Human CRB2 inhibits γ-secretase cleavage of amyloid precursor protein by binding to the presenilin complex

Abstract

Drosophila Crumbs has been reported to attenuate Notch signaling by inhibition of γ-secretase cleavage at the wing margins. γ-Secretase is an intramembrane protease that is responsible for the generation of amyloid-β (Aβ) peptides from the β-amyloid precursor protein (APP). Here, we re-examined γ-secretase inhibition by human CRB2, which is the most abundant Crumbs ortholog in the brain. Transfected CRB2 inhibited proteolytic production of Aβ and APP intracellular domains from APP C-terminal fragments in HEK293 and SH-SY5Y cells. Conversely, knockdown of endogenous CRB2 increased γ-secretase cleavage products in SH-SY5Y cells. CRB2 inhibition of γ-cleavage was also detected in cell-free assays. CRB2 interacted with the γ-secretase complex, but was not a competitive substrate for γ-cleavage. The transmembrane domain of CRB2 was indispensable for inhibition of Aβ generation and mediated CRB2 binding with the γ-secretase complex. In addition, the cytoplasmic domain appeared to play a supportive role in γ-secretase inhibition, whereas mutational disruption of the two protein-binding motifs involved in the formation of cell adhesion complexes did not affect γ-secretase inhibition. Co-overexpression of presenilin-1 or APH-1 abrogated γ-secretase inhibition probably through prevention of the incorporation of CRB2 into the γ-secretase complex. Our results suggest that CRB2 functions as an inhibitory binding protein that is involved in the formation of a mature but inactive pool of the γ-secretase complex. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.