Anthrax Toxin Receptor 1/Tumor Endothelium Marker 8 Mediates Cell Spreading by Coupling Extracellular Ligands to the Actin Cytoskeleton

Abstract

Tumor endothelial marker 8 (TEM8) is induced in tumorassociated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by ␤1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of ␤1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.

The Cytoplasmic Domain of Anthrax Toxin Receptor 1 Affects Binding of the Protective Antigen

“…With potential implications for angiogenesis, a previous study demonstrated that the association of ANTXR1-sv1 with the cytoskeleton mediates cell spreading (49). The cytoskeleton linkage is likely to be functionally important, since spreading was inhibited by pharmacological disruption of the cytoskeleton.…”

“…Both receptors are thought to be involved in cell matrix interactions since the extracellular domain of ANTXR1 was shown to bind collagen type I and to immunoprecipitate with the C5 domain of collagen ␣3, while that of ANTXR2 was shown to bind collagen type IV and laminin (5,15,33,49). ANTXR1 functions as an adhesion molecule, as it was demonstrated to mediate cell spreading via an actin-dependent mechanism (49).…”

Targeting Tumor Endothelial Marker 8 in the Tumor Vasculature of Colorectal Carcinomas in Mice

“…In vitro studies indicate that TEM8 has a role in various endothelial cell functions (4)(5)(6) and the extracellular domain of TEM8 can bind to extracellular matrix proteins (4, 7) as well as the protective antigen of anthrax toxin (8). Expression profiles for TEM8 include both research and clinical samples that provide strong evidence for the increased expression of TEM8 in tumor endothelial cells (3,7,9).…”

The Contribution of Endothelial Marker Proteins in the Determination of Vascular Angiogenic Potential, in Normal Physiological Conditions and in Neoplasia

“…In mice and rat models, iNOS was shown to play a significant role in pathological mechanism of several diseases linked to inflammation [44]. This isoform takes part in the development of arterial hypertension, diabetes mellitus or myocardial infarction [12,23,30,44,53,59]. The isoform of eNOS is present in endothelial cells lining the lumen of both the arterial or venous blood vessels, as well as in capillaries and lymphatic vessels.…”