Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation required the cytoplasmic domain of the mutants and appeared to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins).

in APP 695 (Hardy, 1992). These mutations co-segregate Narasimhan Gautam 8 and with the AD phenotype (Karlinsky et al., 1992), demon- Ikuo Nishimoto 1,2,9strating that V642 mutations in APP are established causes of AD. et al., 1996). Furtherprotein APP. Expression of these mutants causes a more, it has been found that the FAD-associated V642 COS cell NK1 clone to undergo pertussis toxin-sensitive mutants of APP cause cytotoxicity in cultured cells without apoptosis in an FAD trait-linked manner by activating Aβ mediation (Yamatsuji et al., 1996a,b). Thus, the the G protein G o , which consists of Gα o and Gβγ significance, as well as the role, of Aβ deposition for AD subunits. We investigated which subunit was responsdevelopment remains unclear. ible for the induction of apoptosis by V642I APP in NK1In its structure, orientation and localization, APP is cells. In the same system, expression of mutationally similar to cell surface receptors (Kang et al., 1987; Dyrks activated Gα o or Gα i induced little apoptosis. Apoptosis et al., 1988;Weidemann et al., 1989;Schubert et al., by V642I APP was antagonized by the overexpression Ferreira et al., 1993). The cytoplasmic domain of of the carboxy-terminal amino acids 495-689 of the APP binds Fe65 protein, which has a phosphotyrosine-β-adrenergic receptor kinase-1, which blocks the binding domain related to an oncogenic signal transducer, specific functions of Gβγ. Co-transfection of Gβ2γ2Shc (Fiore et al., 1995). It also binds APP-BP1, a gene cDNAs, but not that of other Gβxγz (x ϭ 1-3; z ϭ 2, product similar to AXR1 in Arabidopsis; AXR1 is required 3), induced DNA fragmentation in a manner sensitive for normal response to the plant growth hormone auxin to bcl-2. These data implicate Gβγ as a cell death (Chow et al., 1996). These observations suggest that APP mediator for the FAD-associated mutant of APP.has not only the structure but also the function of a cell Keywords: amyloid precursor protein/apoptosis/ surface receptor. Our own earlier study (Nishimoto et al., βγ complex/familial Alzheimer's disease/G protein 1993) found that APP 695 has an intrinsic G o -stimulating domain at His657-Lys676 and forms a complex with G o through this cytoplasmic domain. It has been confirmed that the synthetic His657-Lys676 peptide activates G o

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SSTR3, a somatostatin (SST) receptor, is an adenylyl cyclase (AC)-inhibiting receptor. To assign the G-protein asubunit (Ga) linked to this receptor, we created a novel reporter system which utilizes the well-established facts that the Cterminal 5 residues of Ga are the receptor contact site and Gets stimulates all subtypes of AC. We constructed chimeric Gag the C-terminal 5 residues of which were replaced with the corresponding C-terminus of each known Ga, and examined which chimera confers SSTR3-induced activation of AC. Cellular transfection of SSTR3 and measurement of SSTdependent AC activity through co-transfected chimeric GOs revealed that SSTR3 recognizes the C-termini of GrXji/ 2 but not of GOQ or Ga z , and those of Ga M and Gai 6 , but not of GOq or Gan. As predicted by the chimeric Gas, SST-bound SSTR3 stimulated polyphosphoinositide turnover only when Gai 6 or Gai4 was co-transfected. We conclude that the chimeric Gag system provides a new approach towards the assignment of Gproteins linked to a given receptor.

APP695 is a transmembrane precursor of Abeta amyloid. In familial Alzheimer's disease (FAD), three mutations V642I/F/G were discovered in APP695, which has been suggested by multiple studies to be a cell surface signaling receptor. We previously reported that normal APP695 encodes a potential GO‐linked receptor with ligand‐regulated function and that expression of the three FAD mutants (FAD‐APPs), not normal APP, induces cellular outputs by GO‐dependent mechanisms. This suggests that FAD‐APPs are constitutively active GO‐linked receptors. Here, we provide direct evidence for this notion. Reconstitution of either recombinant FAD‐APP with GO vesicles induced activation of GO, which was inhibitable by pertussis toxin, sensitive to Mg2+ and proportional in quantity to the reconstituted amounts of FAD‐APP. Consistent with the dominant inheritance of this type of FAD, this function was dominant over normal APP, because little activation was observed in APP695‐GO vesicles. Experiments with antibody competition and sequence deletion indicated that His657‐Lys676 of FAD‐APP, which has been specified as the ligand‐dependent GO‐coupling domain of normal APP, was responsible for this constitutive activation, confirming that the three FAD‐APPs are mutationally activated APP695. This study identifies the intrinsic signaling function of APP to be a novel target of hereditary Alzheimer's disease mutations, providing an in vitro system for the screening of potential FAD inhibitors.

The 14-residue peptide (peptide 14) corresponding to Arg2"0-Lys"23 of the insulin-like growth factor II

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Amyloid precursor protein (APP) has been shown to serve as a G_o-coupled receptor in cell-free systems [Okamoto et al: J Biol Chem 1995;270:4205–4208]. However, it has not been known whether APP exerts intracellular signaling functions in living cells. In this study, we show that stimulation of APP by anti-APP antibody as well as by a mutation found in familial Alzheimer’s disease results in activation of a specific set of mitogen-activated protein kinases in multiple vertebrate cells. We conclude that APP acts as a cell surface receptor of biological relevance that turns on specific Ser/Thr kinases, and suggest that the signaling function of APP is a potential target of familial Alzheimer’s disease mutations.