Two structural and functional domains of MESD required for proper folding and trafficking of LRP5/6

How the endoplasmic reticulum (ER) folding machinery coordinates general and specialized chaperones during protein translation and folding remains an important unanswered question. Here, we show two structural domains in MESD, a specialized chaperone for LRP5/6, carry out dual functions. The chaperone domain forms a complex with the immature receptor, maintaining the β-propeller (BP) domain in an interaction competent state for epidermal growth factor-repeat binding. This promotes proper folding of the BP domain, causing a binding switch from the chaperone domain to the escort domain. The escort complex ensures LRP5/6 safe-trafficking from the ER to the Golgi by preventing premature ligand-binding. Inside the Golgi, the BP domain may contain a histidine switch, regulating MESD dissociation and retrieval. Together, we generate a plausible cell biology picture of the MESD/LRP5/6 pathway, suggesting that it is the specialized chaperones, MESD, that serves as the folding template to drive proper folding and safe trafficking of large multidomain proteins LRP5/6.     

Modulation of LRP6-mediated Wnt signaling by molecular chaperone Mesd

LRP6 is a Wnt coreceptor at the cell surface. Here, we report that a specialized molecular chaperone Mesd modulates LRP6-mediated Wnt signaling and how different LRP6 mutants exhibit differential effects on Wnt signaling. We found that overexpression of increasing amounts of the full-length LRP6 enhances Wnt signaling in a dose dependent manner only in the presence of a co-expression of the molecular chaperone Mesd, which promotes LRP6 folding and maturation to the cell surface. We also demonstrated that LRP6 mutant lacking the intracellular domain impedes LRP6 cell surface expression and Wnt signaling in a dominant-negative fashion by sequestering Mesd from promoting LRP6 folding. Our results present novel mechanisms by which Mesd and LRP6 modulate Wnt signaling.     

Do clustered β-propeller domains within the N-terminus of LRP1 play a functional role?

The six β-propellers located within the N-terminus of low density lipoprotein receptor-related protein 1 (LRP1) are arranged in two clusters that contain two and four β-propellers, respectively. Working with LRP1 deletion mutants, we found that randomly removing large segments of amino acid sequences did not affect the intracellular trafficking of LRP1 as long as the clustered β-propeller domains were retained. However, deletion mutants with crippled β-propeller clusters invariably exhibited retarded exit from the endoplasmic reticulum (ER). To determine potential functions of the clustered β-propellers, we generated a series of deletion mutants in which the β-propellers were systematically removed from the C-terminal end of the second cluster. The resulting minireceptors, designated LRPβ1–6, β1–5, β1–4, β1–3, and β1–2 containing decreasing numbers of the β-propellers, were stably expressed in LRP1-null CHO cells. Binding/degradation assays with receptor-associated protein or α₂-macroglobulin showed that removing one or more β-propellers had little effect on binding or degradation of these ligands. However, minireceptors containing odd number of β-propellers (i.e., LRPβ1–3 and β1–5) showed prolonged retention within the ER and remained endoglycosidase H-sensitive, whereas minireceptors containing even number of β-propellers (i.e., LRPβ1–2, β1–4 and β1–6) exited ER at variable rates. Cell surface biotinylation experiments showed that LRPβ1–3 was absent from the cell surface. Prolonged retention of LRPβ1–3 within the ER was accompanied by increased association with molecular chaperone Grp78/Bip. These results suggest that the clustered β-propellers may play a role in folding and intracellular trafficking of LRP1.     

The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.     

Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein

The low density lipoprotein receptor-related protein (LRP) is a approximately 600-kDa multifunctional endocytic receptor that is highly expressed in the brain. LRP and its ligands apolipoprotein E, alpha2-macroglobulin, and beta-amyloid precursor protein (APP), are genetically linked to Alzheimer disease and are found in characteristic plaque deposits in brains of patients with Alzheimer disease. To identify which extracellular domains of LRP interact with APP, we used minireceptors of each of the individual LRP ligand binding domains and assessed their ability to bind and degrade a soluble APP fragment. LRP minireceptors containing ligand binding domains II and IV, but not I or III, interacted with APP. To test whether APP trafficking is directly related to the rapid endocytosis of LRP, we generated stable Chinese hamster ovary cell lines expressing either a wild-type LRP minireceptor or its endocytosis mutants. Chinese hamster ovary cells stably expressing wild-type LRP minireceptor had less cell surface APP than pcDNA3 vector-transfected cells, whereas those stably expressing endocytosis-defective LRP minireceptors accumulated APP at the cell surface. We also found that the steady-state levels of the amyloid beta-peptides (Abeta) is dictated by the relative expression levels of APP and LRP, probably reflecting the dual roles of LRP in both Abeta production and clearance. Together, these data establish a relationship between LRP rapid endocytosis and APP trafficking and proteolytic processing to generate Abeta.     

Do clustered beta-propeller domains within the N-terminus of LRP1 play a functional role?

The six beta-propellers located within the N-terminus of low density lipoprotein receptor-related protein 1 (LRP1) are arranged in two clusters that contain two and four beta-propellers, respectively. Working with LRP1 deletion mutants, we found that randomly removing large segments of amino acid sequences did not affect the intracellular trafficking of LRP1 as long as the clustered beta-propeller domains were retained. However, deletion mutants with crippled beta-propeller clusters invariably exhibited retarded exit from the endoplasmic reticulum (ER). To determine potential functions of the clustered beta-propellers, we generated a series of deletion mutants in which the beta-propellers were systematically removed from the C-terminal end of the second cluster. The resulting minireceptors, designated LRPbeta1-6, beta1-5, beta1-4, beta1-3, and beta1-2 containing decreasing numbers of the beta-propellers, were stably expressed in LRP1-null CHO cells. Binding/degradation assays with receptor-associated protein or alpha2-macroglobulin showed that removing one or more beta-propellers had little effect on binding or degradation of these ligands. However, minireceptors containing odd number of beta-propellers (i.e., LRPbeta1-3 and beta1-5) showed prolonged retention within the ER and remained endoglycosidase H-sensitive, whereas minireceptors containing even number of beta-propellers (i.e., LRPbeta1-2, beta1-4 and beta1-6) exited ER at variable rates. Cell surface biotinylation experiments showed that LRPbeta1-3 was absent from the cell surface. Prolonged retention of LRPbeta1-3 within the ER was accompanied by increased association with molecular chaperone Grp78/Bip. These results suggest that the clustered beta-propellers may play a role in folding and intracellular trafficking of LRP1.     

Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains

Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts .     

Mutational analysis of the FXNPXY motif within LDL receptor-related protein 1 (LRP1) reveals the functional importance of the tyrosine residues in cell growth regulation and signal transduction

LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.     

Domain interactions of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. alpha-FLAG or alpha-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the alpha-FLAG but not alpha-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the alpha-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor.     

gamma-Secretase-mediated release of the low density lipoprotein receptor-related protein 1B intracellular domain suppresses anchorage-independent growth of neuroglioma cells

The low density lipoprotein receptor related protein 1B (LRP1B) is a large endocytic receptor that was first identified as a candidate tumor suppressor gene. In the current investigation we demonstrate that LRP1B undergoes regulated intramembrane proteolysis in a gamma-secretase-dependent process. The released intracellular domain (ICD) then translocates to the nucleus via a nuclear localization signal that is present within this domain. ICD release first requires shedding of the LRP1B ectodomain, which appears to be catalyzed by a member of the metalloproteinase family. Employing site-directed mutagenesis studies, we identified lysine residues 4432 and 4435 and arginine 4442 as key amino acids important for ectodomain shedding of LRP1B. We also demonstrate that an LRP1B minireceptor as well as the ICD domain alone suppresses anchorage-independent growth of LRP1B-deficient neuroglioma cells (H4 cells). Interestingly, abrogating ectodomain shedding resulted in a loss of the ability of LRP1B minireceptors to suppress anchorage-independent growth. Together, these studies reveal that LRP1B has tumor suppression function that is mediated by proteolytic processing of the receptor resulting in ICD release.     

The C-Terminal Region Mesd Peptide Mimics Full-Length Mesd and Acts as an Inhibitor of Wnt/β-Catenin Signaling in Cancer Cells

While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.     

Striking differences of LDL receptor-related protein 1B expression in mouse and human

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer.     

Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.     

Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6

LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.     

Slow endocytosis of the LDL receptor-related protein 1B: implications for a novel cytoplasmic tail conformation

The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting that LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.     

Proteasome regulates the delivery of LDL receptor-related protein into the degradation pathway

The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin-proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway.     

The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing

The low-density lipoprotein receptor-related protein (LRP) has recently been implicated in numerous intracellular signaling functions, as well as in Alzheimer's disease pathogenesis. Studies have shown that the beta-amyloid precursor protein (APP) interacts with LRP and that this association may impact the production of amyloid beta-protein (Abeta). In this report, we provide evidence that LRP regulates trafficking of intracellular proteins independently of its lipoprotein receptor functions. We show that in the absence of LRP, Abeta production, APP secretion, APP internalization, turnover of full-length APP and stability of APP C-terminal fragments are affected. Importantly, these changes are not APP isoform dependent. Using deletion constructs, the critical region in LRP that modulates APP processing was mapped to a seven peptide domain around the second NPXY domain (residues 4504-4510). Therefore, we propose a model by which LRP functionally modulates APP processing, including those steps critical for Abeta production, through interactions of the cytosolic domains.     

The low density lipoprotein receptor-1, LRP1, interacts with the human frizzled-1 (HFz1) and down-regulates the canonical Wnt signaling pathway

Members of the low density lipoprotein receptor family (LDLR), LRP5/6, were shown to interact with the Frizzled (Fz) receptors and to function as Wnt coreceptors. Here we show that mLRP4T100, a minireceptor of LRP1, another member of the LDLR family, interacts with the human Fz-1 (HFz1), previously shown to serve as a receptor transmitting the canonical Wnt-3a-induced signaling cascade. However, in contrast to LRP5/6, mLRP4T100, as well as the full-length LRP1, did not cooperate with HFz1 in transmitting the Wnt-3a signaling but rather repressed it. mLRP4T100 inhibitory effect was displayed also by endocytosis-defective mLRP4T100 mutants, suggesting that LRP1 repressive effect is not attributable to LRP1-mediated enhanced HFz1 internalization and subsequent degradation. Enforced expression of mLRP4T100 decreased the capacity of HFz1 cysteine-rich domain (CRD) to interact with LRP6, in contrast to HFz1-CRD/Wnt-3a interaction that was not disrupted by overexpressing mLRP4T100. These data suggest that LRP1, by sequestering HFz1, disrupts the receptor/coreceptor complex formation, leading to the repression of the canonical Wnt signaling. Thus, this study implies that the ability to interact with Fz receptors is shared by several members of the LDLR family. However, whereas some members of the LDLR family, such as LRP5/6, interact with Fz and serve as Wnt coreceptors, others negatively regulate Wnt signaling, presumably by sequestering Fz.     

Low density lipoprotein receptor-related protein 1 dependent endosomal trapping and recycling of apolipoprotein E

Background

Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling.

Principal Findings

Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion.

Conclusion

We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.     

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.