C-terminal 37 residues of LRP promote the amyloidogenic processing of APP independent of FE65

The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid β protein (Aβ), a small peptide derived from β- and γ-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that the Low-density lipoprotein receptor-related protein (LRP) plays a pivotal role in the trafficking of APP and generation of Aβ. In particular, we recently showed that the soluble cytoplasmic tail of LRP (LRP-ST) without a membrane tether was sufficient to promote Aβ generation. In this study, we demonstrate that the last 37 residues of LRP cytoplasmic tail (LRP-C37) lacking the NPxY motifs and FE65 binding mediate the core pro-amyloidogenic activity of LRP-ST. Moreover, we show that the conserved dileucine motif within the LRP-C37 region is a key determinant of its Aβ promoting activity. Finally, results from a yeast two-hybrid screen using LRP-C37 region as bait reveal four new LRP-binding proteins implicated in intracellular signalling and membrane protein trafficking. Our findings indicate that the LRP-C37 sequence represents a new protein-binding domain that may be useful as a therapeutic target and tool to lower Aβ generation in AD.


Introduction
Amyloid ␤ protein (A␤) accumulation in the brain is an early toxic event in the pathogenesis of Alzheimer's disease (AD). A␤ is produced by proteolytic processing by ␤and ␥-secretase cleavages of the amyloid precursor protein (APP). The low-density lipoprotein receptor-related protein (LRP) is a large type I transmembrane (TM) protein that functions as a multi-functional endocytosis receptor for a diverse array of extracellular ligands, including lipoproteins, proteases, proteinase inhibitor complexes, matrix proteins, bacterial toxins, viruses, intracellular proteins and growth factors [1]. Within the central nervous system (CNS), LRP is highly expressed in neuronal cell bodies and dendritic processes. LRP is synthesized as a 600-kD precursor protein that is subsequently cleaved in the trans-Golgi compartment by furin to generate a large 515-kD ␣-chain and a smaller 85-kD TM ␤-chain that remain noncovalently linked [2].
Several lines of studies implicate a role for the LRP pathway in AD pathogenesis. First, LRP is genetically associated with late-onset AD in the majority of Caucasian and Asian populations examined [3][4][5][6]. Second, three of its key ligands, apoE, ␣2-macroglobulin and APP are also genetically associated with AD and found in senile plaques in the brains of AD patients along with LRP [7][8][9][10]. Third, LRP mediates the binding and clearance of A␤ complexes bound to apoE or ␣2-macroglobulin in cultured cells and in the brain [11,12]. LRP also directly associates with A␤ and plays a crucial role in brain efflux of A␤ isoforms at the bloodbrain barrier [13]. Finally, circulating secreted LRP can function as a 'peripheral sink' to reduce A␤ levels in brain [13,14]. These findings therefore support a model in which LRP plays an important role in extracellular A␤ uptake and removal.
In an opposing effect, however, LRP also influences APP trafficking and cellular distribution such that processing to A␤ and its extracellular release is enhanced [15][16][17][18][19]. The loss of LRP or treatment of receptor-associated protein (RAP), an antagonist of all known LRP ligands, substantially reduces A␤ release, a phenotype that is reversed when full-length (LRP-FL) or truncated LRP is transfected in LRP-deficient cells [16,17]. Specifically, LRP-CT lacking the extracellular ligand binding regions but containing the TM domain and the cytoplasmic tail (CT) is capable of rescuing amyloidogenic processing of APP and A␤ release in LRP deficient cells [16]. Moreover, LRP soluble tail (LRP-ST) lacking the TM domain and only containing the CT of LRP is sufficient to enhance A␤ secretion [18]. This activity of LRP-ST is achieved by promoting APP/Beta APP clearing enzyme 1 (BACE1) interaction and facilitating the targeting of APP to lipid rafts, cholesterol-rich membrane microdomains enriched in both ␤and ␥-secretase activities [19]. Similar to APP, LRP also undergoes BACE1-mediated ectodomain cleavage and presenilin-dependent intramembrane proteolysis to release the LRP intracellular domain (LICD) [20,21]. Thus, the pro-amyloidogenic action LRP-ST not only reflects an activity of a physiological product but also may in part underlie the manner in which LRP normally promotes A␤ generation.
The short 100 amino acid CT contains two NPxY endocytosis motifs and binds a number of cytoplasmic adaptor and scaffold proteins, such as FE65, Disabled-1 (Dab1), Shc, and JIP-1 and -2, probably through the second NPxY motif [22][23][24]. Of particular significance is FE65, which constitutes a physical link between APP and LRP in regulating sAPP release [24][25][26]. Initially, we had hypothesized that one or more NPxY domains in LRP-ST might underlie the pro-amyloidogenic processing of APP, since all known LRP-interacting cytoplasmic adaptor proteins bind to NPxY-based motifs. In this study, however, we show that the last 37 C-terminal residues of LRP (LRP-C37) robustly promote A␤ production independent of FE65 and constitute a protein-binding domain for several new LRP-binding proteins.

Yeast two-hybrid screen
To subclone the bait constructs for use in the two-hybrid system [30], PCR- and LRP-ST61-97 constructs were tested for self-activation of the His3 reporter gene in the absence of prey by plating transformed yeast on selective dropout plates lacking leucine and tryptophan (SD-LT). A high-stringency protocol was used to screen the cDNA library from 17-day-old mouse embryo fused with the Gal4 transactivation domain constructed in the pACT2 plasmid (Clontech). The yeast two-hybrid screening was performed in AH109 Saccharomyces cerevisiae that contains three reporters ADE2, HIS3 and MEL1. The bait plasmid was initially transformed into AH109, and growth was selected in SD dropout plates lacking leucine (SD-L). This yeast strain expressing LRP-ST61-97 was then used for sequential transformation of the 100 g of cDNA library and plated them on SD-dropout plates lacking adenine, histidine, leucine and tryptophan [30]. Yeast was allowed to grow for 72 hrs at 30°C before His + cells were scored and an X-gal

The C-terminal 37 residues of LRP-ST (ST61-97) are sufficient to robustly enhance A␤ production
We previously reported that deletion of the proximal or distal NPxY domains alone had no effect on the capacity of LRP-ST to promote A␤ production in stably transfected CHO cells.  . 1B and C). Surprisingly, deletion of both NPxY motifs (ST1-97⌬1⌬2) demonstrated that NPxY motifs are not required for LRP-ST-mediated A␤ promotion ( Fig. 1B and C). In fact, the ST1-97⌬1⌬2 mutant elevated A␤ levels beyond that of LRP-ST1-97, suggesting an inhibitory effect of the NPxY motifs in the context of LRP-ST. ST45-97, which contains the second NPxY motif, also elevated A␤ levels as effectively as ST1-97⌬1⌬2 ( Fig. 1B and C). Finally, ST-61-97, the C-terminal 37 residues of LRP (LRP-C37) lacking both NPxY motifs, was sufficient to robustly elevate A␤ secretion by an average of more than fourfold beyond vector controls in multiple experiments ( Fig. 1B and C). The increase in A␤ by ST61-97 was accompanied by a reduction in sAPP␣ without altering total sAPP levels ( Fig. 1B), indicating an increase in ␤-secretase at the expense of ␣-secretase cleavage. Interestingly, ST61-97 transfection also led to a consistent decrease in the level of APP-C-terminal fragment (CTFs) (Fig. 1D)

FE65/APP binding is not required for LRP-ST-mediated increase in A␤
Our finding that the NPxY motifs are not required for LRP-STmediated pro-amyloidogenic activity was surprising, in part, because all known LRP-interacting cytoplasmic proteins are thought to bind through the NPxY motifs [22][23][24]. Of particular significance, FE65 was shown to enhance the interaction between APP and LRP, as the N-and C-terminal domains of FE65 can bind to APP and LRP independently [24][25][26]. To test whether FE65 is required for LRP-ST-mediated effect on A␤ production, we employed the APP-GA mutant in which the Gly737 was mutated to alanine. This mutant was previously shown neither to alter APP endocytosis nor A␤ secretion [31]. However, the APP-GA mutant could not bind FE65 and thus completely failed to transactivate the APP intracellular domain (AICD) in a GAL4-based nuclear reporter assay [32]. Indeed, we confirmed that the APP-GA mutant neither binds FE65 nor alters A␤ production in transient transfection experiments ( Fig. 2A and B). Likewise, cotransfection of APP-GA mutant with ST1-97 or ST61-97 showed a similar elevation in A␤ secretion as seen with wild-type APP (Fig. 2C). Thus, these data clearly demonstrate that LRP-ST neither requires the NPxY motifs nor FE65/APP binding to promote A␤ production.   [33]. To test whether the conserved dileucine sequence might underlie the proamyloidogenic activity of ST61-97, we deleted the sequences DEKR and glutamate-leucine-leucine (ELL) from ST61-97. In transient transfection experiments, the loss of ELL nearly completely abrogated the ST-61-97-mediated A␤ increase (Fig. 3A), indicating that the dileucine motif is a key determinant of the A␤ increasing activity. The deletion of the DEKR sequence only minimally reduced A␤ generation by approximately 25% (not shown). At the same time, however, deletion of the ELL sequence had no effect on the reduction in APP CTFs by ST61-97 (Fig. 3B), suggesting the presence of other protein binding motif(s) within LRP-ST61-97 important for different aspects of APP trafficking.

Identification of LRP-ST61-97 (C37) interacting proteins via yeast two-hybrid screen
The A␤ promoting action of LRP-ST61-97 is likely achieved by the recruitment of one or more as yet unknown interacting proteins. Thus, we next conducted a yeast two-hybrid screen of a mouse E17 cDNA library using the LRP-ST61-97 sequence as bait.
Although two-hybrid screen is a very powerful system to identify novel protein interactions [30], LRP-ST has been shown to selfactivate in a yeast two-hybrid screen [23,24], and therefore, it is not suitable to be used as bait in this assay. Thus, we initially tested whether LRP-ST61-97 might also self-activate. As expected, cotransformation of LRP-ST1-97 and empty prey plasmid pGAD resulted in growth of blue colonies in the presence of X-gal, indicating self-activation (Fig. 4A). However, colonies cotransformed with LRP-61-97 and empty pGAD did not turn blue in the presence of X-gal (Fig. 4B), demonstrating that LRP-ST61-97 does not self-activate and is suitable for use as bait. A conventional two-hybrid screen results in many positive clones, many of which might be false-positives. Therefore, we used a stringent protocol using quadruple dropout plates to screen the cDNA library to minimize false-positive detection. Out of 2 ϫ 10 6 clones screened, we identified 18 colonies that grew in quadruple dropout plates and turned blue in the presence of X-gal. Sequence analysis revealed four partial cDNA inserts fused in frame with Gal4, all of which were represented in multiple colonies. These included Filamin A, a previously uncharacterized SH3/dbl repeat RhoGTPase domain containing protein (shortened as SH3 protein here; Accession: AK030242), Ran-binding protein M (RanBPM) and Snapin. Because it is possible that more than one plasmid may be present in a given yeast colony, the results were reconfirmed after isolation of the plasmids from the yeast. Indeed cotransformation of the aforementioned isolated prey plasmids with LRP-ST61-97 bait plasmid all resulted in growth of colonies with high ␤-gal activity in quadruple dropout plates in the presence of X-gal. However, yeast cotransformed with the prey plasmid and empty bait plasmid failed to grow or show ␤-gal activity under identical conditions (Fig. 5A and B), indicating the specificity of these interactions in the yeast two-hybrid system.

Coimmunoprecipitation of LRP-ST61-97-(C37)-binding proteins to LRP in cultured cells
To demonstrate that the clones identified by two-hybrid screens can interact with LRP in cultured cells, we initially transfected myc-Filamin A or myc-SH3 protein (AK030242) in HEK293T cells, and 1% NP40 lysates were subjected to immunoprecipitation for myc (9E10 antibody). Indeed, 9E10 immune complexes specifically contained not only exogenously transfected LRP-CT but also endogenous LRP ␤-chain only when myc-Filamin A or myc-SH3 protein cDNAs but not when myc empty vector were cotransfected ( Fig. 5C and D). Thus, coimmunoprecipitation experiments confirmed that the clones identified by two-hybrid screens are indeed true binding partners of LRP-C37 region. These results taken together demonstrate that the LRP-C37 region is a novel protein-protein interaction domain of LRP CT that regulates A␤ generation.

Discussion
Accumulation of A␤ is a critical event in the pathogenesis of AD. LRP and three of its key ligands, APP, ␣2-macroglobulin and ApoE are genetically associated or linked to AD [4,7,8,10]. LRP is known to play an important role in extracellular A␤ uptake and removal [11][12][13][14]. At the same time, LRP has been shown to promote A␤ generation by altering the trafficking and processing of APP. Like APP, LRP also undergoes ectodomain cleavage by BACE1 and a phorbol ester-activated protease [20,21]. This, in turn, leads to presenilin-dependent intramembrane proteolysis and release of the LICD [20, 21], a polypeptide that is essentially identical to LRP-ST. Previously, we showed that LRP soluble cytoplasmic tail (LRP-ST) was sufficient to enhance A␤ generation by promoting APP localization to lipid rafts [19]. The CT of LRP has been shown to interact with a number of adapter proteins, including Dab, FE65, JIP-1/2, PSD-95, SEMCAP-1, OMP25 and Shc [22][23][24]. With the exception of FE65 that can bind to both NPxY-based motifs in LRP [26], all other aforementioned proteins only interact with the second NPxY motif. Because LRP CT self-activates in yeast two-hybrid assays, most of these interactions were validated by GST-fusion protein pull-down assays based on yeast two-hybrid screens of homologous CTs of other LDL receptor family members [23,24]. We had initially hypothesized that NPxY motifs within LRP-ST are responsible for increased A␤ generation, since all the known adaptor proteins bind to these motifs. Among all the known LRP-interacting adaptor proteins, FE65 is the only protein that has clearly been demonstrated to simultaneously bind to both LRP and APP and functionally bridge their interaction [24,26]  cells stably transfected with APP [34]. In HEK293T cells, however, conflicting data were reported as to whether FE65 increases or decreases A␤ generation [35,36]. Another study showed that expression of human FE65 in a mouse model of AD led to reduced A␤ load as well reduced APP CTFs and sAPP␣ [37]. While these studies demonstrated that overexpression of FE65 can alter APP processing in different ways, our study clearly indicated that FE65 binding to either APP or LRP-ST are not required for LRP-ST-mediated increase in A␤ production. Instead, we demonstrated that LRP-ST61-97 lacking NPxY motifs constitute the core A␤ promoting region of LRP-ST and that the D/ExxxLL dileucine motif is a key determinant of its proamyloidogenic activity.
Within the C37 region of LRP, it has been shown that the dileucine motif plays a minor role in LRP endocytosis [38]. The triple serine phosphorylation sequence (SRHSLAS) within the C37 region is known to influence the affinity of adaptor proteins to the second NPxY motif [39]. Aside from the preceding observations, we are not aware of any other data regarding the LRP-C37 region. Unlike previous studies that used self-activating sequences surrounding the second NPxY motif as bait [23,24] (Table 1). RanBPM appears to be a multi-functional protein that can bind a number of different cell surface receptors, including ␤2-integrin and Met receptor tyrosine kinase, and mediating their signal transduction events (Table 1) [40,41]. Filamin A is a homodimeric F-actin cross-linking protein involved in organization of the cytoskeleton [42], and therefore, may be involved scaffolding the attachment of LRP to the cytoskeleton (Table 1). Interestingly, mutations in Filamin A result in a variety of human developmental diseases, including neuronal migration disorders [42]. Snapin was originally discovered as a SNAP-25 interacting protein in SNARE complexes that promotes the association of SNAP-25 with synaptotagmin via PKA-mediated phosphorylation [43]. It also interacts with another related SNARE protein expressed in both neuronal and non-neuronal cells, SNAP-23 [44]. Thus, Snapin has been proposed to function in membrane fusion events from ER to plasma membrane and in exocytosis of secretory vesicles (Table 1). Whether any of these aforementioned proteins mediate the LRP-ST-induced A␤ generation requires further detailed investigation. Nonetheless, the specific interactions of LRP to these proteins suggest a new role of the LRP-C37 region in signal transduction and protein trafficking both in the context of the TM receptor as well as the soluble intracellular domain.
The cellular and biochemical mechanisms by which A␤ are generated are critical for designing therapeutic strategies for AD. Inhibiting ␤or ␥-secretase activities are obvious therapeutic strategies. ␥-secretase inhibitors are highly effective in lowering A␤ but also have unintended side effects, such as inhibition of Notch cleavage, an important activity for neurogenesis and maintenance of multiple stem cell populations [45]. Highly specific ␤-secretase inhibitors are not yet available for therapeutic purposes, and the potential side effects are uncertain, especially in light of new observations that BACE1 controls the myelination of CNS and peripheral axons [46]. In this study, we found that