Expression and purification of a recombinant transmembrane domain amyloid precursor protein associated with Alzheimer’s disease

More than half of the mutations of the amyloid precursor protein (APP) discovered in familiar forms of Alzheimer’s disease are located in the transmembrane domain. The pathogenic mutations presumably affect the lateral dimerization of the APP transmembrane domain in the membrane and change the dimer conformation and/or stability. Thus, the mutations cause an alternative APP digestion pattern in the membrane and neurotoxic amyloid β-peptide generation. For the detailed study of the specific protein-protein and protein-lipid interactions of the APP transmembrane domain, an E. coli recombinant expression construct was made. The recombinant protein contains an APP transmembrane domain (APPtm(686–726)) with adjacent extramembrane N and C ends. Here, we report the method of isotope-labeled APPtm expression and purification in quantities necessary for a heteronuclear NMR spectroscopy structure and dynamics study. On the basis of the 1H-15N-HSQC spectra, we developed APPtm(686–726) solubilization conditions in the membrane-emulated milieu detergent micelles and lipid bicelles.     

"Secretase," Alzheimer amyloid protein precursor secreting enzyme is not sequence-specific

The major pathological change in Alzheimer's disease is the deposition of amyloid beta/A4-protein (beta P) in the brain. beta P is derived from a small part of the much larger amyloid protein precursor (APP). In the normal condition, APP is cleaved in the interior of beta P, preventing the formation of beta P, by a hypothetical proteinase "secretase". To characterize this enzyme, APP and mutated APPs were expressed by cDNA transfection in COS-1 cells, a monkey kidney fibroblast derived cell line. The mutant APPs with the mutations of the proposed cleavage site (Gln686-Lys687) were processed in the same way as wild APP. The deleted mutant APP (deletion of Arg676-Asp694) was also cleaved in a similar way to wild APP. The cleavage site of this deletion mutant was located at the 12 amino acid residues from the predicted membrane spanning domain. Hence, "secretase" cleaves APP, depending not on its specific amino acid sequence, but probably on the relative conformation with plasma membrane.     

Characterization of alpha 2,6-sialyltransferase cleavage by Alzheimer's beta -secretase (BACE1)

BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the beta-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu(41). Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu(37) and Gln(38), suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln(38)-Ala(39)-Lys(40), were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu(37) and Gln(38) by BACE1, and then the three-amino acid sequence at the NH(2) terminus is removed by exopeptidase(s) before secretion from the cells.     

Targeting of the dual oxidase 2 N-terminal region to the plasma membrane

Dual oxidase 2 (Duox2) is a cell surface glycoprotein that probably provides thyroperoxidase with the H2O2 required to catalyze thyroid hormone synthesis. No functional H2O2-generating system has yet been obtained after transfecting Duox2 into non-thyroid cell lines, because it is retained in the endoplasmic reticulum (ER). We investigated the level of maturation of various Duox2 truncated proteins in an attempt to identify the region of Duox2 responsible for its remaining in the ER. Duox2-Q686X mutant, corresponding to the N-terminal ectodomain including the first putative transmembrane domain, was expressed in different cell lines. Carbohydrate content analysis revealed that complex type-specific Golgi apparatus (GA) oligosaccharides were present on pig Duox2-Q686X, whereas human truncated Duox2 carried only high mannose-type sugar chains characteristic of the ER. Further characterization using surface biotinylation and flow cytometry assays indicated that pig Duox2-Q686X was present at the plasma membrane, whereas human Duox2-Q686X remained inside the cell. The replacement of the last 90 residues of the human Duox2-Q686X with the pig equivalent region allowed the chimerical peptide to reach the Golgi apparatus. Pig mutants containing the complete first intracellular loop with or without the second transmembrane domain accumulated in the ER. These findings show that 1) the human Duox2-Q686X region encompassing residues 596-685 prevents mutant exportation from the ER and 2) there is a pig Duox2 retention domain in the first intracellular loop. In addition, missense mutations of four cysteines (Cys-351, -370, -568, or -582) completely inhibited the emergence of pig Duox2-Q686X from the ER compartment, indicating their importance in Duox2 maturation.     

Identification of respective lysine donor and glutamine acceptor sites involved in factor XIIIa-catalyzed fibrin α chain cross-linking

Factor XIIIa-catalyzed ε-(γ-glutamyl)-lysyl bonds between glutamine and lysine residues on fibrin α and γ chains stabilize the fibrin clot and protect it from mechanical and proteolytic damage. The cross-linking of γ chains is known to involve the reciprocal linkages between Gln(398) and Lys(406). In α chains, however, the respective lysine and glutamine partners remain largely unknown. Traditional biochemical approaches have only identified the possible lysine donor and glutamine acceptor sites but have failed to define the respective relationships between them. Here, a differential mass spectrometry method was implemented to characterize cross-linked α chain peptides originating from native fibrin. Tryptic digests of fibrin that underwent differential cross-linking conditions were analyzed by high resolution Fourier transform mass spectrometry. Differential intensities associated with monoisotopic masses of cross-linked peptides were selected for further characterization. A fit-for-purpose algorithm was developed to assign cross-linked peptide pairs of fibrin α chains to the monoisotopic masses relying on accurate mass measurement as the primary criterion for identification. Equipped with hypothesized sequences, tandem mass spectrometry was then used to confirm the identities of the cross-linked peptides. In addition to the reciprocal cross-links between Gln(398) and Lys(406) on the γ chains of fibrin (the positive control of the study), nine specific cross-links (Gln(223)-Lys(508), Gln(223)-Lys(539), Gln(237)-Lys(418), Gln(237)-Lys(508), Gln(237)-Lys(539), Gln(237)-Lys(556), Gln(366)-Lys(539), Gln(563)-Lys(539), and Gln(563)-Lys(601)) on the α chains of fibrin were newly identified. These findings provide novel structural details with respect to the α chain cross-linking compared with earlier efforts.     

Intrinsic signaling function of APP as a novel target of three V642 mutations linked to familial Alzheimer's disease.

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.     

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.     

N-cadherin enhances APP dimerization at the extracellular domain and modulates Aβ production

Sequential processing of amyloid precursor protein (APP) by β- and γ-secretase leads to the generation of amyloid-β (Aβ) peptides, which plays a central role in Alzheimer's disease pathogenesis. APP is capable of forming a homodimer through its extracellular domain as well as transmembrane GXXXG motifs. A number of reports have shown that dimerization of APP modulates Aβ production. On the other hand, we have previously reported that N-cadherin-based synaptic contact is tightly linked to Aβ production. In the present report, we investigated the effect of N-cadherin expression on APP dimerization and metabolism. Here, we demonstrate that N-cadherin expression facilitates cis-dimerization of APP. Moreover, N-cadherin expression led to increased production of Aβ as well as soluble APPβ, indicating that β-secretase-mediated cleavage of APP is enhanced. Interestingly, N-cadherin expression affected neither dimerization of C99 nor Aβ production from C99, suggesting that the effect of N-cadherin on APP metabolism is mediated through APP extracellular domain. We confirmed that N-cadherin enhances APP dimerization by a novel luciferase-complementation assay, which could be a platform for drug screening on a high-throughput basis. Taken together, our results suggest that modulation of APP dimerization state could be one of mechanisms, which links synaptic contact and Aβ production.     

Measurement of thermodynamic parameters for hydrophobic mismatch 1: self-association of a transmembrane helix

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.     

Induced Dimerization of the Amyloid Precursor Protein Leads to Decreased Amyloid-�� Protein Production

The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by β- and γ-secretase lead to the generation of the amyloid-β (Aβ) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Aβ generation. However, these studies have relied on APP mutations within the Aβ sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Aβ production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Aβ sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Aβ levels in transfected N2a cells. Similar results were obtained with the direct precursor of β-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Aβ peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect γ-secretase processing and that dimerization is not required for Aβ production.The mechanism of β-amyloid protein (Aβ)² generation from the amyloid precursor protein is of major interest in Alzheimer disease research because Aβ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (1, 2). In the amyloidogenic pathway Aβ is released from the amyloid precursor protein (APP) (3) after sequential cleavages by β-secretase BACE1 (4–6) and by the γ-secretase complex (7, 8). BACE1 cleavage releases the large ectodomain of APP while generating the membrane-anchored C-terminal APP fragment (CTF) of 99 amino acids (C99). Cleavage of β-CTF by γ-secretase leads to the secretion of Aβ peptides of various lengths and the release of the APP intracellular domain (AICD) into the cytosol (9–11). The γ-secretase complex consists of at least four proteins: presenilin, nicastrin, Aph-I, and Pen-2 (12). Presenilin is thought to be the catalytic subunit of the enzyme complex (13), but how the intramembrane scission is carried out remains to be elucidated. Alternatively, APP can first be cleaved in the non-amyloidogenic pathway by α-secretase within the Aβ domain between Lys-16 and Leu-17 (14, 15). This cleavage releases the APP ectodomain (APPsα) while generating the membrane-bound C-terminal fragment (α-CTF) of 83 amino acids (C83). The latter can be further processed by the γ-secretase complex, resulting in the secretion of the small 3-kDa fragment p3 and the release of AICD.APP, a type I transmembrane protein (16) of unclear function, may act as a cell surface receptor (3). APP and its two homologues, APLP1 and APLP2, can dimerize in a homotypic or heterotypic manner and, in so doing, promote intercellular adhesion (17). In vivo interaction of APP, APLP1, and APLP2 was demonstrated by cross-linking studies from brain homogenates (18). To date at least four domains have been reported to promote APP dimerization; that is, the E1 domain containing the N-terminal growth factor-like domain and copper binding domain (17), the E2 domain containing the carbohydrate domain in the APP ectodomain (19), the APP juxtamembrane region (20), and the transmembrane domain (21, 22). In the latter domain the dimerization appears to be mediated by the GXXXG motif near the luminal face of the transmembrane region (21, 23). In addition to promoting cell adhesion, APP dimerization has been proposed to increase susceptibility to cell death (20, 24).Interestingly, by introducing cysteine mutations into the APP juxtamembrane region, it was shown that stable dimers through formation of these disulfide linkages result in significantly enhanced Aβ production (25). This finding is consistent with the observation that stable Aβ dimers are found intracellularly in neurons and in vivo in brain (26). Taken together, these results have led to the idea that APP dimerization can positively regulate Aβ production. However, other laboratories have not been able to confirm some of these observations using slightly different approaches (23, 27).To further address the question of how dimerization of APP affects cleavage by α-, β-, and γ-secretase, we chose to test this with a controlled dimerization system. Accordingly, we engineered a chimeric APP molecule by fusing a portion of the FK506-binding protein (FKBP) to the C terminus of APP such that the addition of the synthetic membrane-permeant bifunctional compound, AP20187, will induce dimerization of the APP-FKBP chimera in a controlled manner by binding to the FKBP domains. Using this system, efficient dimerization of APP up to 70% can be achieved in a time and concentration-dependent fashion. Our studies showed that controlled homodimerization of APP-FKBP leads to decreased total Aβ levels in transfected N2a cells. Homodimerization of the β-CTF/C99 fragment, the direct precursor of γ-secretase cleavage, showed comparable results. In addition, induced dimerization of APP did not lead to a modulation of different Aβ peptides as it was reported for GXXXG mutants within the transmembrane domain of APP (21).     

The E2 domains of APP and APLP1 share a conserved mode of dimerization

Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid β-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, one from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 ? resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.     

Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal transduction

We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two VPAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys(195)). VIP had a lower affinity for R188Q, R188L, K195Q, and K195I VPAC(1) receptors than for VPAC(1) receptors. [Asn(3)] VIP and [Gln(3)] VIP had lower affinities than VIP for VPAC(1) receptors but higher affinities for the mutant receptors; the two basic amino acids facilitated the introduction of the negatively charged aspartate inside the transmembrane domain. The resulting interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [Gln(3)] VIP were partial agonists at VPAC(1) receptors; 2/VIP did not fully activate the K195Q, K195I, R188Q, and R188L VPAC(1) receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn(3)] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q and R188L VPAC(1) receptors; 3/the [Asp(3)] negative charge did not contribute to the recognition of the VIP(1) antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27)] VIP ()/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC(1) receptor, the Asp(3) side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.     

Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.     

Bacterial and cell-free production of APP671-726 containing amyloid precursor protein transmembrane and metal-binding domains

More than half of the mutations associated with familiar Alzheimer’s disease have been found in the transmembrane domain of amyloid precursor protein (APP). These pathogenic mutations presumably influence the APP transmembrane domain structural and dynamic properties and result in its conformational change or/and lateral dimerization. Despite much data about the pathogenesis of Alzheimer’s disease, the initial steps of the pathogenesis remain unclear so far. For the investigation of the molecular basis of Alzheimer’s disease, we selected amyloid precursor protein fragment APP671-726 containing the transmembrane and metal-binding domains. This fragment is the substrate of the γ-secretase complex whose abnormal activity leads to the formation of amyloidogenic Aβ42 peptides. This work for the first time describes a highly effective cell-free APP671-726 production method and improved method of bacterial synthesis. Both methods yield milligram quantities of isotope-labeled protein for structural study by high resolution NMR spectroscopy in membrane mimicking milieus.     

Contribution of transmembrane regions to ATP-gated P2X2 channel permeability dynamics

ATP-gated P2X(2) channels undergo activation-dependent permeability increases as they proceed from the selective I(1) state to the I(2) state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X(2) channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X(2) channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I(1) state to the dilated I(2) state. Initial channel opening to the I(1) state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I(2) state. Ten residues appeared to perturb only the I(1)-I(2) transition (Phe(31), Arg(34), Gln(37), Lys(53), Ile(328), Ile(332), Ser(340), Gly(342), Trp(350), Leu(352)). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X(2) channels.     

The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein.

The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP.     

Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.     

An Alternative Secretase Cleavage Produces Soluble Alzheimer Amyloid Precursor Protein Containing a Potentially Amyloidogenic Sequence

Cell culture studies have shown that the Alzheimer amyloid precursor protein (APP) is secreted after full-length APP is cleaved by a putative secretase at the Lys16-Leu17 bond (secretase cleavage I) of the amyloid peptide sequence. Because this cleavage event is incompatible with amyloid production, it has been assumed that secreted APP cannot serve as a precursor of the amyloid depositions observed in Alzheimer's disease. Here we show that in neuronally differentiated PC12 cells and human kidney 293 cell cultures a portion of the secreted extracytoplasmic APP reacted specifically with both a monoclonal antibody recognizing amyloid protein residues Leu17-Val24 and a polyclonal antiserum directed against amyloid protein residues Ala21-Lys28. Furthermore, this APP failed to react with antisera recognizing the cytoplasmic domain of the full-length protein. These data indicate the presence of an alternative APP secretase cleavage site (secretase cleavage II), C-terminal to the predominant secretase cleavage I. Depending on the exact location of cleavage site II, potentially amyloidogenic secreted APP species may be produced.