The long coiled-coil protein NECC2 is associated to caveolae and modulates NGF/TrkA signaling in PC12 cells [corrected]

Abstract

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.

Figure 1. In silico analysis of rat NECC2 sequence.

A. Schematic representation of the structural and functional motifs predicted in rat NECC2 amino acid sequence. B. Schematic representation of the genomic structure of rat Necc2 coding for the Necc2 isoform containing the HR domain and a newly identified transcript lacking this domain (Necc2α). Arrows indicate the location of the paired primers used to amplify both Necc2 transcripts. C. Standard PCR amplification in PC12 cells shows two PCR products with the expected sizes for the two rat Necc2 transcripts (left panel; primers a and b). Nested PCR amplification of Necc2 transcript (central panel; primer c) or the Necc2α transcript (rightmost panel; primer d) using specific internal reverse primers. Non-DNA samples (C-) are shown as controls for exogenous contamination.

Figure 2. Intracellular distribution of endogenous NECC2 in PC12 cells.

A. Representative confocal images of a PC12 cell immunolabeled with the anti-NECC2 antiserum. NECC2 distributes throughout the cytoplasm and in close apposition to the cell membrane. Specificity of the signal was tested by preadsorption of the anti-NECC2 antibody with excess of antigen. B. PC12 cells were double-stained with antibodies against NECC2 (red) and caveolin-1 or actin (green) (top and bottom panels respectively). Significant overlap between markers at the cell periphery is shown in the binary mask at the rightmost panels. C. Prior to double immunostaining with anti-NECC2 and anti-caveolin-1, PC12 cells were treated with 5 µmol/L of LatB for 30 min at 37°C. Scale bars, 10 µm.

Figure 3. Analysis of the intracellular localization of cMyc-tagged NECC2 variants.

A. Schematic representation of NECC2 constructs and its truncated variants lacking the hydrophobic region (NECC2ΔHR) or containing the first 4 N-terminal coiled-coil regions (NECC2Δ372). B. Confocal images of PC12 cells co-expressing cMyc-NECC2 or its truncated forms and CFP-caveolin-1 (CFP-cav1). Full-length cMyc-NECC2 accumulates in the perinuclear area. NECC2ΔHR and NECC2Δ372 immunosignals accumulate intracellularly and in the proximity of the cell surface. NECC2 immunofluorescence signal strongly overlaps with CFP-caveolin-1 as shown in the binary mask (rightmost panel). Scale bars, 10 µm.

Figure 4. NECC2 associates to caveolae both as an integral membrane protein and a peripheral membrane protein.

A. Immunoblot analysis of whole cell lysates from PC12 cells with the anti-NECC2 antibody. As control, anti-NECC2 antibody was pre-incubated with an excess of antigen. B. Cytosolic (S) and crude membrane (P) fractions from PC12 cells were obtained by subcellular fractionation as described in Methods and subsequent analyzed by immunoblotting. As shown by anti-NECC2 antibody immunolabeling, NECC2 distributes in the cytosol and, to a lesser extent, it also associates with membrane fractions. In contrast, exogenous full-length cMyc-NECC2 distributed to both fractions, with a higher content in the membrane fraction. The distribution of EEA1, TrkA, GM130, actin and caveolin-1 was also analyzed. C. Caveolae-enriched membranes from PC12 cells were isolated by using a detergent-free method based in a discontinuous sucrose gradient (5-35-45%). Distribution of endogenous NECC2 and cMyc-tagged NECC2 variants were assayed by immunoblot. Neither NECC2 nor NECC2ΔHR co-migrated with caveolin-1 and TrkA to the buoyant fraction (fraction 2).

Figure 5. NECC2 expression and distribution are regulated by NGF.

A. Representative confocal images of HA-TrkA transfected PC12 cells under basal conditions or treated with NGF at the indicated time points. After treatment, cells were subjected to double-immunofluorescence using anti-NECC2 and anti-HA antibodies. The colocalization channel was isolated using Imaris 6.4 (Bitplane) and shown alone in the images on the far bottom. Mander’s coefficient A (between NECC2 and TkA) was calculated to quantify the degree of colocalization and represented as the mean ±SEM of at least 5 cells per experimental group. a, P < 0.05 vs. control; b, P < 0.05 vs. 5 min; c, P < 0.05 vs. 30 min (unpaired, 2-tailed t test). B. Representative micrographs of PC12 cells immunolabeled with anti-NECC2 during long-term (1-4 days) stimulation with NGF. NECC2 staining localizes beneath the plasma membrane as well as in puncta/vesicular-like structures in growing neurites and tips. C. Double-immunostaining of NGF-differentiated PC12 cells with anti-NECC2 and anti-caveolin-1 sera. Scale bars, 10 µm. D. Protein extracts from NGF-stimulated PC12 cells during differentiation (1-4 days) were analyzed by immunoblotting using the anti-NECC2 antibody. Quantitative data were represented as ratio of NECC2 vs. Ponceau. The data represent the mean (± SEM) of three independent experiments. a, P < 0.05 vs. corresponding control; b, P < 0.05 vs. 1- or 2-days treated cells (one-way ANOVA followed by Newman–Keuls test).

Figure 6. Reduction of endogenous NECC2 by RNA interference impairs NGF-mediated TrkA signaling.

A. PC12 cells were stably transfected with pEGFP-shRNA or Necc2-shRNA plasmid (siRNA) and whole protein extracts were analyzed for immunoblotting using anti-NECC2 antibody and anti-actin antibody. B and C. PC12 cells overexpressing pEGFP-RNAi (MOCK) or Necc2-shRNA (R1) were grown to 90% confluence and exposed for 4 h to serum-low differentiation media before NGF stimulation for the indicated time points. Cell lysates were subjected to immunoblot with Akt and phospho-Akt (pAkt) antibodies (B) or with ERK and phospho-ERK (pERK) antibodies (C). Quantitative data were represented as ratio of pAkt vs. Akt or pERK vs. ERK, respectively. The data represent the means (± SEM) of three independent experiments. P < 0.05 vs. corresponding control (unpaired, 2-tailed t test).

Figure 7. Overexpression of NECC2 inhibits NGF-mediated TrkA signaling pathway.

A. Representative confocal images of PC12 cells transfected with cMyc-Necc2ΔHR and double-stained with anti-cMyc and anti-TrkA antibodies. Immunofluorescent signals significantly overlap at the cell periphery and intracellularly as shown in the binary mask (right panels). Scale bars, 10 µm. B, C, D and E. PC12 cells transiently transfected with full-length cMyc-Necc2, cMyc-Necc2ΔHR, or the empty vector (mock) were grown to 90% confluence and exposed for 4 h to serum-low differentiation media before NGF stimulation for the indicated time points. Whole cell protein extracts were then subjected to immunoblot with Akt and phospho-Akt (pAkt) antibodies (B and C) or with ERK and phosphor-ERK (pERK) antibodies (D and E). Quantitative data were represented as ratio of pAkt vs. Akt or pERK vs. ERK, respectively. The data represent the means (± SEM) of three independent experiments. P < 0.05 vs. corresponding control (unpaired, 2-tailed t test).