The Carboxyl-Terminal 12 Residues of CMG2 are Crucial for its Function as an Anthrax Toxin Receptor

Capillary morphogenesis gene 2 protein (CMG2) is one of two anthrax toxin receptors; the other is tumor endothelial marker 8 (TEM8). Anthrax toxin protective antigen (PA) binds the vWA domain present in the amino-terminal extracellular portions of both receptors. But the functions of the membrane-proximal extracellular segments and cytoplasmic domains of the receptors are unclear. Two isoforms of CMG2 function as the PA receptors, one 488 residues long, the other 489 residues. Here we report that the unique carboxyl-terminal segments of both forms are crucial for the receptors’ role in intoxication of cells by anthrax toxin. We did this analysis in a functionally receptor-negative cell line. In a two-tier approach, we first derived a receptor that lacks either divergent peptide. Although this receptor bound PA normally, it did not support intoxication of cells, which showed the divergent segments are required for function. We then assessed the significance of each of the unique residues in receptor function by alanine-scanning mutagenesis. These results revealed that all of the divergent residues in both receptors are important. Cells expressing the alanine mutant receptors that did not support intoxication bound PA much less than the normal one. Further, these mutant receptors did not allow formation of SDS-resistant PA heptamer to any appreciable degree. Remarkably, deletion of the entire cytoplasmic domain of CMG2 left the receptor functional. We therefore conclude that the divergent carboxyl-terminal segments of CMG2 are crucial in the context of the native, full-length receptor.