P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

Abstract

Background and purpose: The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7.

Experimental approach: We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging.

Key results: Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation.

Conclusions and implications: We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.

Figure 1

Co-expression of P2X receptors in tsA 201 cells. (A) Cells were co-transfected with DNA encoding P2X2-GFP and P2X4-HA. Cells were fixed, permeabilized and incubated with a mouse monoclonal anti-HA antibody, followed by Cy3-conjugated goat anti-mouse secondary antibody. Cells were imaged by confocal laser scanning microscopy. The left-hand panel shows the GFP signal, the centre panel the Cy3 signal and the right-hand panel the merged signals. Cells expressing GFP and Cy3 completely coincide, indicating that all of the transfected cells express both P2X2 and P2X4. (B) Cells were co-transfected with DNA encoding P2X4-HA and His₁₀-P2X7. Cells were fixed, permeabilized and incubated with mouse monoclonal anti-HA and rabbit polyclonal anti-P2X7 antibodies, followed by either FITC-conjugated goat anti-mouse or Cy3-conjugated goat anti-rabbit secondary antibodies. The left-hand panel shows the FITC signal, the centre panel the Cy3 signal, and the right-hand panel the merged signals. Cells expressing FITC and Cy3 extensively coincide, indicating that the vast majority of transfected cells express both P2X4 and P2X7 receptors. Scale bar, 25 µm. FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HA, haemagglutinin.

Figure 2

In situ proximity ligation assays for P2X subunit interactions. Cells were co-transfected with DNA encoding either P2X2 plus P2X4-HA (A) or P2X4-HA plus P2X7 (B). Transfections also included pEGFP, to identify transfected cells. Cells were permeabilized and incubated with primary antibodies (rabbit polyclonal anti-P2X₂, mouse monoclonal anti-HA (for P2X4) or rabbit polyclonal anti-P2X7) followed by anti-mouse (+) and anti-rabbit (−) proximity ligation secondary antibodies. The proximity ligation assay was then carried out, and treated cells were imaged by confocal laser scanning microscopy. The left-hand panels show the proximity signals, and the right-hand panels the EGFP signals. (C, D) Control experiments in which cells expressing P2X4-HA only (plus EGFP) were probed with anti-P2X2 and anti-HA antibodies (C) or anti-HA and anti-P2X7 antibodies (D). Transfected cells are indicated by the EGFP signal (right-hand panels). There are no corresponding proximity ligation signals (left-hand panels). Scale bar, 20 µm. EGFP, enhanced green fluorescent protein; HA, haemagglutinin.

Figure 3

Co-immunoprecipitation of P2X subunits. (A) P2X2-GFP and P2X4-HA were co-expressed by transient transfection of tsA 201 cells. Crude membrane fractions prepared from the cells were solubilized in 1% CHAPS. P2X2-GFP was immunoprecipitated using a rabbit polyclonal anti-P2X2 antibody, and P2X4-HA was immunoprecipitated using a rabbit polyclonal anti-P2X4 antibody. A rabbit polyclonal anti-His₆ antibody was used as a negative control. Immunoprecipitates were analysed by SDS-PAGE followed by immunoblotting with either mouse monoclonal anti-GFP (P2X2-GFP) or anti-HA (P2X4-HA) antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). (B) P2X4-HA and His₁₀-P2X7 were co-expressed by transient transfection of tsA 201 cells. P2X4-HA was immunoprecipitated from a detergent-solubilized membrane fraction using a rabbit polyclonal anti-P2X4 antibody, and His₁₀-P2X7 was immunoprecipitated using a rabbit polyclonal anti-P2X7 antibody. A rabbit polyclonal anti-P2X2 antibody was used as a negative control. Immunoprecipitates were analysed by SDS-PAGE followed by immunoblotting with either a mouse monoclonal anti-HA antibody (P2X4-HA) or a rabbit polyclonal anti-P2X7 antibody. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate; GFP, green fluorescent protein; HA, haemagglutinin; PAGE, polyacrylamide gel electrophoresis.

Figure 4

Cross-linking analysis of P2X subunits. (A) Detergent extracts of membrane fractions from cells transfected with either P2X2-GFP alone or P2X2-GFP+P2X4-HA were incubated with the chemical cross-linker DSS (0.2 mM, 30 min at room temperature), or with the DMSO vehicle alone, and then subjected to SDS-PAGE followed by immunoblotting with a rabbit polyclonal anti-P2X2 antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). (B) Detergent extracts of membrane fractions from cells transfected with either P2X4-HA alone, P2X2-GFP+P2X4-HA or P2X4-HA+His₁₀-P2X7 were incubated with DSS (4 mM) and then subjected to SDS-PAGE followed by immunoblotting with a mouse monoclonal anti-HA (P2X4-HA) antibody. (C) Detergent extracts of membrane fractions from cells transfected with either His₁₀-P2X7 alone or P2X4-HA+His₁₀-P2X7 were incubated with DSS (0.2 mM) and then subjected to SDS-PAGE followed by immunoblotting with a rabbit polyclonal anti-P2X7 antibody. DSS, disuccinimidyl suberate; GFP, green fluorescent protein; HA, haemagglutinin; PAGE, polyacrylamide gel electrophoresis.

Figure 5

Atomic force microscopy (AFM) imaging of P2X receptors of various subunit compositions. (A) His₆-P2X2 and His₁₀-P2X7 were isolated from detergent extracts of cells through binding of their His tags to Ni²⁺-agarose beads, followed by elution with imidazole. P2X4-HA was isolated through binding of its HA tag to anti-HA immunoaffinity beads, followed by elution with HA peptide. P2X subunits were either expressed alone or in the combinations His₆-P2X2+P2X4-HA (with pull-down of His₆-P2X2) or P2X4-HA+His₁₀-P2X7 (with pull-down of His₁₀-P2X7). Eluted samples were subjected to SDS-PAGE followed by immunoblotting with either a rabbit polyclonal anti-P2X2 antibody, a mouse monoclonal anti-HA (P2X4-HA) antibody or a rabbit anti-P2X7 antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). (B) Low-magnification AFM images of a sample isolated from cells expressing P2X4-HA. The arrowheads indicate single receptors; arrows indicate double receptors. A colour-height scale is shown at the right. Scale bars, 100 nm. (C) Gallery of zoomed images of double receptors present in samples isolated from cells expressing all five subunit combinations. A colour-height scale is shown at the right. Scale bar, 50 nm. HA, haemagglutinin; PAGE, polyacrylamide gel electrophoresis.