Malvankar, S. R.; Wolfe, M. S.

{gamma}-Secretase is an intramembrane protease complex, with nearly 150 substrates that are cleaved within their transmembrane domains (TMD). Amyloid Precursor Protein (APP) is the most widely studied, as processive proteolysis by {gamma}{gamma}secretase releases the amyloid-{beta}-peptide implicated in the pathogenesis of Alzheimer's disease. In contrast, proteolysis of other substrates has been little explored. The only known sequence specificity rule for {gamma}-secretase cleavage is for APP, in which phenylalanine is not tolerated at P2' with respect to any step in processive proteolysis. Recently, we found this specificity rule applies to the initial cleavage of Notch1 substrate as well. In this study, we examined the site of initial cleavage by {gamma}-secretase and explored the phenylalanine rule for two other {gamma}-secretase substrates: neuregulin1 (NRG1) and E-cadherin (CDH1). Upon incubation of recombinant substrates with purified protease complex, followed by mass spectroscopy (MS) and immunoblot analysis, initial cleavage products for NRG1 and CDH1 were identified. Two cleavage sites were observed in the NRG1 TMD, one of which matched that seen previously. However, the observed single CDH1 TMD cleavage site differed from the reported cytosolic cleavage site. Phenylalanine mutants of NRG1 and CDH1 in the P2' position relative to the first {gamma}-secretase cleavage site showed a shift in the cleavage site, along with reduction in total C-terminal and N-terminal products, compared to that seen with wild-type substrates. Taking together, these findings clarify the initial cleavage sites of NRG1 and CDH1 and support the intolerance of Phe at P2' position as a general rule for {gamma}-secretase substrates.