Binding of filamentous actin to anthrax toxin receptor 1 decreases its association with protective antigen

Abstract

ANTXR1 is a type I membrane protein that binds the protective antigen (PA) component of anthrax toxin. The cytosolic domain of ANTXR1 has a novel actin-binding region that influences the interaction of the ectodomain with PA. Here, we have investigated features of the cytosolic domain of ANTXR1 that reduce the association of the receptor with PA. We mutated a stretch of conserved acidic amino acids adjacent to the actin-binding region and found that the mutation increased the affinity for monomeric actin in vitro. ANTXR1 bearing this mutation exhibited increased association with the cytoskeleton and bound less PA compared to the wild-type receptor, confirming the inverse correlation between the two interactions. To determine whether binding of actin is sufficient to regulate the ectodomain, we replaced the actin-binding region of ANTXR1 with that from the yeast protein abp140 and with the WH2 domain of WAVE2. Although both of these domains bound monomeric actin in vitro, only the sequence from abp140 reduced binding of PA to a hybrid receptor. The actin binding regions of ANTXR1 and abp140, but not the WH2 domain, colocalized with actin stress fibers, which suggested that filamentous actin regulates ANTXR1. Consistent with this notion, disruption of actin filaments using latrunculin A increased the amount of PA bound to cells. This work provides evidence that cytoskeletal dynamics regulate ANTXR1 function.

Figure 1

Model of how the cytosolic tail of ANTXR1 affects binding of PA. Binding of actin to the tail of ANTXR1-sv1 alters the packing of the transmembrane domains of a receptor dimer causing the VWA/I domains to close. ANTXR1-sv2 lacks an actin-binding region, which allows the transmembrane domains to cross at a different position. This alters how the extracellular domains interact, causing the VWA/I domains to open.

Figure 2

Mutation of an acidic region within the cytoplasmic domain of ANTXR1-sv1 decreases PA-binding and increases actin-binding. (A) Cells expressing ANTXR1-sv1-HA, ANTXR1-sv1-Y383C-HA, or the corresponding acidic region mutants (ARM) were exposed to furin-resistant PA for 2 h. Cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis using α-PA antibody. Surface expression of the HA-tagged receptors was determined by exposing the cells to a membrane impermeable biotinylation reagent, precipitating biotinylated proteins with streptavidin-agarose, and performing a Western blot assay using anti-HA antibody. Blots are representative of three independent experiments. (B) F-actin association assays of ANTXR1-sv1-HA and ANTXR1-sv1-Y383C-HA receptors expressed in HeLa cells. Cells were lysed and centrifuged for 30 min, and the ratio of the amount of receptors that sedimented with the cytoskeleton to the amount that remained in the supernatant fraction was quantified. Error bars indicate the standard error of the mean of three independent experiments. (C) A biotinylated peptide corresponding to amino acids 360–420 of ANTXR1-sv1 (sv1_360–420) and a similar peptide containing the acidic region mutation were bound to streptavidin agarose beads and incubated with purified monomeric β-actin. Proteins were eluted from beads and subjected to SDS-PAGE and Western blot analysis for β-actin (top) or biotin (bottom). Blots are representative of three independent experiments.

Figure 3

Cytosolic tails influence binding of PA to ANTXR1 hybrid receptors. (A) Comparison of sequences of hybrid receptor tails with the region of ANTXR1-sv1 encompassing the actin-binding region and adjacent acidic region. The acidic region mutation (ARM) is underlined. The cytosolic domain of ANTXR1 consists of amino acids 348–564 (not shown). The actin-binding region of ANTXR1-sv1 was replaced with the WH2 domain of WAVE2 or the actin-binding region of abp140 (ABP). (B) Cells expressing indicated receptors were incubated at 4°C with a furin-resistant form of PA and the amount of bound PA was assessed by Western blotting (top). Actin was used as a loading control (bottom). Surface expression of the HA-tagged receptors was determined by exposing the cells to a membrane impermeable biotinylation reagent, precipitating biotinylated proteins with streptavidin-agarose, and performing a Western blot assay using anti-HA antibody (middle). Blots are representative of three independent experiments. (C) Association of indicated receptors with the F-actin cytoskeleton was determined by lysing cells and sedimenting the cytoskeleton. The amount of each receptor found in the cytoskeleton pellet and the supernatant was determined by Western blot assays. The ratio of the amount of each receptor localized in pellet fractions to the amount that remained in supernatant was determined. Error bars indicate the standard error of the mean of three independent experiments.

Figure 4

Actin-binding regions of ANTXR1-sv1 and abp140 colocalize with actin stress fibers. Confocal micrographs of HeLa cells transfected with EGFP fusion proteins of the isolated actin-binding regions of ANTXR1-sv1, WH2, or abp140 with or without the acidic region mutation (ARM) (green). Cells were stained with AlexaFluor 555 phalloidin (red) to visualize actin, and DAPI (blue) to visualize nuclei. Right panels show merged images with colocalization displayed in yellow. Images are representative of three independent experiments.

Figure 5

The actin-binding regions of ANTXR1-sv1 and abp140 bind F-actin. (A) GST fusion proteins containing the actin-binding region of ANTXR1-sv1, the WH2 domain of WAVE2, or the actin-binding region of abp140 were incubated with cell lysates and the mixtures were centrifuged to pellet the F-actin cytoskeleton. The pellet and supernatant fractions were subjected to SDS-PAGE and Western blot analysis with an α-GST antibody. (B) GST fusion proteins were bound to glutathione sepharose and incubated with purified monomeric β-actin. Bound proteins were eluted and subjected to SDS-PAGE and Western blot analysis to detect monomeric actin. Blots are representative of three independent experiments.

Figure 6

Binding of actin filaments to ANTXR1-sv1 diminishes PA-binding. CHOR1.1 cells stably expressing ANTXR1-sv1 or ANTXR1-sv1-Y383C or ANTXR1-sv1_ARM were treated with DMSO vehicle or with the actin filament disrupting agent latrunculin A for 2 h. Furin-resistant PA was incubated with cells for 2 h and the amount of PA that bound cells was assessed by Western blotting. Receptor surface expression was assessed using a biotinylation assay. Blots are representative of three independent experiments.