Recent studies have discovered two cell surface-expressed anthrax toxin receptors (ATRs), originally named TEM8 and CMG-2. The latter receptor, CMG-2, was first discovered in my laboratory in a screen for genes regulating the process of capillary tube morphogenesis in three-dimensional collagen matrices. Intriguingly, both receptors were identified as upregulated genes during the process of new blood vessel formation (i.e. angiogenesis). In addition, recent work in collaboration with my laboratory has revealed that CMG-2 is mutated in two familial human diseases, juvenile hyaline fibromatosis and infantile systemic hyalinosis. CMG-2 shows characteristics of a type I transmembrane receptor with a von Willebrand factor (vWF) type A domain in its extracellular domain and potential cytoskeletal interacting domains in its cytoplasmic tail. The vWF domain of CMG-2 was shown by us to bind the extracellular matrix ligands laminin and collagen type IV and recently was shown to bind protective antigen (PA83), the known ATR binding component of the tripartite anthrax toxin. We hypothesize that CMG-2 associates with co-receptors such as integrins to mediate its normal function in extracellular matrix homeostasis as well as its function as an ATR. Clearly, molecular studies involving structure-function analyses of the novel alternatively spliced receptor, CMG-2, are required to elucidate the mechanisms underlying its ability to serve as an ATR. Also, it is critical to identify CMG-2 binding proteins, such as cell surface co-receptors and cytosolic binding partners, which regulate its function. Such studies are critical toward the development of novel therapeutic agents that interfere with the ability of anthrax toxin to exert its toxic effects. We present preliminary data showing the novel involvement of cw integrins in endothelial cell adhesion and spreading on protective antigen-coated wells (mimicking the presentation of extracellular matrix ligands). These data strongly suggest that integrin co-receptors may play a role in the binding, processing and internalization of protective antigen.
The specific aims of this application are:
Specific Aim #1. To perform structure-function analyses with CMG-2 splice variants, mutants, and domains (i.e. extracellular versus cytoplasmic) to determine which domains are involved in protective antigen binding, proteolytic processing and internalization.
Specific Aim #2. To identify CMG-2 co-receptors, such as integrins, which are involved in its ability to regulate protective antigen binding, proteolytic processing and internalization. ? ? ?
