Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder in which amyloid β plaques are a pathological characteristic. Little is known about the physiological functions of amyloid β precursor protein (APP). Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1, members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease (PMD) and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

Keywords: Alzheimer’s disease; Amyloid precursor protein; Cell death; Pelizaeus-Merzbacher disease; Yeast two-hybrid screening.

Fig. 1

Screened products of APP_ex-pGBKT7. A QDO/X/A plates co-transformed with APP_ex-pGBKT7 and its screened products. The red circles indicated yeast colonies. B Gene and protein names of screened products.

Fig. 2

Co-immunoprecipitation of full-length APP with full-length PLP1, full-length KCTD16, and full-length FLRT in HEK293T cells. A Cell lysates were immunoprecipitated with an anti-EGFP antibody for mutated PLP1 and probed with an anti-mCherry antibody for APP (130 kD). B Cell lysates were immunoprecipitated with an anti-mCherry antibody for APP and probed with an anti-EGFP antibody for mutated PLP1 (55 kD). C Cell lysates were immunoprecipitated with an anti-EGFP antibody for wild-type PLP1 and probed with an anti-mCherry antibody for APP (130 kD). D Cell lysates were immunoprecipitated with an anti-mCherry antibody for APP and probed with an anti-EGFP antibody for wild-type PLP1 (57.5 kD). E Cell lysates were immunoprecipitated with an anti-EGFP antibody for KCTD16 and probed with an anti-mCherry antibody for APP (130 kD). F Cell lysates were immunoprecipitated with an anti-mCherry antibody for APP and probed with an anti-EGFP antibody for KCTD16 (76 kD). G Cell lysates were immunoprecipitated with an anti-EGFP antibody for FLRT3 and probed with an anti-mCherry antibody for APP (130 kD). H Cell lysates were immunoprecipitated with an anti-mCherry antibody for APP and probed with an anti-EGFP antibody for FLRT3 (130 kD). I Cell lysates were immunoprecipitated with an anti-mCherry antibody for APP and probed with an anti-EGFP antibody for FLRT1 (130 kD).

Fig. 3

Localization of ectodomain proteins in HEK293T cells. HEK293T cells were transfected with ecto-FLRT3-EGFP (A), ecto-APP-mCherry (B), and ecto-mPLP1-EGFP (C) and immunostained. Nuclei were stained with DAPI.

Fig. 4

Co-immunoprecipitation of the APP extracellular domain with the FLRT3 extracellular domain or the mPLP1 extracellular domain in HEK293T cells. A Cell lysates were immunoprecipitated with an anti-EGFP antibody for FLRT3 extracellular domain and probed with an anti-mCherry antibody for APP extracellular domain. B Cell lysates were immunoprecipitated with an anti-mCherry antibody for APP extracellular domain and probed with an anti-EGFP antibody for FLRT3 extracellular domain. C Cell lysates were immunoprecipitated with an anti-His antibody for PLP1 extracellular domain and probed with an anti-mCherry antibody for APP extracellular domain. D Cell lysates were immunoprecipitated with an anti-c-myc antibody for APP extracellular domain and probed with an anti-EGFP antibody for PLP1 extracellular domain (32 kD).