Repulsive guidance molecule (RGM) is usually a membrane-bound protein that was

Repulsive guidance molecule (RGM) is usually a membrane-bound protein that was originally identified as an axon guidance molecule in the chick retinotectal system. we propose that this peptide is usually a promising lead in finding reagents capable of inhibiting RGMa signaling. Introduction Repulsive guidance molecule (RGM) is usually a cell membrane-associated glycosylphosphatidylinositol (GPI)-anchored glycoprotein that was originally identified AWD 131-138 as an axon repellent in the chick retinotectal system [1] [2]. This protein contains an N-terminal signal AWD 131-138 peptide an Arg-Gly-Asp (RGD) site a partial von Willebrand type-D (vWD) domain name and a hydrophobic domain name of unknown function [3]. It also has a putative proteolytic site and the cleaved carboxy-terminal mature protein is usually a 33-kDa Mmp2 fragment [4] [5]. In vertebrates at least 3 homologues of RGM RGMa RGMb (DRAGON) and RGMc (hemojuvelin HJV HFE2) have been identified [6] [7]. RGMa is the most closely related to chick RGM with 80% homology. Although overexpression or downregulation of RGM in chick tectum results in pathfinding and mapping errors RGMa mutant mice show normal retinal axon projection patterns in the superior colliculus [6]. In contrast another study showed that RGMa functions as an axon guidance molecule in the developing mouse hippocampus [8]. In addition 50 of the homozygous mutant mice show defects in cephalic neural tube closure [6]. Moreover both and studies have shown that RGMa inhibits axon growth [9]. RGMa expression is usually induced in adult rats with a spinal cord injury (SCI) around the lesion site while loss of function by local treatment with a neutralizing antibody to RGMa significantly promotes axon regeneration after SCI. Neogenin the receptor for all of the RGM homologues is usually a single membrane-embedded protein originally isolated from chick cerebellum AWD 131-138 as a homolog of deleted in colorectal cancer a receptor for AWD 131-138 AWD 131-138 the axon guidance cue netrin-1 [10]. Neogenin contains 4 immunoglobulin-(IgG) like and 6 fibronectin type III-(FN III)-like domains in its extracellular region. Both netrin-1 and RGM bind to the FN III-like domain name of neogenin but the binding affinity of RGM for neogenin is much higher than that of netrin-1 for neogenin [10] [11]. Unfortunately information around the binding site on RGM for neogenin has been lacking. In this study we identified that the region of RGMa made up of aa 259-295 critically regulates the conversation between RGMa and neogenin. Results from enzyme-linked immunosorbent assays (ELISAs) revealed that this RGMa aa 284-293 directly bind to neogenin’s extracellular domain name. Furthermore this peptide attenuated RGMa-induced growth cone collapse in mouse cortical neurons. Results C-terminus of RGMa binds to neogenin First we sought to determine whether the binding sites for neogenin are located in the amino- (N-) or carboxyl- (C-) terminus of RGMa. Deletion constructs were made consisting of Myc-tagged human full-length RGMa (FL-RGMa-Myc) N-terminal region of RGMa (N-RGMa-Myc) and C-terminal region of RGMa without a GPI anchor domain name (C-RGMa-Myc) (Fig. 1A). HEK293T cells were transiently co-transfected with a control or VSV-G-tagged full-length neogenin constructs together with each of the RGMa constructs and the cell lysates were immunoprecipitated with an anti-VSV-G antibody. Expression of FL-RGMa N-RGMa and C-RGMa was observed at approximately 55 27 and 37 kDa respectively in the whole cell lysates (Fig. 1B right panel) although multiple bands could be seen in each lane as previously reported [2] [9]. These multiple bands may be due to the posttranslational modifications in the expressed proteins because RGMa is considered to have three asparagine-linked glycosylation sites (two in the N-terminus and one in the C-terminus) [12]. FL-RGMa-Myc and C-RGMa-Myc AWD 131-138 co-immunoprecipitated with VSVG-Neogenin (Fig. 1B); however N-RGMa did not. These results show that this C-terminus but not the N-terminus of RGMa binds to neogenin. Physique 1 The RGMa domain name required for binding to neogenin is within aa 259-295. Neogenin-binding site resides between the vWD and the hydrophobic domain name of RGMa To more precisely determine the neogenin-binding site of RGMa expression vectors.