The Journal of Neuroscience, January 26, 2005, ():
The Nogo-66 Receptor Homolog NgR2 Is a Sialic Acid-Dependent Receptor Selective for Myelin-Associated Glycoprotein
J. Neurosci. Venkatesh et al.
25: 808
Supplemental data
Files in this Data Supplement:
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Supplementary Figure S1
Comparison of MAG-Fc and AP-MAG binding to NgR2 and NgR1. A: Semi-quantitative analysis of MAG-Fc binding to NgR1 and NgR2 transiently expressed in COS-7 cells. Serial dilutions (0-80 nM) of MAG-Fc (pre-clustered with an anti-human Fc-AP conjugated antibody) were washed on transiently transfected COS-7 cells. To compare the relative binding strength to NgR1 and NgR2, the AP reaction was developed and followed over time. After ~1h, labeling of NgR2 transfected cells incubated with MAG-Fc at 5 nM started to become detectable and the AP reaction was stopped in all wells. In three independent experiments (of a total of three experiments) MAG-Fc bound NgR2 more strongly than NgR1. MAG-Fc binding to NgR2 at a concentration of 5-10 nM is comparable to MAG-Fc binding to NgR1 at a concentration of 40-80 nM. Thus, MAG-Fc binds approximately 4-8 fold stronger to NgR2 than to NgR1. B: Binding of AP-MAG (17 and 33 nM) to transiently transfected COS-7 cells. Consistent with a previous study (Barton et al., 2003), AP-MAG binds to NgR1 but not to NgR2. Thus, N-terminal tagging of MAG with AP selectively abolishes binding to NgR2. Consistent with this finding, we show that the structural basis of NgR1 and NgR2 to support MAG binding is distinct (see figure 8 for details). Scale bar is equal to 10 mm (A and B).
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Supplementary Figure S2
Soluble NgR1 does not interact with NgR2. Affinity precipitation experiments, using NgR1-Fc (NgR-Fc, R&D Systems) or anti-human IgG did not reveal any interaction between NgR1 and NgR2. Western blot analysis of lysates of Ad-NgR2 transduced cells (Inp.) revealed robust expression of NgR2. As a control, we show that NgR1-Fc allows selective precipitation of AP-Nogo66 but not AP-NiG.
