Presenilin, the proposed catalytic subunit of the large, multimeric γ-secretase complex, has been shown to cleave transmembrane proteins following removal of their extracellular domains, or ectodomains. In the case of APP, for example, γ-secretase action follows that of α- or β-secretase (see ARF related news story), which lop off different amounts of the N-terminal head of the protein.

Together with Kim, first author Kwang-Mook Jung and coworkers noticed that phorbol esters, which are known to promote the shedding of ectodomains from many transmembrane receptors, promotes the release of an N-terminal fragment from p75NTR, leaving behind about half the molecule (34 kDa). To test if this C-terminus is susceptible to γ-secretase cleavage, Jung added phorbol ester and a γ-secretase inhibitor, Compound E, to HEK 293 cells expressing p75. This resulted in a massive accumulation of the C-terminal fragment, suggesting that the secretase is involved in processing the cleaved receptor. In fact, even in the absence of the ester, Jung found that Compound E caused a slow accumulation of the C-terminal fragment, indicating that p75NTR ectodomain shedding occurs constitutively. To conclusively establish the role of γ-secretase in p75 processing, Jung coexpressed dominant-negative forms of both presenilins (PS1 and PS2) in cells expressing the receptor. This dramatically raised the levels of the 34 kDa fragment, which were even further elevated by phorbol esters.

But Jung and colleagues realized that part of the picture was missing. In the case of APP and Notch, γ-secretase activity releases a soluble intracellular domain, which can translocate to the nucleus. If the secretase similarly cleaved p75NTR, then it should release a soluble intracellular domain (ICD) of about 25 kDa in size, and yet the authors failed to detect this fragment. Concluding that it may be getting rapidly metabolized, Jung added MG132, a proteasome inhibitor, to phorbol ester-treated cells. Under these conditions the ICD was readily detectable, suggesting that the proteasome normally degrades it.

This finding paved the way for Jung and colleagues to purify and analyze the ICD by mass spectroscopy. This revealed that the γ-secretase cleavage site is between p75 amino acids valine 235 and valine 236. This site comes after an alanine-alanine-valine-valine sequence, which is very similar to the glycine-glycine-valine-valine motif that precedes the Aβ 40 cleavage site in APP.

What these findings mean for neurotrophin signaling and for γ-secretase as a potential therapeutic target for Alzheimer's disease (AD) remains to be seen. But p75NTR is involved in a diverse array of processes including cell migration and death, and elongation of neuronal axons. It also serves as a receptor for nerve growth factor, the loss of which induces AD pathology in mice (see ARF related news story). These associations have prompted Kim and colleagues to warn that "altered proteolytic processing of p75NTR may contribute to the selective dysfunction and/or degeneration of p75-expressing neurons in AD," and that "therapeutic reagents based on general inhibition of γ-secretase activity may potentially affect p75 signaling and p75-associated cholinergic neuronal survival and differentiation." In support of this, the authors found that p75ICD, unlike the full-length receptor, fails to interact with its tyrosine receptor kinase partner TrkA.-Tom Fagan.

Reference:
Jung K-M, Tan S, Landman N, Petrova K, Murray S, Lewis R, Kim PK, Kim DS, Ryu SH, Chao MV, Kim T-W. Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor. J Biol Chem. 2003 Aug 11 [Epub ahead of print] Abstract