Hemojuvelin (HJV for humans, and Hjv for mouse), 2 also called repulsive guidance molecule c (RGMc), is a glycosylphosphatidylinositol-linked membrane protein. It belongs to the highly conserved RGM family that also contains RGMa and RGMb. Although RGMa and RGMb are predominantly expressed in the developing and adult central nervous system and are essential for neural development (1), HJV is expressed mainly in the liver, skeletal muscle, and heart and is essential for iron homeostasis (2). Mutations in the HJV gene in humans markedly reduce hepcidin expression in the liver and result in juvenile hemochromatosis (JH), the most severe form of iron overload disorder (2). Hepcidin is a 25-amino acid peptide hormone that inhibits the iron efflux from enterocytes and macrophages into circulation by binding to and targeting ferroportin, the only known iron exporter, for degradation (3). It is synthesized in hepatocytes as an 84-amino acid prepropeptide that contains an N-terminal 24-amino acid signal sequence, a 35-amino acid proregion, and a C-terminal 25-amino acid bioactive peptide. After post-translational processing, the bioactive C-terminal 25-amino acid peptide is secreted into the circulation as a mature form to regulate iron homeostasis (4). Consistently, low hepatic hepcidin expression and a marked iron overload were also observed in Hjv knock-out (Hjv Ϫ/Ϫ ) mice (5, 6). Further studies in mice with tissue-specific Hjv knockdown demonstrate that only the hepatic Hjv is indispensable for hepcidin expression and iron homeostasis (7,8).HJV, in the liver, acts as a co-receptor for BMP6 to stimulate hepcidin expression through the BMP signaling pathway (9 -11). BMP signaling is initiated upon the binding of BMP ligands to type-I and type-II BMP receptors on the cell surface. Upon BMP binding, the type-II receptors phosphorylate the type-I receptors, leading to the phosphorylation of SMAD1/5/8 in the cytoplasm. The phosphorylated SMADs form heteromeric complexes with SMAD4 and then translocate to the nucleus where they induce the transcription of target genes. HJV most likely uses two type-I BMP receptors, ALK2 and ALK3, to induce hepcidin expression, because liver-specific deletion of either Alk3 or (to a lesser extent) Alk2 causes iron overload in mice (12).Structural studies of the HJV ectodomain demonstrate that it can simultaneously bind BMP2 and neogenin with nanomolar affinities through its N-terminal portion (amino acids 1-145) and C-terminal portion (amino acids 146 -401), respectively, and identify the key residues in these molecules that are responsible for these interactions (13,14). Neogenin is a ubiquitously expressed type-I transmembrane protein that contains four immunoglobulin (Ig)-like domains and six fibronectin III (FNIII) domains in its large extracellular region. HJV specifically binds to the FNIII 5-6 subdomains (15). However, the precise role of neogenin in HJV induction of hepcidin expression is still unclear, largely because of lack of an appropriate animal model. In a hepatoma cell line ...