Characterization and Expression of a Novel Alternatively Spliced Human Angiopoietin-2

Abstract Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2443 (Ang2443), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96–148) is absent in Ang2443 because of alternative splicing of the gene. Like Ang2, recombinant Ang2443expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2443 binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2443 on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression ofAng2 443 mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2 443 mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2 443 mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2443 is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.