Myelin-associated glycoprotein (MAG) is definitely a sialic acid binding Ig-family lectin

Myelin-associated glycoprotein (MAG) is definitely a sialic acid binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. (NT) and MCM7 C-terminal (CT)-cap GBR-12935 2HCl domains. The LRR cluster is usually connected through a stalk region to a membrane lipid anchor. The CT-cap domain name and stalk region of NgR2 but not NgR1 are sufficient for MAG binding and when expressed in neurons exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66 OMgp and MAG. Deletion of disulfide loop Cys309-Cys336 of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgROMNI) in which Cys309-Cys336 is deleted and followed by a 13 amino acid MAG binding motif of the NgR2 stalk shows superior binding of OMgp Nogo-66 and MAG compared to wild-type NgR1 or NgR2. Soluble NgROMNI (NgROMNI-Fc) binds strongly to membrane bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus NgROMNI-Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration. is necessary for growth cone collapse in response to acutely offered myelin inhibitors (Kim et al. 2004 Chivatakarn et al. 2007 but is usually dispensable for neurite outgrowth inhibition on substrate-bound Nogo-66 (Zheng et al. 2005 MAG or OMgp (Venkatesh et al. 2007 Chivatakarn et al. 2007 Williams et al. 2008 Mechanistically this apparent dichotomy of the role of NgR1 in neuronal growth inhibitory responses is usually poorly comprehended. Physiological signaling limits experience-dependent plasticity in the visual cortex (McGee et al. 2005 and in the adult hippocampus regulates activity-dependent synaptic strength and dendritic spine morphology (Lee et al. 2008 Following CNS injury limits axon collateral sprouting but not long-distance regenerative growth of severed corticospinal tract fibers (Kim et al. 2004 Zheng et al. 2005 Cafferty and Strittmatter 2006 MAG is usually a member of the siglec family of sialic acid binding Ig-lectins and employs neuronal cell type-specific mechanisms to mediate growth inhibition. Cerebellar granule neurons (CGNs) but not dorsal root ganglion (DRG) neurons deficient for complex gangliosides are more resistant to MAG inhibition. In retinal ganglion cells (RGCs) hippocampal and DRG neurons functional depletion of gangliosides or NgR1 alone is not sufficient to attenuate MAG inhibition. Simultaneous loss of terminal sialic acids and NgR1 however significantly attenuates MAG inhibition (Mehta et al. 2007 Venkatesh et al. 2007 A receptor complex comprised of NgR1 Lingo-1 and p75 or TROY has been implicated in signaling Nogo-66 OMgp and MAG inhibition of neurite outgrowth (Yiu and He 2006 is usually important for growth inhibition of DRG neurons but neither nor is necessary for MAG inhibition of CGNs or RGCs (Zheng et al. 2005 Venkatesh et al. 2007 MAG-induced repulsive growth cone steering requires the presence of an arginine-glycine-aspartate (RGD) dependent conversation with neuronal β1-integrin (Goh et al. 2008 The ligand-binding GBR-12935 2HCl domain name (LBD) of NgR1 is composed of 8.5 canonical LRRs flanked by cysteine-rich LRR-NT and LRR-CT cap domains. The LBD GBR-12935 2HCl harbors overlapping yet distinct binding pouches for Nogo OMgp and MAG (Lauren et al. 2007 In soluble form the NgR1 LBD (NgR1(310)) has CNS myelin inhibitor antagonistic properties (Fournier et al. 2002 He et al. 2003 Zheng et al. 2005 Liu et al. 2002 Following spinal cord injury NgR1(310)-Fc promotes sprouting and regenerative growth of severed corticospinal and raphespinal fibers (Li et al. 2004 Wang et al. 2006 Here we define the structural basis of the MAG association with NgR1 and NgR2 and develop GBR-12935 2HCl a soluble chimeric Nogo receptor variant with potent CNS myelin antagonistic properties. EXPERIMENTAL PROCEDURES Recombinant DNA constructs Chimeric receptors were generated by PCR using rat NgR1 NgR2 or NgR3 cDNA themes and put together in the expression vector pMT21 (Venkatesh et al. 2005 To fuse PCR-amplified receptor fragments either endogenous restriction enzyme sites or newly-introduced restriction sites were used that resulted in either no amino acid substitution or conservative substitutions. None of the conserved leucine or phenylalanine residues critical for the tertiary structure of the LRR cluster or cysteine residues in the LRRNT- and LRRCT-cap domains implicated in disulfide bonds were altered. N-terminal NgR1 and NgR2 deletion mutants were fused to the transmission sequence of peptidylglycine.