Boca,an endoplasmic reticulum protein required for wingless signaling and trafficking of LDL receptor family members in Drosophila

The maturation of cell surface receptors through the secretory pathway often requires chaperones that aid in protein folding and trafficking from one organelle to another. Here we describe boca, an evolutionarily conserved gene in Drosophila melanogaster, which encodes an endoplasmic reticulum protein that is specifically required for the intracellular trafficking of members of the low-density lipoprotein family of receptors (LDLRs). Two LDLRs in flies, Arrow, which is required for Wingless signal transduction, and Yolkless, which is required for yolk protein uptake during oogenesis, both require boca function. Consequently, boca is an essential component of the Wingless pathway but is more generally required for the activities of multiple LDL receptor family members.     

Cloning of a novel member of the low-density lipoprotein receptor family

A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.     

Structural characterization of the Boca/Mesd maturation factors for LDL-receptor-type β propeller domains

Folding and trafficking of low-density lipoprotein receptor (LDLR) family members, which play essential roles in development and homeostasis, are mediated by specific chaperones. The Boca/Mesd chaperone family specifically promotes folding and trafficking of the YWTD β propeller-EGF domain pair found in the ectodomain of all LDLR members. Limited proteolysis, NMR spectroscopy, analytical ultracentrifugation, and X-ray crystallography were used to define a conserved core composed of a structured domain that is preceded by a disordered N-terminal region. High-resolution structures of the ordered domain were determined for homologous proteins from three metazoans. Seven independent protomers reveal a novel ferrodoxin-like superfamily fold with two distinct β sheet topologies. A conserved hydrophobic surface forms a dimer interface in each crystal, but these differ substantially at the atomic level, indicative of nonspecific hydrophobic interactions that may play a role in the chaperone activity of the Boca/Mesd family.     

The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits

ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.     

Characterization of residues in the cytoplasmic domain of the LDL receptor required for exit from the endoplasmic reticulum

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE₈₁₄ and/or ExD₈₃₇ had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization.     

Plasma PCSK9 preferentially reduces liver LDL receptors in mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the expression of LDL receptor (LDLR) protein. Gain-of-function mutations in PCSK9 cause hypercholesterolemia, and loss-of-function mutations result in lower plasma LDL-cholesterol. Here, we investigate the kinetics and metabolism of circulating PCSK9 relative to tissue levels of LDLRs. The administration of recombinant human PCSK9 (32 microg) to mice by a single injection reduced hepatic LDLRs by approximately 90% within 60 min, and the receptor levels returned to normal within 6 h. The half-life of the PCSK9 was estimated to be approximately 5 min. Continuous infusion of PCSK9 (32 microg/h) into wild-type mice caused a approximately 90% reduction in hepatic LDLRs within 2 h and no associated change in the level of LDLR in the adrenals. Parallel studies were performed using a catalytically inactive form of PCSK9, PCSK9(S386A), and similar results were obtained. Infusion of PCSK9(D374Y), a gain-of-function mutation, resulted in accelerated clearance of the mutant PCSK9 and a greater reduction in hepatic LDLRs. Combined, these data suggest that exogenously administrated PCSK9 in plasma preferentially reduces LDLR protein levels in liver at concentrations found in human plasma and that PCSK9's action on the LDLR is not dependent on catalytic activity in vivo.     

Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members

Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.     

Molecular characterization of the first avian LDL receptor: role in sterol metabolism of ovarian follicular cells

Low levels of expression and sluggish sterol-mediated regulation have been likely reasons for the failure to molecularly characterize a bona fide LDL receptor (LDLR) in egg-laying species to date. The overall structure of the chicken LDLR, delineated here by cDNA cloning, has been conserved in evolution, since hallmark properties of mammalian LDLRs are already present in the avian protein. The chicken receptor appears to prefer LDL over VLDL as ligand, in compliance with its main role in providing lipoprotein-derived cholesterol for steroid production in ovarian follicular cells. This is also compatible with the fact that estrogen administration increased hepatic LDLR expression in roosters despite dramatically stimulated VLDL production. In cultured chicken embryo fibroblasts, expression of the receptor was induced by incubation with cholesterol synthesis inhibitors such as a statin. Furthermore, preincubation of induced cells with a specific anti-receptor antibody blocks LDL endocytosis, demonstrating that the receptor is ligand-endocytosis competent. Finally, the distribution of LDLRs among the extraoocytic cell populations lends support to a three-cell model for estrogen production within the ovarian follicle. In summary, the molecular characterization of the first avian LDLR reveals novel information about evolutionary, structural, and functional aspects of members of the supergene family of LDLR-related proteins.     

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 μg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30 min and the degradation of hepatic LDLR reached the maximum 2 h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5 min of protein injection and internalized PCSK9 was only barely detectable within 1 h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.     

Solution structure of the LDL receptor EGF-AB pair: a paradigm for the assembly of tandem calcium binding EGF domains.

BACKGROUND: From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. RESULTS: Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. CONCLUSIONS: The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.     

Coaxing the LDL receptor family into the fold

Low-density-lipoprotein (LDL) receptor family members control diverse developmental and physiological pathways. In this issue of Cell, both Culi and Mann and Hsieh et al. report on Boca/MESD, a highly conserved chaperone required for transport of LDLR family proteins to the cell surface. Together with recent insights into the atomic structure of the LDL receptor, they shed new light on the synthesis and trafficking of this important class of multifunctional receptors.     

A complete NMR spectral assignment of the conserved region of the MESD protein, MESD(12-155)

The low-density lipoprotein (LDL) receptor family members control diverse developmental and physiological pathways. Mesoderm development (MESD) protein is a 195-residue protein that functions as a specialized molecular chaperone to promote the proper folding of the six-bladed β-propeller/EGF modules of the LDL receptor family members. Here we report a complete NMR spectral assignment of the most conserved region of MESD protein, MESD(12-155).     

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.     

The first epidermal growth factor-like domain of the low-density lipoprotein receptor contains a noncanonical calcium binding site

Removal of cholesterol-containing particles from the circulation is mediated by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the receptor-ligand complex is endocytosed, and the ligand is released. The important biological role of the LDL receptor (LDLR) has been highlighted by the identification of more than 400 LDLR mutations that are associated with familial hypercholesterolemia. The extracellular region of the LDLR is modular in nature and principally comprises multiple copies of ligand binding, epidermal growth factor-like (EGF), and YWTD-type domains. This report describes characterization of the calcium binding properties of the tandem pair of EGF domains. While only the C-terminal EGF module contains the consensus sequence associated with calcium binding, a noncanonical calcium binding site in the N-terminal domain has been revealed using solution NMR spectroscopy. The calcium dissociation constants for the N- and C-terminal sites have been measured under physiologically relevant pH and ionic strength conditions using a combination of solution NMR, intrinsic protein fluorescence, and chromophoric chelator methods to be approximately 50 microM and approximately 10-20 microM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity within the LDLR gene family. Comparison of the K(d) for the C-terminal site with the calcium concentration in late vesicles indicates that the binding properties of this module may be tuned to titrate upon endocytosis of the LDL receptor-ligand complex, and thus calcium binding may play a role in the ligand dissociation process.     

Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.     

Ligand-binding domains in vitellogenin receptors and other LDL-receptor family members share a common ancestral ordering of cysteine-rich repeats

Insect vitellogenin and yolk protein receptors (VgR/YPR) are newly discovered members of the low-density lipoprotein receptor (LDLR) family, which is characterized by a highly conserved arrangement of repetitive modular elements homologous to functionally unrelated proteins. The insect VgR/YPRs are unique in having two clusters of complement-type cysteine-rich (class A) repeats or modules, with five modules in the first cluster and seven in the second cluster, unlike classical LDLRs which have a single seven-module cluster, vertebrate VgRs and very low density lipoprotein receptors (VLDLR) which have a single eight-module cluster, and LDLR-related proteins (LRPs) and megalins which have four clusters of 2–7, 8, 10, and 11 modules. Alignment of clusters across subfamilies by conventional alignment programs is problematic because of the repetitive nature of the component modules which may have undergone rearrangements, duplications, and deletions during evolution. To circumvent this problem, we ``fingerprinted' each class A module in the different clusters by identifying those amino acids that are both relatively conserved and relatively unique within the cluster. Intercluster reciprocal comparisons of fingerprints and aligned sequences allowed us to distinguish four cohorts of modules reflecting shared recent ancestry. All but two of the 57 modules examined could be assigned to one of these four cohorts designated A, B, C, and D. Alignment of clusters based on modular cohorts revealed that all clusters are derived from a single primordial cluster of at least seven modules with a consensus arrangement of CDCADBC. All extant clusters examined are consistent with this consensus, though none matches it perfectly. This analysis also revealed that the eight-module clusters in vertebrate VgRs, insect VgR/YPRs, and LRP/megalins are not directly homologous with one another. Assignment of modules to cohorts permitted us to properly align 32 class A clusters from all four LDLR subfamilies for phylogenetic analysis. The results revealed that smaller one-cluster and two-cluster members of the family did not originate from the breakup of a large two-cluster or four-cluster receptor. Similarly, the LRP/megalins did not arise from the duplication of a two-cluster insect VgR/YPR-like progenitor. Rather, it appears that the multicluster receptors were independently constructed from the same single-cluster ancestor. Received: 16 January 1997 / Accepted: 21 August 1997     

Insect vitellogenin/lipophorin receptors: Molecular structures, role in oogenesis, and regulatory mechanisms

Insect vitellogenin and lipophorin receptors (VgRs/LpRs) belong to the low-density lipoprotein receptor (LDLR) gene superfamily and play a critical role in oocyte development by mediating endocytosis of the major yolk protein precursors Vg and Lp, respectively. Precursor Vg and Lp are synthesized, in the majority of insects, extraovarially in the fat body and are internalized by competent oocytes through membrane-bound receptors (i.e., VgRs and LpRs, respectively). Structural analysis reveals that insect VgRs/LpRs and all other LDLR family receptors share a group of five structural domains: clusters of cysteine-rich repeats constituting the ligand-binding domain (LBD), epidermal growth factor (EGF)-precursor homology domain that mediates the acid-dependent dissociation of ligands, an O-linked sugar domain of unknown function, a transmembrane domain anchoring the receptor in the plasma membrane, and a cytoplasmic domain that mediates the clustering of the receptor into the coated pits. The sequence analysis indicates that insect VgRs harbor two LBDs with five repeats in the first and eight repeats in the second domain as compared to LpRs which have a single 8-repeat LBD. Moreover, the cytoplasmic domain of all insect VgRs contains a LI internalization signal instead of the NPXY motif found in LpRs and in the majority of other LDLR family receptors. The exception is that of Solenopsis invicta VgR, which also contains an NPXY motif in addition to LI signal. Cockroach VgRs still harbor another motif, NPTF, which is also believed to be a functional internalization signal. The expression studies clearly demonstrate that insect VgRs are ovary-bound receptors of the LDLR family as compared to LpRs, which are transcribed in a wide range of tissues including ovary, fat body, midgut, brain, testis, Malpighian tubules, and muscles. VgR/LpR mRNA and the protein were detected in the germarium, suggesting that the genes involved in receptor-endocytotic machinery are specifically expressed long before they are functionally required.     

Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulse-chase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.     

Blockade of cholesterol absorption by ezetimibe reveals a complex homeostatic network in enterocytes

Enterocyte cholesterol homeostasis reflects aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Cholesterol synthesis and LDL uptake are coordinately regulated by sterol regulatory element-binding proteins (SREBP), whereas sterol efflux is regulated by liver X receptors (LXR). How these processes are coordinately regulated in enterocytes, the site of cholesterol absorption, is not well understood. Here, we treat mice with ezetimibe to investigate the effect of blocking cholesterol absorption on intestinal SREBPs, LXRs, and their effectors. Ezetimibe increased nuclear SREBP-2 8-fold. HMG-CoA reductase (HMGR) and LDL receptor (LDLR) mRNA levels increased less than 3-fold, whereas their protein levels increased 30- and 10-fold, respectively. Expression of inducible degrader of LDLR (IDOL), an LXR-regulated gene that degrades LDLRs, was reduced 50% by ezetimibe. Coadministration of ezetimibe with the LXR agonist T0901317 abolished the reduction in IDOL and prevented the increase in LDLR protein. Ezetimibe-stimulated LDLR expression was independent of proprotein convertase subtilisin/kexin type 9 (PSCK9), a protein that degrades LDLRs. To maintain cholesterol homeostasis in the face of ezetimibe, enterocytes boost LDL uptake by increasing LDLR number, and they boost sterol synthesis by increasing HMGR and other cholesterologenic genes. These studies reveal a hitherto undescribed homeostatic network in enterocytes triggered by blockade of cholesterol absorption.