Member Of Low-density Lipoprotein Receptor Family Research Articles

Differential Effects of Platelet Factor 4 (CXCL4) and Its Non-Allelic Variant (CXCL4L1) on Cultured Human Vascular Smooth Muscle Cells.

Platelet factor 4 (CXCL4) is a chemokine abundantly stored in platelets. Upon injury and during atherosclerosis, CXCL4 is transported through the vessel wall where it modulates the function of vascular smooth muscle cells (VSMCs) by affecting proliferation, migration, gene expression and cytokine release. Variant CXCL4L1 is distinct from CXCL4 in function and expression pattern, despite a minor three-amino acid difference. Here, the effects of CXCL4 and CXCL4L1 on the phenotype and function of human VSMCs were compared in vitro. VSMCs were found to constitutively express CXCL4L1 and only exogenously added CXCL4 was internalized by VSMCs. Pre-treatment with heparin completely blocked CXCL4 uptake. A role of the putative CXCL4 receptors CXCR3 and DARC in endocytosis was excluded, but LDL receptor family members appeared to be involved in the uptake of CXCL4. Incubation of VSMCs with both CXCL4 and CXCL4L1 resulted in decreased expression of contractile marker genes and increased mRNA levels of KLF4 and NLRP3 transcription factors, yet only CXCL4 stimulated proliferation and calcification of VSMCs. In conclusion, CXCL4 and CXCL4L1 both modulate gene expression, yet only CXCL4 increases the division rate and formation of calcium-phosphate crystals in VSMCs. CXCL4 and CXCL4L1 may play distinct roles during vascular remodeling in which CXCL4 induces proliferation and calcification while endogenously expressed CXCL4L1 governs cellular homeostasis. The latter notion remains a subject for future investigation.

Abstract 102: Apolipoprotein E Receptor 2 In Endothelium Affords Protection From Both Atherosclerosis And Insulin Resistance

Introduction: Apolipoprotein E Receptor 2 (ApoER2) is an LDL receptor family member that mediates the actions of apolipoprotein E (apoE) and other ligands. The LRP8 gene that encodes ApoER2 is a major gene locus for premature atherosclerosis and myocardial infarction. Studies were designed to determine how ApoER2 in endothelial cells impacts cardiovascular and metabolic health. Hypothesis: Based on prior cell culture studies, the hypothesis was that endothelial ApoER2 is atheroprotective. How endothelial ApoER2 impacts adiposity or glucose homeostasis is more difficult to predict. Methods: In vivo studies were done in control ApoER2 floxed mice (ApoER2 fl/fl ) and mice deficient in ApoER2 in endothelial cells (ApoER2 ΔEC ). Studies of endothelial cell insulin uptake and transcytosis were performed in human aortic and skeletal muscle endothelial cells (HAEC and HSMEC). Results: Atherosclerotic lesion severity on LDLR -/- background was increased in ApoER2 ΔEC mice compared to ApoER2 fl/fl , and this was not related to differences in circulating lipids. Intravital microscopy revealed that leukocyte-endothelial cell adhesion is increased in ApoER2 ΔEC , and vascular TNFα and IL-1β expression was exaggerated. There was no impact of endothelial ApoER2 silencing on adiposity. However, standard chow-fed ApoER2 ΔEC mice displayed marked glucose intolerance and insulin resistance. Both pancreatic insulin secretion and hepatic insulin sensitivity were normal. Alternatively, in ApoER2 ΔEC mice skeletal muscle glucose disposal was decreased by 41%, and this was due to a 55% fall in insulin delivery to muscle. Using human apoE3 ex vivo, apoE stimulated a 279% increase in insulin uptake in HAEC, and the finding was confirmed in HSMEC. Insulin transcytosis rose 209% with apoE, and apoE enhancement of insulin transport was fully ApoER2-dependent. Conclusions: Endothelial ApoER2 affords atheroprotection by decreasing leukocyte-endothelial cell adhesion and inflammatory gene expression in the vessel wall. Endothelial ApoER2 also has antidiabetic action, and this is due to the promotion of endothelial insulin transport to skeletal muscle. Targeting of the related mechanisms may provide protection from both cardiovascular and metabolic disease.

The role and mechanism of ubiquitin ligase IDOL in cholesterol metabolism

<p indent="0mm">Cholesterol homeostasis plays an important role in maintaining proper cellular and systemic functions. A means for the cell to acquire cholesterol is low-density lipoprotein receptor (LDLR)-mediated uptake of low-density lipoprotein. Dysfunction of LDLR leads to hypercholesterolemia that ultimately contributes to cardiovascular disease such as atherosclerosis. The stability of LDLR protein is regulated by proprotein convertase subtilisin/kexin type 9 and inducible degrader of the LDLR (IDOL). As an E3 ubiquitin ligase, IDOL is subjected to transcriptional regulation by liver X receptor. It can ubiquitylate LDLR and leads to its degradation in the lysosome. IDOL also mediates the degradation of other LDLR family members including very low-density lipoprotein receptor and apolipoprotein E receptor 2, thus regulating apolipoprotein E levels in the brain. This review summarizes the most recent knowledge about IDOL, from the structure, the mechanism on regulating LDLR, to the transcriptional and post-transcriptional regulation of IDOL, as well as its roles in cardiovascular disease and Alzheimer’s disease. These research advances may provide potential therapeutic targets for treating the aforementioned diseases.

LRP1 regulates food intake and energy balance in GABAergic neurons independently of leptin action.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a member of LDL receptor family that plays a key role in systemic glucose and lipid homeostasis. LRP1 also regulates energy balance in the hypothalamus by mediating leptin's anorexigenic action, although the underlying neurocircuitry involved is still unclear. Because GABAergic neurons are a major mediator of hypothalamic leptin action, we studied the role of GABAergic LRP1 in energy balance and leptin action using mice lacking LRP1 in Vgat- or AgRP-expressing neurons (Vgat-Cre; LRP1loxP/loxP or AgRP-Cre; LRP1loxP/loxP). Here, we show that LRP1 deficiency in GABAergic neurons results in severe obesity in male and female mice fed a normal-chow diet. This effect is most likely due to increased food intake and decreased energy expenditure and locomotor activity. Increased adiposity in GABAergic neuron-specific LRP1-deficient mice is accompanied by hyperleptinemia and hyperinsulinemia. Insulin resistance and glucose intolerance in these mice are occurred without change in body weight. Importantly, LRP1 in GABAergic neurons is not required for leptin action, as evidenced by normal leptin's anorexigenic action and leptin-induced hypothalamic Stat3 phosphorylation. In contrast, LRP1 deficiency in AgRP neurons has no effect on adiposity and caloric intake. In conclusion, our data identify GABAergic neurons as a key neurocircuitry that underpins LRP1-dependent regulation of systemic energy balance and body-weight homeostasis. We further find that the GABAergic LRP1 signaling pathway modulates food intake and energy expenditure independently of leptin signaling and AgRP neurons.

A Novel Platelet-Targeting Fibrinolytic Strategy: Endocytosed Urokinase By Megakaryocytes Is Stored in α-Granules and Is Functional In Vivo in a Murine Model of Thrombosis

We have been interested in developing fibrinolytic agents that have a prolonged half-life and can selectively prevent new thrombi from forming without destabilizing established hemostatic clots. We believe that platelet-targeting of urokinase plasminogen activator (uPA) might be able to achieve this goal. Two approaches had been tried in the past: In the first, we had generated chimeric drugs consisting of a platelet-targeting scFvs fused to a thrombin activatable low molecular weight (LMW) uPA (uPA-T). Infused scFv/uPA-T bound to circulating platelets that targeted the fusion within nascent thrombi where significant amounts of thrombin are generated to activate surface-bound scFv/uPA-T. In contrast, mature thrombi are spared because scFv/uPA-T platelets are not activated on the "shell", nor do they penetrate the "core" where thrombin might be present. Murine studies affirmed αIIbβ3-directed scFv/uPA-T provided thromboprophylaxis, but therapeutic doses caused significant thrombocytopenia in murine and baboon models. We therefore considered a second approach: ectopic storage of uPA during megakaryopoiesis. We found that scuPA was stored in platelet α-granules of transgenic mice that ectopically express single-chain uPA (scuPA) and did not cause systemic fibrinolysis. Infusion of such "scuPA platelets" into wildtype mice was highly effective at preventing new thrombi from developing. We now wish to develop this strategy further by taking advantage of ongoing efforts by others to generate in vitro-grown megakaryocytes (Mks) and platelets that can be modified to express uPA near the point-of-care and then infused into patients, bypassing the need to establish uPA-expressing hematopoietic cell lines. We have previously shown that Mks express low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) during their maturation, whereas the platelets that are released do not. We now asked whether in vitro-grown Mks beginning with CD34+ hematopoietic cells would endocytose uPA as seen in other cell types. We show that Mks internalize and store scuPA (scu-Mks, Fig. 1A), as well as LMW uPA and uPA-T (not shown) after overnight incubation. Endocytosed uPA is found within membrane bound structures that partly colocalize with von Willebrand factor (VWF)-positive granules, suggesting uPA is sorted to α-granules (Fig. 1A). Uptake is blocked by receptor-associated protein (RAP), which inhibits endocytosis by LDL receptor family members, including LRP1. We then studied whether platelets with endocytosed scuPA (scuPA-Plts) prevent nascent thrombus development in immunodeficient NOD-scid IL2rγnull (NSG) mice that are also homozygous for VWFR1326H (a single amino acid substitution that switches species selectivity of VWF so that it binds human platelet glycoprotein (GP) Ib/IX receptor rather than mouse GPIb/IX). These mice show a mild bleeding diathesis in a Rose Bengal photochemical carotid artery injury model unless they are infused with human Mks, which we have shown go on to release functional platelets in the recipient animal in its pulmonary capillary bed over the ensuing several hours (Fig. 1B). Thus, when 106 Mks not exposed to uPA were infused so that ~1-10% of circulating platelets were human, thrombi developed in this model; however similar infusion of scuPA-Mks did not occlude (Fig. 1B). In tail clip studies in the same genotypic mice, where the mice were first corrected with human platelets (Fig. 1C) followed 10 min later by scuPA-Mks, rebleeding did not develop when given a similar dose of scuPA-Mks that prevented thrombosis in the photochemical injury model. These studies suggest that Mks internalize biologically relevant concentrations of uPA through a process likely to involve LRP1. Whether ex vivo loading of Mks with uPA can serve as model for point-of-care therapeutics for thromboprophylaxis and diverse other hematologic and non-hematologic indications should be explored. Disclosures No relevant conflicts of interest to declare.

A Novel Approach for Generating Platelet-Delivered FVIII: Role of Transient LRP1 Expression during Megakaryopoiesis

Human platelets endocytose coagulation factor (F) V into their α-granules, in part, via low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). In contrast to humans, mouse FV is endogenously expressed during megakaryopoiesis and stored in α-granules. The FV-related coagulation factor FVIII is not endogenously expressed in megakaryocytes (Mks) and thought not to be endocytosed by human or murine Mks/platelets. The lack of FVIII endocytosis is surprising as LRP1 on multiple cell types is involved in FVIII uptake and clearance. We and others have shown that FVIII can be ectopically expressed during human or murine megakaryopoiesis and stored in α-granules. These platelets are effective in the delivery of FVIII to sites of vascular injury and improve outcome in hemophilia A (HA) mice even in the presence of circulating inhibitors. Thus, it has been proposed that lentiviral bone marrow gene therapy to ectopically express FVIII during megakaryopoiesis may be a curative strategy especially for patients with HA and intractable inhibitors. However, we have shown that pFVIII has a toxic effect on Mks during intracellular processing in the endoplasmic reticulum/Golgi, limiting platelet yield and pFVIII levels. We have recently shown that exogenous FV can be taken up by in vitro differentiated CD34+- and by induced pluripotent stem cell (iPSC)-derived Mks and asked if the same can be done with FVIII. We found that Mks can take up full-length FVIII (Advate) (Fig. 1A), B-domainless (BD) FVIII (Xyntha, not shown) and BD FVIIIR1645H (not shown), a FVIII mutant we have shown is particularly effective when released by platelets. Uptake is half maximum at 0.25 IU/ml following overnight incubation. Endocytosed FVIII colocalizes with labeled fibrinogen uptake (Fig. 1A), supporting its localization to α-granules, and this FVIII uptake can be blocked by receptor-associated protein (RAP), a blocker of LDL receptor family members, including LRP1 (Fig. 1A) or by including FVIII's carrier protein von Willebrand factor (VWF) (Fig. 1B). To test the biological efficacy of endocytosed pFVIII, we took advantage of our previous studies showing that infused Mks into mice release highly functional platelets after becoming entrapped in the lungs. To focus our studies on the released human platelets and the hemostatic efficacy of the endocytosed pFVIII, we used immunodeficient NOD-scid IL2rgnull (NSG) mice that were also FVIII-deficient and that only expressed mutant VWF that binds human, but not mouse, platelet glycoprotein Ib/IX (VWFR1326H) (N/8/V mice). We infused 1x106 human FVIII-endocytosed Mks into these N/8/V mice resulting in ~1-10% being human platelets in recipient mice. We studied hemostatic efficacy by rotational thromboelastography (ROTEM) and demonstrated that the in vivo-released FVIII from released human platelets within the N/8/V blood corrected hemostasis in this system (Fig. 1C). These FVIII-containing platelets fully corrected clotting as well in a FeCl3 carotid artery injury model (Fig. 1D). In summary, we found that in vitro-grown human Mks can endocytose FVIII from the media into their α-granules in sufficient amounts to have potential clinical application in the care of HA patients. This endocytosis is likely via LRP1. FVIII is not found normally in platelets likely because of a combination of the following: 1) in plasma, FVIII it is bound to VWF, 2) circulating platelets also lack LRP1, and 3) in the marrow, there is little or no free FVIII for endocytosis by LRP1-positive Mks. We propose that endocytosed FVIII by Mks can be an important clinical application of commercial, in vitro-grown Mks for patients with HA and inhibitors who need supplemental long-lasting, hemostatic support to their emicizumab without the thrombotic risks of FVIII-bypassing agents. Disclosures Sabatino: Spark Therapeutics: Patents &amp; Royalties. Camire:Pfizer: Research Funding.

The Roles of Low-Density Lipoprotein Receptor-Related Proteins 5, 6, and 8 in Cancer: A Review.

Low-density lipoprotein receptor (LDLR) has been an object of research since the 1970s because of its role in various cell functions. The LDLR family members include LRP5, LRP6, and LRP8. Even though LRP5, 6, and 8 are in the same family, intriguingly, these three proteins have various roles in physiological events, as well as in regulating different mechanisms in various kinds of cancers. LRP5, LRP6, and LRP8 have been shown to play important roles in a broad panel of cancers. LRP5 is highly expressed in many tissues and is involved in the modulation of glucose-induced insulin secretion, bone development, and cholesterol metabolism, as well as cancer progression. Recently, LRP5 has also been shown to play a role in chondroblastic subtype of osteosarcoma (OS) and prostate cancer and also in noncancer case such as osteoporosis. LRP6, which has been previously discovered to share the same structures as LRP5, has also been associated with many cancer progressions such as human triple negative breast cancer (TNBC), primary chronic lymphocytic leukemia (CLL), nonsmall cell lung cancer (NSCL), lung squamous cell carcinoma (LSCC), and hepatocellular carcinoma (HCC). In addition to its role in cancer progression, LRP8 (apolipoprotein E receptor 2 [APOER2]) has also been demonstrated to regulate canonical Wnt/β-catenin signaling pathway whereby this pathway plays a role in cell migration and development. Therefore, this review aimed to elucidate the role of LRP 5, 6, and 8 in regulating the cancer progression.

Intravenously Administered Novel Liposomes, DCL64, Deliver Oligonucleotides to Cerebellar Purkinje Cells.

Cerebellar Purkinje cells (PCs) show conspicuous damages in many ataxic disorders. Targeted delivery of short nucleic acids, such as antisense oligonucleotides, to PCs may be a potential treatment for ataxic disorders, especially spinocerebellar ataxias (SCAs), which are mostly caused by a gain of toxic function of the mutant RNA or protein. However, oligonucleotides do not cross the blood-brain barrier (BBB), necessitating direct delivery into the central nervous system (CNS) through intra-thecal, intra-cisternal, intra-cerebral ventricular, or stereotactic parenchymal administration. We have developed a novel liposome (100 to 200nm in diameter) formulation, DCL64, composed of dipalmitoyl-phosphatidylcholine, cholesterol, and poloxamer L64, which incorporates oligonucleotides efficiently (≥ 70%). Confocal microscopy showed that DCL64 was selectively taken up by brain microvascular endothelial cells by interacting with low-density lipoprotein receptor (LDLr) family members on cell surface, but not with other types of lipid receptors such as caveolin or scavenger receptor class B type 1. LDLr family members are implicated in brain microvascular endothelial cell endocytosis/transcytosis, and are abundantly localized on cerebellar PCs. Intravenous administration of DCL64 in normal mice showed distribution of oligonucleotides to the brain, preferentially in PCs. Mice that received DCL64 showed no adverse effect on hematological, hepatic, and renal functions in blood tests, and no histopathological abnormalities in major organs. These studies suggest that DCL64 delivers oligonucleotides to PCs across the BBB via intravenous injection with no detectable adverse effects. This property potentially makes DCL64 particularly attractive as a delivery vehicle in treatments of SCAs.

LRP1 Regulates Adiposity by Controlling Energy Balance in MC4R Neurons

Hypothalamic low-density lipoprotein receptor-related protein 1 (LRP1), a member of LDL receptor family, plays a pivotal role in the regulation of food intake and body-weight homeostasis. However, the role of LRP1 in MC4R-expressing neurons remains unknown. This study was to determine whether deleting LRP1 from MC4R-expressing neurons affects adiposity and energy balance in mice. Here we show that MC4R neuron-specific LRP1 deletion results in decreased body weight in mice fed a normal chow diet (control 30.4±0.8g vs. MC4R-LRP1 deficient mice 27.2±0.5g, p&amp;lt;0.05). Consistent with this, MRI analysis indicated that fat mass in MC4R-LRP1 deficient mice significantly decreased by ∼64% without changes in lean mass, compared with control mice. This occurred despite the fact that MC4R-LRP1 deficient mice are hyperphagic (control 3.99±0.17g vs. MC4R-LRP1 deficient mice 4.71±0.18g, p&amp;lt;0.05). In addition, MTII’s ability to suppress food intake was impaired in MC4R-LRP1 deficient mice. These effects are mainly due to increased energy metabolism, as evidenced by the fact that oxygen consumption for 24 hours increased in MC4R-LRP1 deficient mice compared with control mice (control mice 6002±287 ml/kg/hour vs. MC4R-LRP1 deficient mice 7121±1947 ml/kg/hour, p&amp;lt;0.01), along with exhibiting insulin sensitivity and glucose tolerance. However, locomotor activity was normal in these mice. The current data demonstrate that LRP1 signaling in MC4R neurons is required for the homeostatic regulation of energy expenditure and adiposity. Thus, LRP1 is a key player of energy balance in MC4R-expressing neurons. Disclosure M. Kang: None. J.A. Seo: None. H. Cho: None. H. Lee: None. Y. Kim: None.

Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein

Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.

Lipophorin receptor regulates Nilaparvata lugens fecundity by promoting lipid accumulation and vitellogenin biosynthesis

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens. The deduced amino acid sequence of NlLpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction.

Degradation routes of trafficking-defective VLDLR mutants associated with Dysequilibrium syndrome

Low density lipoprotein receptor (LDLR) family members are involved in signaling in the developing brain. Previously we have reported that missense mutations in the Very Low Density Lipoprotein Receptor gene (VLDLR), causing Dysequilibrium syndrome (DES), disrupt ligand-binding, due to endoplasmic reticulum (ER) retention of the mutants. We explored the degradation routes of these VLDLR mutants in cultured cells. Our results indicate that VLDLR mutants are retained in the ER for prolonged periods which could be facilitated by association with the ER-resident chaperone calnexin. The mutants were prone to aggregation and capable of eliciting ER stress. The VLDLR mutants were found to be degraded predominantly by the proteasomal pathway, since ubiquitinated VLDLR was found to accumulate in response to proteasomal inhibition. Further, the mutants were found to interact with the ER degradation adaptor protein SEL1L. The degradation of VLDLR wild type and mutant were delayed in CRISPR/Cas9 edited SEL1L knock-out cells which was reversed by exogenous expression of SEL1L. In summary, ER retention of pathogenic VLDLR mutants involves binding to calnexin, elevated ER stress, and delayed degradation which is dependent on SEL1L. Since core LDLR family members share common structural domains, common mechanisms may be involved in their ER processing.

How multi-scale structural biology elucidated context-dependent variability in ectodomain conformation along withthe ligand capture and release cycle for LDLR family members.

The low-density lipoprotein receptor (LDLR) and its homologs capture and internalize lipoproteins into the cell. Due to the fact that LDLR family members possess a modular ectodomain that undergoes dynamic conformational changes, multi-scale structural analysis has been performed so as to understand the ligand capture and release mechanism. For example, crystallographic analyses have provided models for boththe entire ectodomain and high-resolution structures of individual modules. In addition, nuclear magnetic resonance spectroscopic analyses have shown the rigidity and flexibility of inter-module linkers to restrict the mobility of ectodomain. Accumulated structural data suggest that the ectodomains of LDLR family members are flexible at the cell surface and switch between two metastable conformations, that is, the extended and contracted conformations. Recent structural analysis of ApoER2, a close homolog of LDLR, raised the possibility that the receptor binds with the ligand in the contracted conformation. After transport to an endosome by endocytosis, the receptor undergoes a conformational change to the closed conformation for completion of ligand release. In contrast, LDLR has been reported to adopt the extended conformation when it binds with a inhibitory regulator that recruits LDLR toward the degradation pathway. These findings support a mechanism of different ectodomain conformations for binding the ligand versus binding the regulatory protein. In this review, I provide an overview of studies that analyze the structural and biophysical properties of the ectodomains of LDLR family members and discuss a hypothetical model for ligand uptake and receptor recycling that integrates the known ectodomain conformational variability.

Interaction of Fibrin with the Very Low-Density Lipoprotein (VLDL) Receptor: Further Characterization and Localization of the VLDL Receptor-Binding Site in Fibrin βN-Domains.

Our recent study revealed that fibrin and the very low-density lipoprotein receptor (VLDLR) interact with each other through a pair of fibrin βN-domains and CR domains of the receptor and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major objectives of this study were to further clarify the molecular mechanism of fibrin-VLDLR interaction and to identify amino acid residues in the βN-domains involved in this interaction. Our binding experiments with the (β15-66)2 fragment, which corresponds to a pair of fibrin βN-domains, and the VLDLR(1-8) fragment, consisting of eight CR domains of VLDLR, revealed that interaction between them strongly depends on ionic strength and chemical modification of all Lys or Arg residues in (β15-66)2 results in abrogation of this interaction. To identify which of these residues are involved in the interaction, we mutated all Lys or Arg residues in each of the three positively charged Lys/Arg clusters of the (β15-66)2 fragment, as well as single Arg17 and Arg30, and tested the affinity of the mutants obtained for VLDLR(1-8) by an enzyme-linked immunosorbent assay and surface plasmon resonance. The experiments revealed that the second and third Lys/Arg clusters make the major contribution to this interaction while the contribution of the first cluster is moderate. The results obtained suggest that interaction between fibrin and the VLDL receptor employs the "double-Lys/Arg" recognition mode previously proposed for the interaction of the LDL receptor family members with their ligands. They also provide valuable information for the development of highly specific peptide-based inhibitors of fibrin-VLDLR interaction.

Structural basis for ligand capture and release by the endocytic receptor ApoER2.

Apolipoprotein E receptor 2 (ApoER2) is a close homologue of low‐density lipoprotein receptor (LDLR) that mediates the endocytosis of ligands, including LDL particles. LDLR family members have been presumed to explore a large conformational space to capture ligands in the extended conformation at the cell surface. Ligands are subsequently released through a pH‐titrated structural transition to a self‐docked, contracted‐closed conformation. In addition to lipoprotein uptake, ApoER2 is implicated in signal transduction during brain development through capture of the extracellular protein reelin. From crystallographic analysis, we determine that the full‐length ApoER2 ectodomain adopts an intermediate contracted‐open conformation when complexed with the signaling‐competent reelin fragment, and we identify a previously unappreciated auxiliary low‐affinity binding interface. Based on mutational analyses, we propose that the pH shift during endocytosis weakens the affinity of the auxiliary interface and destabilizes the ligand–receptor complex. Furthermore, this study elucidates that the contracted‐open conformation of ligand‐bound ApoER2 at neutral pH resembles the contracted‐closed conformation of ligand‐unbound LDLR at acidic pH in a manner suggestive of being primed for ligand release even prior to internalization.

Expression of a recombinant full-length LRP1B receptor in human non-small cell lung cancer cells confirms the postulated growth-suppressing function of this large LDL receptor family member.

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B), a member of the LDL receptor family, is frequently inactivated in multiple malignancies including lung cancer. LRP1B is therefore considered as a putative tumor suppressor. Due to its large size (4599 amino acids), until now only minireceptors or receptor fragments have been successfully cloned. To assess the effect of LRP1B on the proliferation of non-small cell lung cancer cells, we constructed and expressed a transfection vector containing the 13.800 bp full-length murine Lrp1b cDNA using a PCR-based cloning strategy. Expression of LRP1B was analyzed by quantitative RT-PCR (qRT-PCR) using primers specific for human LRP1B or mouse Lrp1b. Effective expression of the full length receptor was demonstrated by the appearance of a single 600 kDa band on Western Blots of HEK 293 cells. Overexpression of Lrp1b in non-small cell lung cancer cells with low or absent endogenous LRP1B expression significantly reduced cellular proliferation compared to empty vector-transfected control cells. Conversely, in Calu-1 cells, which express higher endogenous levels of the receptor, siRNA-mediated LRP1B knockdown significantly enhanced cellular proliferation. Taken together, these findings demonstrate that, consistent with the postulated tumor suppressor function, overexpression of full-length Lrp1b leads to impaired cellular proliferation, while LRP1B knockdown has the opposite effect. The recombinant Lrp1b construct represents a valuable tool to unravel the largely unknown physiological role of LRP1B and its potential functions in cancer pathogenesis.

Molecular Characterization and Function Analysis of the Vitellogenin Receptor from the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae).

Developing oocytes accumulate plentiful yolk protein during oogenesis through receptor-mediated endocytosis. The vitellogenin receptor (VgR), belonging to the low-density lipoprotein receptor (LDLR) family, regulates the absorption of yolk protein. In this work, the full-length vitellogenin receptor (HaVgR) in the cotton bollworm Helicoverpa armigera was identified, encoding a 1817 residue protein. Sequence alignment revealed that the sequence of HaVgR contained all of the conservative structural motifs of LDLR family members, and phylogenetic analysis indicated that HaVgR had a high identity among Lepidoptera and was distinct from that of other insects. Consistent with other insects, HaVgR was specifically expressed in ovarian tissue. The developmental expression pattern showed that HaVgR was first transcribed in the newly metamorphosed female adults, reached a peak in 2-day-old adults and then declined. Western blot analysis also revealed an ovarian-specific and developing expression pattern, which was consistent with the HaVgR mRNA transcription. Moreover, RNAi-mediated HaVgR knockdown strongly reduced the VgR expression in both the mRNA and protein levels, which inhibited the yolk protein deposition in the ovaries, led to the dramatic accumulation of vitellogenin and the up-regulation of HaVg expression in hemolymph, and eventually resulted in a declined fecundity. Together, all of these findings demonstrate that HaVgR is a specific receptor in uptake and transportation of yolk protein for the maturation of oocytes and that it plays a critical role in female reproduction.

Abstract 98: Antiphospholipid Antibodies Induce Thrombosis by Activating Endothelial PP2A via ApoER2-Dab2-PSD95 Complex Formation

In the antiphospholipid syndrome (APS), patients generate antiphospholipid antibodies (aPL) that promote thrombosis. We previous showed that aPL recognition of the endothelial cell surface protein β2-Glycoprotein I (β2GPI) causes β2GPI to interact with the LDL receptor family member ApoER2 and thereby antagonize endothelial NO synthase (eNOS). The decrease in bioavailable NO then leads to exaggerated thrombus formation. In the present work we sought to determine how aPL and the β2GPI-ApoER2 tandem antagonize eNOS. Initial experiments employed co-immunoprecipitation, RNAi-based gene silencing and adenoviral introduction of mutant proteins into human aortic endothelial cells. We discovered that in contrast to normal human IgG from healthy subjects (NHIgG), aPL invoke the formation of a complex between the cytoplasmic tail of ApoER2 and Dab-2 and PSD95, and that the formation of the complex is required for eNOS antagonism. We also found that ApoER2-Dab2-PSD95 complex formation in response to aPL potently activates the serine/threonine phosphatase PP2A, which in turn dephosphorylates eNOS-S1179, thereby extinguishing eNOS enzymatic activity. Furthermore, we found that upon aPL treatment, the PP2A catalytic and regulatory subunits are recruited to the ApoER2-Dab2-PSD95 complex. To test if these processes are operative in APS-related thrombosis in vivo, intravital microscopy of the mesenteric microcirculation was employed to evaluate thrombus formation in mice. In wild-type mice, aPL administration caused exaggerated thrombus formation compared to treatment with NHIgG. In contrast, ApoER2 did not promote thrombosis in knock-in ApoER2-EIG mice harboring a mutant receptor incapable of interacting with Dab-2. Moreover, in wild-type mice aPL treatment caused a dramatic increase in PP2A activity in the aorta, and administration of the PP2A inhibitor Endothall fully prevented thrombus formation induced by aPL. Having revealed the molecular underpinnings of the disorder, current treatment of APS with anticoagulation, which is often ineffective and fraught with complications, can potentially be replaced by new mechanism-based therapies targeting ApoER2 complex formation or PP2A.

Abstract 142: Characterizing the Mechanisms of Factor VIII Binding to Low-density Lipoprotein Receptor-related Protein 1

Hemophilia A is a bleeding disorder caused by a deficiency in coagulation factor VIII (fVIII) that affects 1 in 5,000 males. Current prophylactic replacement therapy, while effective, is difficult to maintain due to cost and frequency of injections. Hepatic clearance of fVIII is mediated by the LDL receptor-related protein 1 (LRP1), a member of the LDL receptor family. While it is well established that fVIII binds LRP1, the molecular details of this interaction are unclear. The goal of this project is to better define the molecular interactions between fVIII and LRP1, particularly to determine the regions of LRP1 that are responsible for binding and clearance and if activation of fVIII is required to bind LRP1. We mapped the binding of fVIII to the Cluster II and IV regions of LRP1, with higher affinity for cluster IV. This is in contrast to many LRP1 ligands that bind to multiple regions with similar affinity. The canonical model of LDL receptor family member binding involves lysine residues on the ligand docking in acidic pockets on the receptor. We examined the binding of fVIII to LRP1 using surface plasmon resonance and found that increasing ionic strength, which disrupts the charged interactions required between lysine residues and the acidic pockets, did not alter the affinity of fVIII to LRP1. These results suggest that fVIII may bind to LRP1 via a different mode of binding than the canonical model. A recent publication raised the possibility that LRP1-mediated clearance of fVIII may first require its activation. To test this hypothesis, we compared the clearance of wild-type fVIII with a mutant fVIII in which the thrombin cleavage sites were converted to alanine. Both fVIII molecules were cleared from the circulation with a fast and slow phase, with no significant difference between WT and mutant fVIII. These results suggest that activation of fVIII is not essential for clearance in vivo . Together these data reveal important insights into the binding mechanisms between fVIII and LRP1 and provide insight into ways to extend fVIII half-life in vivo and improve prophylactic replacement therapy.

Human extravillous trophoblasts bind but do not internalize antiphospholipid antibodies

IntroductionObstetric morbidity in women with antiphospholipid antibodies (aPLs) may reflect the adverse effects of aPLs on placental cells such as extravillous trophoblasts and the syncytiotrophoblast. Antiphospholipid antibodies may affect the syncytiotrophoblast after being internalised by members of the Low-density lipoprotein receptor (LDLR) family and the antigen of aPLs, β2 glycoprotein I. AimThis study aimed to determine whether aPL internalization was a mechanism by which aPLs adversely affect extravillous trophoblasts. Methodof Study: Fluorescently-labelled monoclonal aPLs IIC5 or ID2 were incubated with first trimester extravillous trophoblast outgrowths and visualized by microscopy. The subcellular expression of β2 glycoprotein I and LDLR family members was investigated by live/permeabilised immunocytochemistry. ResultsUnlike the syncytiotrophoblast of anchoring villi, monoclonal aPLs were not internalised by extravillous trophoblasts, which expressed LDLR family members intracellularly. The aPL IIC5 bound to the surface of extravillous trophoblasts in a pattern similar to the extracellular expression of β2 glycoprotein I. ConclusionsThe mechanisms of action of aPLs are different in extravillous trophoblasts and the syncytiotrophoblast. The interaction of aPLs with the extravillous trophoblast surface, which may involve β2 glycoprotein I, is consistent with reports that aPLs trigger intracellular signaling cascades through cell-surface receptors.