Abstract 816: Identification of a Receptor Necessary for Nogo-B Stimulated Chemotaxis of Endothelial Cells
Nogo isoforms are part of a superfamily of proteins called reticulons. Nogo-A is a myelin associated inhibitor of axonal sprouting, Nogo-B is expressed in many cells in culture and the primary Nogo isoform expressed in blood vessels. Nogo-A and -B isoforms have a common amino terminus for the first 184 amino acids and contain a conserved reticulon homology domain (RHD). A 66 amino acid loop domain termed Nogo-66 in the RHD can interact with a GPI linked cell surface receptor, NgR, and this receptor mediates, in part, the inhibitory function of Nogo-A on neuronal outgrowth. In contrast with Nogo-A, the amino terminus of Nogo-B promotes the adhesion and chemotaxis of endothelial cells and negatively regulates platelet derived growth factor induced chemotaxis in smooth muscle cells. The ability of soluble AmNogo-B, but not Nogo-66, to induce chemotaxis and the specific binding of alkaline phosphatase (AP) fusion protein expressing the amino terminus of Nogo-B (AP-AmNogo-B) to endothelial cells and Nogo-A/B knock-out mouse smooth muscle cells suggests that AmNogo-B may interact with a unique receptor. The receptor for the amino terminus of Nogo-B that mediates vascular function is unknown. Here we identify a novel Nogo-B receptor (NgBR) specific for AmNogo-B binding from human heart cDNA library using expression cloning approach. Structure-function analysis of the amino terminus of Nogo-A and -B defines the unique domain of Nogo-B necessary for binding to NgBR. We developed a polyclonal antibody directed at the putative ectodomain of NgBR. Northern blot and Western blot analysis show that NgBR protein is highly expressed in heart, liver, kidney and pancreas. Furthermore, NgBR co-localizes with the ligand Nogo-B during angiogenesis in vivo and mediates chemotaxis in a heterologous expression system and chemotaxis and three-dimensional tube formation of endothelial cells. Mechanistically, siRNA mediated knockdown of endogenous NgBR blocks endothelial cell morphogenesis and chemotaxis stimulated by AmNogo-B. Thus, identification of this new receptor may lead to the discovery of agonists or antagonists of this pathway to regulate vascular remodeling and angiogenesis.