Substrate Sequence Influences γ-Secretase Modulator Activity, Role of the Transmembrane Domain of the Amyloid Precursor Protein

A subset of non-steroidal anti-inflammatory drugs modulates the gamma cleavage site in the amyloid precursor protein (APP) to selectively reduce production of A beta 42. It is unclear precisely how these gamma-secretase modulators (GSMs) act to preferentially spare A beta 40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on gamma-cleavage of artificial constructs containing several gamma-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. gamma-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to gamma-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on gamma-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the gamma-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.