Binding of F-spondin to amyloid-beta precursor protein: a candidate amyloid-beta precursor protein ligand that modulates amyloid-beta precursor protein cleavage - PubMed (original) (raw)

Binding of F-spondin to amyloid-beta precursor protein: a candidate amyloid-beta precursor protein ligand that modulates amyloid-beta precursor protein cleavage

Angela Ho et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Amyloid-beta precursor protein (APP), a type I membrane protein, is physiologically processed by alpha- or beta-secretases that cleave APP N-terminal to the transmembrane region. Extracellular alpha-/beta-cleavage of APP generates a large secreted N-terminal fragment, and a smaller cellular C-terminal fragment. Subsequent gamma-secretase cleavage in the transmembrane region of the C-terminal fragment induces secretion of small extracellular peptides, including Abeta40 and Abeta42, which are instrumental in the pathogenesis of Alzheimer's disease, and intracellular release of a cytoplasmic tail fragment. Although APP resembles a cell-surface receptor, no functionally active extracellular ligand for APP that might regulate its proteolytic processing has been described. We now show that F-spondin, a secreted signaling molecule implicated in neuronal development and repair, binds to the conserved central extracellular domain of APP and inhibits beta-secretase cleavage of APP. Our data indicate that F-spondin may be an endogenous regulator of APP cleavage, and suggest that the extracellular domains of APP are potential drug targets for interfering with beta-secretase cleavage.

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Figures

Fig. 1.

Fig. 1.

Binding of F-spondin to immobilized APP. (A) Domain structure of APP (Upper) and diagram of various APP vectors used for the present study (Lower). N-terminally, APP is composed of a signal peptide (SP), a CRD, a zinc-binding motif, acidic sequence regions, and a Kunitz domain. The center of APP is occupied by a large domain that contains no cysteine residues (referred to as CAPPD) and a short linker sequence that includes the cleavage sites for α- and β-secretases. C-terminally, APP contains a transmembrane region and a cytoplasmic tail. The constructs used here include Ig-fusion proteins of the entire extracellular region or CRD alone (Ig-APP.1 or Ig-APP.2 respectively), a GST-CAPPD fusion protein, and expression vectors that encode full-length APP, or APP in which the CRD or part of the CAPPD were deleted marked by dashed lines (pCMV-APPΔ1 or 2, respectively). Nonneuronal APP contains an alternatively spliced Kunitz domain that is absent from all APP constructs used here. (B) Affinity chromatography of secreted myc-tagged recombinant F-spondin pull-downs on immobilized APP proteins. Ig- and GST-fusion proteins of various fragments of APP, as indicated in A, were used to affinity-purify secreted myc-tagged F-spondin produced in the supernatant of transfected COS cells.

Fig. 2.

Fig. 2.

Binding of APP to immobilized F-spondin. (A) Domain structure of F-spondin (Upper) and constructs of F-spondin included in the various Ig-fusion vectors used for the present study (Lower). F-spondin is composed of an N-terminal signal peptide (SP), reelin-like and spondin domains, and six C-terminal thrombospondin repeats. The positions of the two N-glycosylation sites are indicated. (B) Pulldown of full-length APP695. APP695 was solubilized with 1% Triton X-100 from transfected COS cells and was bound to immobilized Ig-F spondin proteins containing full-length or parts of F-spondin (see A). (C) Pulldown of APP deletion mutants (see Fig. 1_A_ for extent of the deletions) with full-length Ig-F spondin. (D) Comparison of the ability of immobilized full-length F-spondin to affinity-purified APP, APLP1, and APLP2 expressed in transfected COS cells and visualized with antibodies to the C termini of indicated proteins.

Fig. 3.

Fig. 3.

Lack of an interaction of APP with Mindin. (A) Domain structure of F-spondin with Mindin. SP, signal peptide, a spondin-like domain; TSR, thrombospondin repeat. (B) Pulldown of myc-tagged Mindin with immobilized GST-CAPPD fusion protein. (C) Pulldown of APP with a Ig-Mindin fusion protein.

Fig. 4.

Fig. 4.

F-spondin inhibits cleavage of APP by BACE 1. (A) Immunoblot of HEK293 cells that were transfected without or with BACE 1, Ig-C, or Ig-F spondin as indicated. Experiments were carried out in triplicate to ensure reproducibility. Numbers on the left indicate positions of molecular mass markers. Note that BACE 1 cotransfection promotes production of two C-terminal fragments, termed CTFβ1 and CTFβ2. (B) Quantification of the results shown in A. Relative levels of full-length APP and of both CTFs were quantified using 125I-labeled secondary antibodies and PhosphorImager detection. Data shown are means ± SEM derived by dividing for each sample the signal for CTFβ1 or CTFβ2 by the APP signal.

Fig. 5.

Fig. 5.

Titration of F-spondin-mediated inhibition of APP cleavage by BACE 1. (A) Relative levels of proteins expressed in an experiment similar to that described in Fig. 4. Increasing amounts of Ig-F spondin plasmid were cotransfected with constant amounts of APP and BACE 1. The levels of full-length APP and the CTFs of APP and of F-spondin were quantified by immunoblotting and are shown in arbitrary units. (B) Ratio of CTF to full-length APP as a function of increasing amount of F-spondin. CTF levels were corrected for APP expression. Data shown are means ± SEM from a representative experiment (n = 3) independently repeated multiple times.

Fig. 6.

Fig. 6.

Effect of F-spondin on APP-dependent transactivation of Gal4-Tip60-mediated transcription. (A) F-spondin inhibits APP-dependent transactivation. A constant amount of Gal4-Tip60, Fe65, and APP was cotransfected with increasing amounts of Ig-F spondin. Note that, without F-spondin, APP causes a strong stimulation of Gal4-Tip60-dependent transcription as described (7). F-spondin dramatically inhibits APP-dependent transactivation of transcription by Gal4-Tip60, such that even low concentrations of F-spondin (<100 ng of transfected plasmid) almost completely block the response. (B) Comparison of the effects of multiple Ig-fusion proteins on the APP-dependent transactivation of Gal4-Tip60. All proteins were expressed with 50 ng of cotransfected plasmids. (C) Increasing concentrations of APP are unable to rescue the F-spondin-dependent inhibition of APP-dependent transactivation of Gal4-Tip60. Constant amounts of Gal4-Tip60, Fe65, and Ig-C, Ig-N1β-1, or Ig-F spondin were cotransfected with increasing concentrations of APP. The bell-shaped dose–response curve under control conditions as reported (7) is probably due to dilution of transcription factors by increasing amounts of APP. Nevertheless, even at high concentrations of APP, F-spondin induces a relative inhibition of transactivation.

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