The intracellular domain of CMG2 and its role in anthrax toxin receptor activity

Date
2012
Authors
Jimenez, Rodolfo, Jr.
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Abstract

Anthrax toxin is known to bind to two receptors: tumor endothelial marker 8 (ANTRX1/TEM8) and capillary morphogenesis gene 2 protein (ANTRX2/CMG2). Both are similar in that they are both type 1 single pass integral membrane proteins and have similar topology: von Williebrand factor A (vWa) domain, proximal membrane domain, and a cytoplasmic domain. Over all there is about a 40% sequence similarity between the two, but the vWa domains share a 60% sequence identity. Both receptors' vWa domains contain a metal ion dependent adhesion site (MIDAS), which is crucial for toxin binding. It is without doubt that manipulation of the extracellular portion of the receptors can lead to a malfunction in anthrax toxicity. The opposite has long been thought for the cytoplasimc domain. But recent studies have shown that this is not the case. The following work focuses on the characterization of the role of the cytoplasmic domain in anthrax toxicity. One approach we took to investigate the receptors' cytoplasmic domains role was through a yeast 2 hybrid system, where I used the cyotplasmic domains of both TEM8 and CMG2 as the bait proteins for the assay. With the assay we identified that eukaryotic initiation factor 4 gamma 2 (eIF4gamma2) as an interacting partner with CMG2, and when knocked down does cause slight resistance to toxicity. The other approach we took was a mutational analysis one. With the discovery of CMG2-480 as a non-functioning receptor we first decided to concentrate on the role that the terminal residues of both CMG2-488 and CMG2-489 had in toxicity. When either of these residues was removed the receptor was killed. Additionally, a point mutational analysis showed that each of the residues, for both CMG2-488 and CMG2-489, was necessary for toxicity to take place. With CMG2-480 being nonfunctional we added the last 12 and 13 residues of CMG2-488 and CMG2-489, respectively, in an attempt to restore function. Unfortunately none of these additions lead to restoration of the receptors functionality. These findings help in reversing the notion that the cytoplasmic domain of the anthrax toxin receptors is a non-factor in athrax toxicity.

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Integrative Biology