Glycosylation regulates pannexin intermixing and cellular localization - PubMed (original) (raw)

Glycosylation regulates pannexin intermixing and cellular localization

Silvia Penuela et al. Mol Biol Cell. 2009 Oct.

Abstract

The pannexin family of mammalian proteins, composed of Panx1, Panx2, and Panx3, has been postulated to be a new class of single-membrane channels with functional similarities to connexin gap junction proteins. In this study, immunolabeling and coimmunoprecipitation assays revealed that Panx1 can interact with Panx2 and to a lesser extent, with Panx3 in a glycosylation-dependent manner. Panx2 strongly interacts with the core and high-mannose species of Panx1 but not with Panx3. Biotinylation and dye uptake assays indicated that all three pannexins, as well as the N-glycosylation-defective mutants of Panx1 and Panx3, can traffic to the cell surface and form functional single-membrane channels. Interestingly, Panx2, which is also a glycoprotein and seems to only be glycosylated to a high-mannose form, is more abundant in intracellular compartments, except when coexpressed with Panx1, when its cell surface distribution increases by twofold. Functional assays indicated that the combination of Panx1 and Panx2 results in compromised channel function, whereas coexpressing Panx1 and Panx3 does not affect the incidence of dye uptake in 293T cells. Collectively, these results reveal that the functional state and cellular distribution of mouse pannexins are regulated by their glycosylation status and interactions among pannexin family members.

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Figures

Figure 1.

Figure 1.

Transmembrane topology of pannexin family members and peptides used for antibody generation. Toppred algorithms predicted a similar membrane tetra spanning topology for all three mouse pannexin proteins. Numbers indicate the amino acid position of transmembrane domains. _N_-Glycosylation consensus sites are depicted in red. Peptides from different domains of the three pannexins (green residues) were used to generate rabbit polyclonal antibodies (CT, carboxy terminus, EL, extracellular loop; and IL, intracellular loop, followed by the first amino acid position of the peptide).

Figure 2.

Figure 2.

Characterization of pannexin-specific antibodies. Panx1, Panx2, and Panx3 were localized in transfected 293T cells by using various anti-pannexin affinity-purified antibodies denoted by CT, carboxy terminus; EL, extracellular loop; and IL, intracellular loop, followed by the first amino acid position of the peptide (A). As a control, preimmune Panx2 sera revealed no significant labeling (inset). Bars, 20 μm. Immunoblotting of cell lysates from pannexin-expressing 293T cells (B) revealed similar banding profiles for the two Panx1 antibodies (∼41–47 kDa), as well as the three Panx3 antibodies (doublet ∼43 kDa). The anti-Panx2 rabbit antibody (Zymed Laboratories/Invitrogen) identified an ∼80-kDa band (sometimes resolved as a doublet), and this antibody was chosen along with CT-395 and CT-379 for further immunoblotting assays. Peptide preadsorption assays revealed that all antibodies were specific for pannexins. β-Actin was used as loading control.

Figure 3.

Figure 3.

Pannexin family members are diversely glycosylated. Cell lysates from 293T cells expressing Panx1, Panx2, and Panx3 were treated with _N_-glycosidase F (A). Gel banding patterns revealed band shifts for each of the pannexins at both the 15- and 60-min time points, suggesting that each pannexin has a single glycan chain. _N_-Glycosidase F (A) and Endo H (B) enzymatic digestion revealed that Panx2 only exists as core (Gly0) and high mannose (Gly1) species, whereas Panx1 and Panx3 also exist as complex glycoprotein species (Gly2). Panx1 also exists in an unglycosylated state (Gly0). GAPDH was used as loading control.

Figure 4.

Figure 4.

Glycosylated and unglycosylated pannexin species reach the cell surface. Cell surface biotinylation of 293T cells expressing Panx1 (A), Panx3 (B), Panx1N254Q (C), Panx3N71Q (D), Panx2 (E), or cells expressing both Panx1 and Panx2 (F) revealed that all pannexin species and their glycosylation-defective mutants have at least some capacity to traffic to the cell surface. Coexpression of Panx1 and Panx2 caused over a twofold increase in the cell surface biotinylation of Panx2 (G) (p < 0.05, n =3 independent transfections). Lysates with (+) or without (−) biotin were precipitated with NeutrAvidin beads and run in parallel with control lysates. GAPDH was used as a control for biotin internalization.

Figure 5.

Figure 5.

Coexpression of Panx1, but not Panx3, increased the cell surface localization of Panx2. Ectopic Panx2 was localized to intracellular compartments in NRK and 293T cells with some, but limited, evidence for its localization at the cell surface (A). Peptide preadsorption competed the antibody binding for Panx2, whereas some nonspecific nuclear labeling remained (A). In NRK cells, the coexpression of Panx1 or Panx1N254Q with Panx2 induced an increase in the cell surface population of Panx2. The subcellular localization of Panx2 did not change when coexpressed with Panx3 (B). Bars, 20 μm.

Figure 6.

Figure 6.

Colocalization of Panx1 and Panx3. Double immunofluorescent labeling revealed that endogenous Panx1 and Panx3 partially colocalized in MC3T3-E1 cells (A). Coexpression of Panx1 or Panx1N254Q with Panx3 in NRK cells revealed colocalization of these pannexins and an increase in the cell surface distribution of the _N_-glycosylation mutant Panx1N254Q (B), compared with the more intracellular profile observed for Panx1N254Q when expressed alone (B; inset). Bars, 20 μm.

Figure 7.

Figure 7.

Interactions among pannexin family members are regulated by glycosylation. An interaction between the Gly0 and Gly1 species of Panx1 with Panx2 was revealed when lysates from 293T cells expressing both pannexins were immunoprecipitated (IP) with Panx1 (A) or Panx2 (B) antibodies and immunoblotted for Panx1 and Panx2. (An occasional band seen below Gly0 seems to be a proteolytic product). Coimmunoprecipitation assays revealed an interaction between Panx1 and Panx3 that was more pronounced when the Panx1 glycosylation-deficient mutant was coexpressed with Panx3 (C). Panx2 and Panx3 did not interact when coexpressed in 293T cells (C). The reciprocal IP for Panx1 and Panx3 was performed with equivalent results (D). Twenty micrograms of the corresponding protein lysates were run in parallel to assess the expression levels of each pannexin.

Figure 8.

Figure 8.

Cell surface localization of Panx3-GFP was evident when coexpressed with Panx1 in NRK cells. When Panx3-GFP was coexpressed with Panx1N254Q it exhibited a similar intracellular localization profile (A; arrows) as when it was expressed alone (A; inset). Bars, 20 μm (A). Antibodies to Panx3 coimmunoprecipitated the Gly0 and Gly1 species of Panx1 and Panx3-GFP (B) in 293T cells. The same blot was probed sequentially with Panx3 after Panx1, by using different secondary antibodies (see Materials and Methods). Twenty micrograms of the corresponding protein lysates were run in parallel to assess pannexin expression levels.

Figure 9.

Figure 9.

Panx2, but not Panx3, intermixing with Panx1 affects dye uptake. 293T cells expressing Panx1, Panx2, or Panx3 were active in sulforhodamine dye uptake, whereas cells expressing GFP exhibited negligible dye uptake (A). The expression of Panx2 together with Panx1 reduced the incidence of dye uptake compared with Panx1 or Panx2 alone (B), whereas mixing Panx1 and Panx3 had no functional effect (C). Parallel experiments using dextran rhodamine dye revealed no uptake (data not shown). Experiments are composites of four independent transfections and assessed by one-way ANOVA followed by a Tukey test, p < 0.01. Bars indicate SEM, and letters depict statistical significance among groups. Bars, 20 μm.

Figure 10.

Figure 10.

Glycosylation-defective mutants of Panx1 and Panx3 can form functional single-membrane channels. Uptake of sulforhodamine B dye by 293T cells expressing pannexins, _N_-glycosylation–defective mutants, or combinations of pannexins was assessed (A). Dextran-rhodamine dye uptake was used as a negative control to assess damaged cells (data not shown). Significant dye uptake was observed in cells expressing Panx1, Panx3, and their corresponding _N_-glycosylation–deficient mutants but not in any cells coexpressing pannexins or pannexin mutants together with Panx3-GFP (B). n =3 (one-way ANOVA, p < 0.001). Bars indicate SEM, and letters depict statistical significance among groups (C). Bars, 20 μm.

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