Neogenin interacts with hemojuvelin through its two membrane-proximal fibronectin type III domains.
Hemojuvelin is a recently identified iron-regulatory protein that plays an important role in affecting the expression of hepcidin, a key iron regulatory hormone. Although the underlying mechanism of this process is not clear, several hemojuvelin-binding proteins, including the cell surface receptor neogenin and bone morphogenetic protein (BMP) cytokines, have been identified. The ectodomain of neogenin is composed of four immunoglobulin-like (Ig) domains followed by six fibronectin type III-like (FNIII) domains. Here we report expression of soluble versions of hemojuvelin and neogenin for biochemical characterization of their interaction and the interaction of HJV with BMP-2. Hemojuvelin normally undergoes an autocatalytic cleavage, and as in vivo, recombinant hemojuvelin exists as a mixture of cleaved and uncleaved forms. Neogenin binds to cleaved and noncleaved hemojuvelin, as verified by its binding to an uncleaved mutant hemojuvelin. We localized the hemojuvelin binding site on neogenin to the membrane-proximal fifth and sixth FNIII domains and the juxtamembrane linker and showed that a fragment containing only this region binds 2-3 orders of magnitude more tightly than the entire neogenin ectodomain. Binding to the most membrane-proximal region of neogenin may play a role in regulating the levels of soluble and membrane-bound forms of hemojuvelin, which in turn would influence the amount of free BMP-2 available for binding to its receptors and triggering transcription of the hepcidin gene. Our finding that BMP-2 and neogenin bind simultaneously to hemojuvelin raises the possibility that neogenin is part of a multiprotein complex at the hepatocyte membrane involving BMP, its receptors, and hemojuvelin.
Yang
Fan
F
Graduate Option in Chemistry, California Institute of Technology, Pasadena, California 91125, USA.
West
Anthony P
AP
Allendorph
George P
GP
Choe
Senyon
S
Bjorkman
Pamela J
PJ
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058851:GPI-Linked Proteins; D000071020:Hemochromatosis Protein; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D016212:Transforming Growth Factor beta / Q000378:metabolism
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005353:Fibronectins; D058851:GPI-Linked Proteins; C481006:HJV protein, human; D000071020:Hemochromatosis Protein; D008565:Membrane Proteins; D010446:Peptide Fragments; D016212:Transforming Growth Factor beta; C091279:neogenin