Abstract
Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75(NTR), best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75(NTR) ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75(NTR) are γ-secretase substrates. However, dimerization is not a requirement for p75(NTR) cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75(NTR) interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75(NTR) transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75(NTR). Nonetheless, heteromer interactions of p75(NTR) with TrkA increase full-length p75(NTR) homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75(NTR) transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.
Highlights
Cleavage of transmembrane receptors by ␥-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function
Together these results suggest that p75 neurotrophin receptor (p75NTR) forms dimeric complexes that can be measured by FRET methods that are not influenced by the intracellular domain
As the previous experiment had demonstrated that p75NTR dimers undergo RIP, these results suggest that a dimeric substrate is not a requirement for the ␥-secretase complex to interact with and cleave p75NTR
Summary
Construct Design—p75NTR expression constructs used a modified pCDNA3 (Invitrogen) backbone. P75NTR peptide coding sequences were amplified under standard PCR conditions with 5Ј EcoRV and 3Ј NheI restriction sites incorporated into the respective primers. P75NTR coding sequences were cloned between the EcoRV and NheI restriction sites of the pCDNA-CFP or pCDNA3-YFP vector to generate in-frame FRET-capable fusion proteins. Immunofluorescence and Microscopic FRET Analysis—For immunofluorescence imaging, HEK293 cells were plated at ϳ25% confluence onto polyornithine-coated 12-mm coverslips and transiently transfected with wild type or YFP-tagged p75NTR expression constructs as described above. The FRET between p75NTR proteins in single cells was measured as sensitized acceptor emission using the three-cube method, as previously described [37, 38]. The models were prepared using PyMOL [43]
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