FIGURE 3.

p75^NTR interacts predominantly with intracellular LMM LINGO-1. A, LMM LINGO-1 is selectively sensitive to endoglycosidase H (EndoH) activity. HEK293 cells were transiently transfected with LINGO-1 plasmid, LINGO-1 was immunoprecipitated, and immunoprecipitates were exposed to endoglycosidase H prior to immunoblot (IB) analysis. Endoglycosidase H exposure converted 90-kDa LMM LINGO-1 to an 80-kDa product, whereas 100-kDa HMM LINGO-1 exhibited only limited conversion to a slightly smaller form. B, only HMM LINGO-1 can traffic to the cell surface. HEK293 cells were transiently transfected with p75^NTR and C-terminal V5-tagged LINGO-1 or LINGO-1 with most of its ICD deleted, and cell-surface proteins were biotinylated. Cell-surface proteins isolated by streptavidin (Streptav) pulldown (PD) and total cell proteins (whole cell lysate (WCL)) were assessed by immunoblot analysis. Only HMM LINGO-1 species were detected in the surface-expressed fraction. No LMM LINGO-1 species were detected in the surface-expressed fraction. C, p75^NTR interacts exclusively with LMM LINGO-1. HEK293 cells were transiently transfected with plasmids encoding Myc-tagged p75^NTR, FLAG-tagged NgR, and V5-tagged LINGO-1 bearing wild-type or mutant C-terminal sequences. LINGO-1 in detergent extracts of cells was examined by immunoblot analysis with anti-V5 antibody either for whole cell lysates or following immunoprecipitation (IP) of NgR and p75^NTR with antibodies against FLAG and Myc epitope tags, respectively. NgR co-immunoprecipitated with both HMM LINGO-1 and LMM LINGO-1, whereas p75^NTR co-immunoprecipitated with only LMM LINGO-1. D, confocal immunofluorescence detection of p75^NTR (red) and LINGO-1 (green) co-localization in transfected HEK293 cells. LINGO-1·p75^NTR co-localization was restricted to intracellular puncta. Nuclei were stained with DAPI (blue).