV4 at the plasma membrane. Ubiquitination of TRPV4 is dramatically increased by the HECT (homologous to E6-AP carboxyl terminus)-family ubiquitin ligase AIP4 without inducing degradation of this channel. Instead, AIP4 promotes the endocytosis of TRPV4 and decreases its amount at the plasma membrane. Consequently, the basal activity of TRPV4 is reduced despite an overall increase in TRPV4 levels. This mode of regulation is not limited to TRPV4. TRPC4, another member of the TRP channel family, is also strongly ubiquitinated in the presence of AIP4, leading to the increased intracellular localization of TRPC4 and the reduction of its basal activity. However, ubiquitination of several other TRP channels is not affected by AIP4, demonstrating that AIP4-mediated regulation is a unique property of select TRP channels.

Figure 1

The AIP4 ubiquitin ligase increases multiubiquitination of TRPV4. (A) HEK 293T cells were transfected with plasmids encoding TRPV4-His, FLAG-ubiquitin, and three different ubiquitin ligases of the HECT family, as indicated. The TRPV4-His protein purified on Ni-NTA beads was analyzed by Western blotting for ubiquitin conjugation using α-FLAG antibodies. (B) The in vivo ubiquitination assay was performed as above, using constructs, as indicated, to compare the effects of AIP4 WT and AIP4 DN on the ubiquitination of TRPV4. (C) The cells were transfected as in (B), except that the plasmid encoding FLAG-ubiquitin was omitted. Ubiquitinated TRPV4 was detected with α-ubiquitin P4D1 antibodies. (D) The TRPV4-His protein purified from transfected HEK 293T cells was analyzed by Western blotting using monoclonal antibodies P4D1, which recognize all ubiquitinated proteins, and FK1, which recognize only polyubiquitinated proteins. As control, His-tagged c-Jun was purified from cells overexpressing AIP4 WT or MEKK1 CA. A nonspecific band is marked with an asterisk. (E) HEK 293T cells were transfected with the indicated plasmids and used for an in vivo ubiquitination assay. The ubiquitinated TRPV4 was detected with α-FLAG antibodies. The bars represent the level of TRPV4 ubiquitination in MEKK1 CA-expressing cells (lanes 5–7). For quantifications, the amount of ubiquitinated TRPV4 was normalized to the amount of the non-ubiquitinated form; the signal in lane 8 was treated as background. The depletion of endogenous AIP4 by RNAi (lane 7 in comparison to lane 5) is shown in the bottom panel.

Figure 2

The N-terminus, but not the C-terminus, is required for the ubiquitination of TRPV4. (A) The sequence alignment between extended PY motifs of ENaC channel subunits: α (Swissprot accession number P37088), β (P51168) and γ (P51170) and a similar region found in the N-terminus of murine TRPV4 (Q9EPK8). (B) HEK 293T cells were transfected with plasmids encoding V5/His-tagged WT TRPV4 and TRPV4 mutants lacking either the PY-like motif (ΔPY) or the whole C-terminus (ΔC), along with plasmids encoding myc-AIP4 and FLAG-ubiquitin, as indicated. The cell lysates were incubated with Ni-NTA beads and the purified proteins were resolved on SDS–PAGE. Following Western blotting, V5/His-tagged TRPV4 proteins were detected with α-V5 antibodies and their ubiquitin-conjugated fractions with α-FLAG antibodies. (C) HEK 293T cells were transfected with plasmids encoding FLAG/His₉-tagged N-terminus of TRPV4 (F9-N-TRPV4) or similarly tagged C-terminus of TRPV4 (F9-TRPV4-C), along with the plasmid encoding myc-AIP4, as indicated. Cell lysates were treated as in (B). The purified N- and C-termini of TRPV4 were detected with α-FLAG antibodies and the conjugated endogenous ubiquitin with P4D1 α-ubiquitin antibodies.

Figure 3

AIP4 does not accelerate the degradation of TRPV4. (A) HEK 293T cells were transfected with plasmids encoding TRPV4-V5/His, GFP, and myc-AIP4 WT or dominant-negative (DN), as indicated. Shown are steady-state levels of TRPV4 in comparison to cotransfected GFP and to actin. (B) The cells transfected as above, with and without myc-AIP4 WT, were treated with 30 μg/ml cycloheximide for 0–10 h. The cell lysates were resolved on SDS–PAGE and the protein turnover was analyzed by Western blotting with α-V5, α-GFP, α-myc, and α-actin antibodies. The amount of cell lysates loaded on gels was adjusted for the initial difference in the TRPV4-V5/His protein levels between myc-AIP4-expressing and control cells. (C) The graph depicts the fraction of the remaining TRPV4-V5/His protein at 2–10 h after cycloheximide treatment for each condition analyzed in (B). Note that, for clarity, the error bars (s.e.m., n=3) point to opposite directions for each curve. (D) HEK 293T cells were transfected with plasmids encoding TRPV4-His and FLAG-ubiquitin, either with or without a plasmid encoding myc-AIP4 WT, as indicated. Cells were treated with 12 μM MG132 or 200 μM chloroquine for 200 min. The lysates were incubated with Ni-NTA beads and the purified proteins analyzed by Western blotting. TRPV4 was detected with α-TRPV4 antibodies and the ubiquitin conjugated to TRPV4 with α-FLAG antibodies.

Figure 4

AIP4 decreases the amount of TRPV4 at the plasma membrane. (A) In the representative experiment shown, HEK 293T cells were transfected with plasmids encoding C-terminally V5-tagged TRPV4 (TRPV4-V5 C-term), TRPV4 tagged with V5 in the first extracellular loop (TRPV4-V5 loop), GFP and myc-tagged AIP4 (0.1 or 0.5 μg), as indicated. The transfected cells were incubated with α-V5 antibodies at 4°C, washed, and fixed. The amount of V5 epitope associated with the plasma membrane was measured by ELISA (AP activity). The total amounts of proteins were determined by Western blotting. Results obtained for cells not expressing any V5-tagged protein (lane 1) were treated as background. Results obtained for cells expressing TRPV4-V5 loop in the absence of myc-AIP4 (lane 3) were normalized to 100%. Ratios of AP activity to the total amount of V5 epitope (AP/total V5), and to the total amount of GFP (AP/GFP) are shown in the table. (B) Statistical analysis of six independent experiments, similar to the one presented in (A). The ratios of surface TRPV4 (measured by ELISA) to total TRPV4, as well as coexpressed GFP (analyzed by Western blotting), in the presence of myc-AIP4 (AIP4) relative to control (Con), are depicted. Statistical analysis was performed with one sample t-test; the calculated P-values are indicated. (C) HEK 293T cells expressing TRPV4-V5 loop, with and without myc-AIP4, were incubated with α-V5 antibodies at 4°C, washed, returned to the incubator for 0, 10, 20, or 45 min, and subsequently fixed. The amount of V5 epitope present at the plasma membrane was measured by ELISA (left panel). For both control and myc-AIP4-expressing cells, the AP activity at time 0 was set to 100%. A statistical analysis (means and s.e.m., n=3) of the amount of TRPV4 at the cell surface at the 20-min time point relative to time 0 is shown on the right. The P-value was calculated with a paired t-test. (D) Confocal immunofluorescence analysis of HEK 293T cells expressing TRPV4-V5 loop and myc-AIP4. The cells were incubated with α-V5 antibodies at 4°C, washed, returned to the incubator for 20 min, and subsequently fixed. The bound α-V5 antibody was detected with a Cy3-conjugated donkey α-mouse IgG (a; red). The fixed cells were also stained with α-AIP4 antibodies followed by Alexa488-conjugated donkey α-goat IgG (b; green). The overlay of both pictures is shown in panel c. The colocalization of TRPV4-V5 loop and myc-AIP4 in vesicular structures is best seen at the top section of the cells, as shown in the insets (slightly enlarged relative to main pictures). The arrows in the overlay picture point to several vesicles, where TRPV4-V5 loop and mycAIP4 colocalize. (E) Depletion of endogenous AIP4 in HEK 293T cells by coexpression of the α-AIP4 shRNA construct (AIP4 RNAi) increases the surface localization of TRPV4. A representative experiment is shown. Surface expression of TRPV4-V5 loop was measured by ELISA; total levels of proteins were analyzed by Western blotting. (F) Measurement of the ratio of surface TRPV4 to its total levels in cells depleted of AIP4 (AIP4 RNAi) relative to control cells (Con). Depicted are means and s.e.m. from four experiments. The P-value was calculated with one sample t-test. (G) Depletion of AIP4 results in decreased levels of internalized TRPV4, as measured at 20-min chase time point. Depicted are means and s.e.m. from three experiments performed and analyzed as in (C). A plasmid devoid of the α-AIP4 shRNA sequence was transfected as control.

Figure 5

AIP4 ubiquitin ligase is involved in the ubiquitination of both TRPV4 and TRPC4. HEK 293T cells were transfected with plasmids encoding V5/His-tagged TRPC4, TRPV1, or TRPV4 along with plasmids encoding FLAG-ubiquitin and either catalytically active (WT) or inactive (DN) versions of myc-AIP4. The TRP channels were purified from cell lysates on Ni-NTA beads and resolved on SDS–PAGE. Following Western blotting, V5-tagged TRP channels were detected using α-V5 antibodies and the ubiquitin conjugates with α-FLAG antibodies.

Figure 6

Basal TRPC4 activity and surface expression are reduced upon coexpression with AIP4. (A–C) Mn²⁺ influx through TRPC4-YFP was detected with the Mn²⁺-quench technique in fura 2-loaded HEK 293 cells expressing either TRPC4-YFP alone (A; 1.5 μg of cDNA plasmid and 0.5 μg of pcDNA3) or TRPC4-YFP together with AIP4 (B; 0.5 μg AIP4-encoding cDNA plasmid instead of pcDNA3). (A, B) Representative data sets depict means (black symbols)±s.e.m. of n=6 independent transfection experiments containing data from a total number of 267–282 cells. The Ca²⁺-independent fluorescence intensity was measured by exciting the probe at 358 nm, which corresponds to the isosbestic point of fura 2 in our system. Background intensities and contaminating YFP signals were subtracted before the analysis. (C) Statistical analysis of the data shown in (A, B). The Mn²⁺-induced loss of 358-nm-excited fura 2 fluorescence (ΔF₃₅₈) was calculated over a 1-min interval before (Basal) and after (CCh) the addition of 100 μM carbachol and expressed as percentage of the initial intensity. The indicated P-values were calculated by unpaired Student's t-test (n=6). (D) Confocal live cell microscopy shows the typical distribution pattern of TRPC4-YFP without (left panel) and with coexpressed AIP4 (right panel).