Figure 3.
Structural basis of the NgR1–FGF2 association.
A
, Chimeric Nogo receptors were expressed on the surface of COS-7 cells and assayed for binding of AP-FGF2, AP-Nogo-66-myc, or AP-OMgp. NgR1 sequences are labeled in red and non-NgR1 sequences are labeled in gray. LRR, Leucine-rich repeats 1–8; NT, LRRNT cap domain; CT, LRRCT cap domain. Full-length NgR1 (construct I), but not NgR3 (construct II), supports binding of FGF2, Nogo-66, and OMgp. The NgR1 LRR cluster (composed of domains LRRNT-LRR-LRRCT) fused to the stalk region of NgR3 is sufficient to confer high-affinity ligand binding (see construct III). Construct IV, composed of the NgR3 LRR cluster and the NgR1 stalk region, does not support binding of FGF2, Nogo-66, or OMgp. Furthermore, the NgR1 LRRNT cap domain and the first three LRRs (construct V) and the NgR1 LRRCT distal portion and stalk region (construct VI) are dispensable for AP-FGF2, AP-Nogo-66-myc, or AP-OMgp binding. The NgR1 LRRCT distal region and stalk are not sufficient to support ligand binding (construct VII). Cell surface expression of chimeric Nogo receptor constructs was confirmed by ICC under nonpermeabilizing conditions. Scale bars:
A
,
D
, 30 μm.
B
, Quantification of ligand binding to chimeric Nogo receptors, normalized to wild-type NgR1 binding (=100%). Of note, the molecular basis for AP-FGF2-myc, AP-Nogo-66, and AP-OMgp is very similar.
C
, An NgR1-Fc pull-down (PD) assay was used for affinity precipitation of AP-Nogo-66-myc in the presence of increasing concentrations of AP-FGF2. AP-FGF2 competes with AP-Nogo-66-myc for NgR1 binding.
D
, Binding of AP-FGF2 to full-length NgR1 and NgR1Δstalk (Δstalk) transiently expressed on COS-7 cells. Binding of AP-FGF2 to wild-type NgR1 was normalized to 100%, and no significant change in AP-FGF2 binding was observed after deletion of residues T373–G448 of the NgR1 stalk (110 ± 9%).
E
, Experiments with PC12 cells stably expressing either NgR1 or NgR1Δstalk revealed that the NgR1 stalk region T373–G448 is important for the inhibition of FGF2-elicited PC12 cell differentiation. *p < 0.05, NgR1 versus vector. Error bars indicate SEM.