Association of p75(NTR) with caveolin and localization of neurotrophin-induced sphingomyelin hydrolysis to caveolae

Abstract

Caveolae are plasma membrane microdomains that are enriched in caveolin, the structural protein of caveolae, sphingomyelin, and other signaling molecules. We previously suggested that neurotrophin-induced p75(NTR)-dependent sphingomyelin hydrolysis may be localized to the plasma membrane. Therefore, we examined if caveolae were a major site of p75(NTR)-dependent sphingomyelin hydrolysis in p75(NTR)-NIH 3T3 fibroblasts. Caveolin-enriched membranes (CEMs) were prepared by either detergent or detergent-free extraction and separated from noncaveolar membranes by centrifugation through sucrose gradients. Immunoblot analysis of the individual gradient fractions indicated that caveolin and p75(NTR) were enriched in CEMs. The localization of p75(NTR) to CEMs was not an artifact of receptor overexpression in the fibroblasts because a similar distribution of p75(NTR) was evident from PC12 cells, which endogenously express p75(NTR). In the p75(NTR) fibroblasts, nerve growth factor induced a time-dependent hydrolysis of sphingomyelin only in CEMs with no hydrolysis detected in noncaveolar membranes. Intriguingly, endogenous p75(NTR) was found to co-immunoprecipitate with caveolin, suggesting that p75(NTR) may associate with caveolin in vivo. This interaction was confirmed in vitro by the co-immunoprecipitation of a glutathione S-transferase fusion protein expressing the cytoplasmic domain of p75(NTR) with caveolin. Collectively, these results demonstrate that neurotrophin-induced p75(NTR)-dependent sphingomyelin hydrolysis localizes to CEMs and suggest that the interaction of p75(NTR) with caveolin may affect signaling through p75(NTR).