Figure 1. Transmembrane Cys²⁵⁷ mediates formation of disulphide-linked p75^NTR dimers.

(A) Cell surface expression of disulphide-linked p75^NTR dimers (dim) and monomers (mon) in transfected COS-7 cells visualized by Neutravidin probing of p75^NTR immunoprecipitates under non-reducing (−DTT) and reducing (+DTT) conditions. In non-reducing conditions, p75^NTR dimers run somewhat higher, and monomers lower, than their predicted molecular weights.

(B) Cell surface expression of disulphide-linked p75^NTR dimers (dim) and monomers (mon) in COS-7 cells transfected with different amounts of wild type p75^NTR. Immunoprecipitates were electrophoresed under non-reducing conditions and p75^NTR was visualized by Neutravidin probing of p75^NTR immunoprecipitates. Results are expressed as mean dimer/monomer ratio.

(C) Alignment of p75^NTR transmembrane domain sequences from vertebrate and invertebrate species.

(D) Cell surface expression of endogenous disulphide-linked p75^NTR dimers (dim) and monomers (mon) in PC12 cells, RN22 Schwannoma cells and SCG sympathetic neurons visualized by Neutravidin probing of p75^NTR immunoprecipitates. Control, untransfected COS cells.

(E) Expression of endogenous disulphide-linked p75^NTR dimers (dim) and monomers (mon) in extracts of newborn rat cerebellum (cblm), cortex (ctx) and hippocampus (hc) visualized by immunoblotting of p75^NTR immunoprecipitates under reducing and non-reducing conditions. The control lane represents a mock immunoprecipitate of the cblm extract.

(F) Binding of ¹²⁵I-NGF to wild type and C257A p75^NTR analyzed by chemical crosslinking. Samples were run under reducing conditions. For each construct, binding counts were normalized to levels of expression as assessed by immunoblotting (IB). Results are expressed as mean ± SD of three independent determinations.

(G) γ-secretase-dependent intramembrane cleavage of C257A p75^NTR following stimulation with PMA in transfected COS cells. The proteasome inhibitor epoxomycin was used to prevent degradation of CTF and ICD fragments. Mutant and wild type (wt) p75^NTR molecules were recovered by immunoprecipitation and visualized by immunoblotting. The γ-secretase inhibitor DAPT blocked the generation of p75^NTR intracellular domain (ICD). CTF, carboxy terminal fragment.