Article Figures & Data
Figures
- Fig. 1.
AC6 interacts with 4.1G in vivo. (A) Intracellular distribution of GFP-AC6-FL and FLAG-4.1G-FL. HEK293 cells stably expressing GFP-AC6-FL were transiently expressed with FLAG-4.1G-FL. The cells were colabeled with rabbit anti-GFP antibody (green) and mouse anti-FLAG antibody (clone M2, red). The boxed area indicates the enlarged region. One of the representative 10 images is shown. Scale bar, 10 μm. (B) Fluorescence intensity (F.I.) profile plots from (a) to (a’) shown in (A). Arrowheads indicate the position of the cell periphery. (C) Interaction of GFP-AC6-FL and endogenous 4.1G in HEK293 cells. Parental HEK293 cells or HEK293 cells stably expressing GFP-AC6-FL (GFP-AC6-FL/HEK293) were subjected to a coimmunoprecipitation assay. Mouse anti-GFP antibody (mFX73) or mouse anti-DYKDDDDK antibody as a negative control was used for immunoprecipitation. Both immunoprecipitants (IP) and whole lysates were immunoblotted (IB) with rabbit anti-4.1G or rabbit anti-GFP antibody. Representative immunoblots of five independent experiments are shown.
- Fig. 2.
The N-terminus of AC6 directly binds to the FERM domain of 4.1G. (A) Schematic of the domain structure of AC6. AC6 consists of five domains: the N-terminus (N), the first six-transmembrane domain (TM1), the first cytoplasmic loop (C1), the second six-transmembrane domain (TM2), and the second cytoplasmic loop (C2). Intracellular subdomains of AC6 were fused with maltose binding protein (MBP) to produce MBP-AC6-N, MBP-AC6-C1, and MBP-AC6-C2, respectively. (B) Schematic of the domain structure of 4.1G. 4.1G is composed of four major domains: the headpiece (HP), the 4.1-ezrin-moesin-radixin domain (FERM), the spectrin-actin binding domain (SABD), and the carboxy-terminal domain (CTD). Subdomains of 4.1G were FLAG-tagged to produce FLAG-4.1G-HP, FLAG-4.1G-FERM, and FLAG-4.1G-SABD/CTD, respectively. (C) In vitro pull-down assay. MBP or MBP-AC6 proteins were incubated with FLAG-4.1G proteins and further incubated with amylose resin beads. Proteins bound to the amylose resin were separated by SDS-PAGE and detected by immunoblotting (IB) by using mouse anti-DYKDDDDK antibody (upper). MBP or MBP-AC6 proteins purified by amylose resin beads were stained with CBB (lower). (-): MBP (43 kDa); N: MBP-AC6-N (59 kDa); C1: MBP-AC6-C1 (82 kDa); and C2: MBP-AC6-C2 (68 kDa). Arrowheads indicate the apparent molecular weight of FLAG-4.1G-HP (26 kDa), -FERM (36 kDa), and -SABD/CTD (58 kDa). Representative immunoblots of three independent experiments are shown.
- Fig. 3.
AC6-N is associated with the plasma membrane via 4.1G. (A) Intracellular distribution of GFP-AC6-N and FLAG-4.1G-FL. HEK293 cells transiently expressed with GFP-AC6-N and FLAG-4.1G-FL were labeled with rabbit anti-GFP (green) and mouse anti-FLAG (clone M2, red) antibodies. The boxed areas indicate the enlarged regions. One of the representative 16 images is shown. Scale bar, 10 μm. (B) Fluorescence intensity (F.I.) profile plots from (a) to (a’) shown in (A). Arrowheads indicate the position of the cell periphery. (C) Plasma membrane distribution of GPF-AC6-N. HEK293 cells expressing both GPF-AC6-N and DsRed-TMD were also transfected with (lower) or without (upper) 4.1G-siRNA (4.1G-si). The cells fixed with 4% PFA were observed by confocal microscopy without immunolabeling. One representative image from each transfection is shown. The boxed areas indicate the enlarged regions. Scale bar, 10 μm. (D) Quantification of the fluorescence intensity of GFP-AC6-N distributed at DsRed-TMD-labeled plasma membrane (PM) region in (C). n = 16 (control) or 23 (4.1G-si). Data are presented as the means ± S.D. Student’s t test was performed for statistical analysis. (E) Knockdown efficiency of 4.1G-si in HEK293 cells. The cell lysates were immunoblotted (IB) with rabbit anti-4.1G or mouse anti-GAPDH antibody. Representative immunoblots of three independent experiments are shown.
- Fig. 4.
4.1G does not affect the plasma membrane distribution of GPF-AC6-FL. (A) Intracellular distribution of GFP-AC6-FL. HEK293 cells transiently expressed with both GFP-AC6-FL and DsRed-TMD were further transfected with (lower) or without (upper) of 4.1G-siRNA (4.1G-si). The cells fixed with 4% PFA were observed by confocal microscopy without immunolabeling. One representative image from each transfection is shown. The boxed areas indicate the enlarged regions. Scale bar, 10 μm. (B) Quantification of the fluorescence intensity of GFP-AC6-FL distributed at DsRed-TMD-labeled plasma membrane (PM) region in (A). n = 22 (control) or 30 (4.1G-si). Data are presented as the means ± S.D. Student’s t test was performed for statistical analysis.
- Fig. 5.
The three consecutive arginine sequence in the N-terminus of AC6 is essential for binding to the FERM domain of 4.1G. (A) Amino acid sequences of AC6-Ns. Triple-arginine and triple-alanine sequences in AC6-N and AC6-N-3A are underlined. Amino acid residues are numbered above the sequences. (B) In vitro pull-down assay of MBP-AC6-Ns and FLAG-4.1G-FERM. FLAG-4.1G-FERM that bound to MBP-AC6-Ns (212 nM) were visualized by SDS-PAGE and immunoblotting (IB) using mouse anti-DYKDDDDK antibody (upper). FLAG-4.1G-FERM proteins contained in the lysates were immunoblotted with anti-DYKDDDDK antibody (middle). MBP and MBP-AC6-Ns pulled down by amylose resin beads were separated by SDS-PAGE and visualized by CBB staining (lower). MBP (-) (43 kDa), MBP-AC6-N (59 kDa), MBP-AC6-N-3A (59 kDa), and FLAG-4.1G-FERM (36 kDa). Representative immunoblots of three independent experiments are shown. (C) Plasma membrane distribution of GPF-AC6-N or -3A. HEK293 cells were expressed with GPF-AC6-N or -3A in the presence of DsRed-TMD. The cells fixed with 4% PFA were observed by confocal microscopy without immunolabeling. One representative image from each transfection is shown. The boxed areas indicate the enlarged regions. Scale bar, 10 μm. (D) Quantification of the fluorescence intensities of GFP-AC6-N or -3A distributed at DsRed-TMD-labeled plasma membrane (PM) region in (C). n = 26 (WT) or 26 (3A). Data are presented as the means ± S.D. Student’s t test was performed for statistical analysis.
- Fig. 6.
The association of AC6-N with the plasma membrane is critical for AC6 activity. (A and B) Effect of (A) overexpression of GFP-AC6-Ns or (B) knockdown of 4.1G in forskolin-induced cAMP production. HEK293 cells were exogenously expressed with (A) GFP-AC6-N or -3A or (B) transfected with 4.1G-shRNA (4.1G-sh). The cells were treated with 10 μM forskolin (FK) in the presence of 100 μM IBMX for 10 minutes at 37°C. (C and D) Effect of (C) overexpression of GFP-AC6-Ns or (D) knockdown of 4.1G in PTH-(1-34)-induced cAMP production. HEK293 cells stably expressing HA-PTHR were transiently expressed with (C) GFP-AC6-N or -3A or (D) transfected with 4.1G-sh. The cells were treated with 10 nM PTH-(1-34) in the presence of 100 μM IBMX for 14 minutes at 37°C. The intracellular cAMP concentration was analyzed by AlphaScreen cAMP assay as described in Materials and Methods. (A and C) Data are presented as the means ± S.E.M., n = 3; one-way analysis of variance followed by Tukey’s tests were performed within the drug-treated cells. (B and D) Data are presented as the means ± S.E.M., n = 3, Student’s t tests were performed between the drug-treated cells.
- Fig. 7.
The expression of endogenous AC6 protein is not altered by AC6-N-expression or 4.1G-knockdown. (A) Expression of endogenous AC6 protein in HEK293 cells transiently expressing GFP, GFP-AC6-N, or GFP-AC6-N-3A was detected by Western blotting. The samples were immunoblotted (IB) with rabbit anti-AC6 antibody, rabbit anti-GFP antibody, or mouse anti-GAPDH antibody. (B) Quantification of (A). Data are presented as the means ± S.D. from three independent experiments. (C) Expression of endogenous AC6 protein in HEK293 cells transfected with 4.1G siRNA cocktail. The samples were immunoblotted (IB) with rabbit anti-AC6 antibody, rabbit anti-4.1G antibody, or mouse anti-GAPDH antibody. (D) Quantification of (C). Data are presented as the means ± S.D. from two independent experiments.
- Fig. 8.
Theoretical models. (A) In normal condition, the three consecutive arginine sequence (RRR) in the N-terminus of AC6 is associated with the plasma membrane via 4.1G, and AC6 activity is restricted. (B) When the N-terminus of AC6 is dissociated from the plasma membrane (e.g., lower expression level of 4.1G or overexpression of AC6-N), AC6 activity is high.
Additional Files
Data Supplement
- Supplemental Figures -
Supplementary Figure 1 - Identification of FLAG-4.1G proteins in E. coli, and exogenously expressed GFP-AC6 proteins and FLAG-4.1G-FL protein in HEK293 cells.
Supplementary Figure 2 - Amino acid sequences of the intracellular subdomains of human AC6.
Supplementary Figure 3 - The binding affinity of MBP-AC6-N and FLAG-4.1G-FERM proteins.
Supplementary Figure 4 - Intracellular localization of GFP-AC6-Ns and FLAG-4.1G-FL.
Supplementary Figure 5 - The three consecutive positive charges in the N-terminus of AC6 are essential for binding to the FERM domain of 4.1G.
Supplementary Figure 6 - Binding of the N-termini of MBP-ACs with FLAG-4.1G-FERM.
Supplementary Figure 7 - A dose-dependent increase in cAMP production by PTH-(1-34).
- Supplemental Figures -
In this issue
Activity of AC6 Is Suppressed by Direct Binding of 4.1G
