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Dudley K Strickland - One of the best experts on this subject based on the ideXlab platform.
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generation of a potent low density Lipoprotein Receptor related protein 1 lrp1 antagonist by engineering a stable form of the Receptor associated protein rap d3 domain
Journal of Biological Chemistry, 2015Co-Authors: Joni M Prasad, Mary Migliorini, Rebeca Galisteo, Dudley K StricklandAbstract:Abstract The low density Lipoprotein Receptor-related protein 1 (LRP1) is a member of the low density Lipoprotein Receptor family and plays important roles in a number of physiological and pathological processes. Expression of LRP1 requires the Receptor-associated protein (RAP), a molecular chaperone that binds LRP1 and other low density Lipoprotein Receptor family members in the endoplasmic reticulum and traffics with them to the Golgi where the acidic environment causes its dissociation. Exogenously added RAP is a potent LRP1 antagonist and binds to LRP1 on the cell surface, preventing ligands from binding. Following endocytosis, RAP dissociates in the acidic endosome, allowing LRP1 to recycle back to the cell surface. The acid-induced dissociation of RAP is mediated by its D3 domain, a relatively unstable three-helical bundle that denatures at pH <6.2 due to protonation of key histidine residues on helices 2 and 3. To develop an LRP1 inhibitor that does not dissociate at low pH, we introduced a disulfide bond between the second and third helices in the RAP D3 domain. By combining this disulfide bond with elimination of key histidine residues, we generated a stable RAP molecule that is resistant to both pH- and heat-induced denaturation. This molecule bound to LRP1 with high affinity at both neutral and acidic pH and proved to be a potent inhibitor of LRP1 function both in vitro and in vivo, suggesting that our stable RAP molecule may be useful in multiple pathological settings where LRP1 blockade has been shown to be effective.
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low density Lipoprotein Receptor related protein 1 lrp1 regulates thrombospondin 2 tsp2 enhancement of notch3 signaling
Journal of Biological Chemistry, 2010Co-Authors: He Meng, Xiaojie Zhang, Soo Jung Lee, Dudley K Strickland, Daniel A Lawrence, Michael M WangAbstract:Intracellular trafficking of Notch and Notch ligands modulates signaling, suggesting that choreography of ligand and Receptor translocation is essential for optimal Notch activity. Indeed, a major model for Notch signaling posits that Notch trans-endocytosis into the ligand-expressing (signal sending) cell is a key driving force for Notch signal transduction. The extracellular protein thrombospondin-2 (TSP2) enhances Notch signaling and binds to both Jagged1 and Notch3 ectodomains, potentially bridging two essential extracellular components of Notch signaling. We investigated the role of low density Lipoprotein Receptor-related protein-1 (LRP1), a TSP2 Receptor, in the regulation of Notch3 signaling. TSP2 potentiation of Notch is blocked by the Receptor-associated protein (an inhibitor of low density Lipoprotein Receptor-related protein function) and requires LRP1 expression in the signal-sending cell. TSP2 stimulates Notch3 endocytosis into wild type fibroblasts but not LRP1-deficient fibroblasts. Finally, recombinant Notch3 and Jagged1 interact with the LRP1 85-kDa B-chain, a subunit that lacks known ligand binding function. Our data suggest that LRP1 and TSP2 stimulate Notch activity by driving trans-endocytosis of the Notch ectodomain into the signal-sending cell and demonstrate a novel, non-cell autonomous function of LRP1 in cell-cell signaling.
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the low density Lipoprotein Receptor related protein 1 mediates tissue type plasminogen activator induced microglial activation in the ischemic brain
American Journal of Pathology, 2009Co-Authors: Chen Zhang, Dudley K Strickland, Manuel YepesAbstract:Microglia are the immune cells of the central nervous system (CNS) that become activated in response to pathological situations such as cerebral ischemia. Tissue-type plasminogen activator (tPA) is a serine proteinase that is found in the intravascular space and the CNS. The low-density Lipoprotein Receptor-related protein 1 (LRP1) is a member of the low-density Lipoprotein Receptor gene family found in neurons, astrocytes, and microglia. The present study investigated whether the interaction between tPA and microglial LRP1 plays a role in cerebral ischemia-induced microglial activation. We found that middle cerebral artery occlusion (MCAO) induces microglial activation in both wild-type and plasminogen-deficient (Plg−/−) mice. In contrast, MCAO-induced microglial activation is significantly decreased in tPA-deficient (tPA−/−) mice and in mice that lack LRP1 in microglial cells (macLRP−). We observed a significant increase in microglial activation when tPA−/− mice received treatment with murine tPA after MCAO. In contrast, treatment of macLRP− mice with tPA did not have an effect on the extent of microglial activation. Finally, both the volume of the ischemic lesion as well as inducible nitric oxide synthase production were significantly decreased in macLRP− mice and macLRP− microglia. In summary, our results indicate that the interaction between tPA and LRP1 induces microglial activation with the generation of an inflammatory response in the ischemic brain, suggesting a cytokine-like role for tPA in the CNS.
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the low density Lipoprotein Receptor related protein mediates fibronectin catabolism and inhibits fibronectin accumulation on cell surfaces
Journal of Biological Chemistry, 2002Co-Authors: Ana M. Salicioni, Dudley K Strickland, Kellie S Mizelle, Elena Loukinova, Irina Mikhailenko, Steven L GoniasAbstract:Abstract Low density Lipoprotein Receptor-related protein (LRP) is a member of the low density Lipoprotein Receptor family, which functions as an endocytic Receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, Receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded 125I-Fn at an increased rate, compared with MEF-2 cells. 125I-Fn degradation by MEF-1 cells was inhibited by RAP. These results demonstrate that LRP functions as a catabolic Receptor for Fn. The function of LRP in Fn degradation and the ability of LRP to regulate levels of other plasma membrane proteins represent possible mechanisms whereby LRP prevents Fn accumulation on cell surfaces.
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expression of the very low density Lipoprotein Receptor vldl r an apoLipoprotein e Receptor in the central nervous system and in alzheimer s disease
Journal of Neuropathology and Experimental Neurology, 1996Co-Authors: R H Christie, Dudley K Strickland, Haeyoung Chung, G W Rebeck, Bradley T HymanAbstract:The very low density Lipoprotein Receptor (VLDL-r) is a cell-surface molecule specialized for the internalization of multiple diverse ligands, including apoLipoprotein E (apoE)-containing Lipoprotein particles, via clathrin-coated pits. Its structure is similar to the low-density Lipoprotein Receptor (LDL-r), although the two have substantially different systemic distributions and regulatory pathways. The present work examines the distribution of VLDL-r in the central nervous system (CNS) and in relation to senile plaques in Alzheimer disease (AD). VLDL-r is present on resting and activated microglia, particularly those associated with senile plaques (SPs). VLDL-r immunoreactivity is also found in cortical neurons. Two exons of VLDL-r mRNA are differentially spliced in the mature Receptor mRNA. One set of splice forms gives rise to Receptors containing (or lacking) an extracellular O-linked glycosylation domain near the transmembrane portion of the molecule. The other set of splice forms appears to be brain-specific, and is responsible for the presence or absence of one of the cysteine-rich repeat regions in the binding region of the molecule. Ratios of the Receptor variants generated from these splice forms do not differ substantially across different cortical areas or in AD. We hypothesize that VLDL-r might contribute to metabolism of apoE and apoE/A beta complexes in the brain. Further characterizations of apoE Receptors in Alzheimer brain may help lay the groundwork for understanding the role of apoE in the CNS and in the pathophysiology of AD.
Joachim Herz - One of the best experts on this subject based on the ideXlab platform.
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amyloid precursor protein regulates brain apoLipoprotein e and cholesterol metabolism through Lipoprotein Receptor lrp1
Neuron, 2007Co-Authors: Qiang Liu, Joachim Herz, Celina V Zerbinatti, Juan Zhang, Hyang Sook Hoe, Baiping Wang, Sarah L Cole, Louis J MugliaAbstract:Summary Mutations in the amyloid precursor protein (APP) cause early-onset Alzheimer's disease (AD), but the only genetic risk factor for late-onset AD is the ɛ4 allele of apoLipoprotein E (apoE), a major cholesterol carrier. Using Cre-lox conditional knockout mice, we demonstrate that Lipoprotein Receptor LRP1 expression regulates apoE and cholesterol levels within the CNS. We also found that deletion of APP and its homolog APLP2 , or components of the γ-secretase complex, significantly enhanced the expression and function of LRP1, which was reversed by forced expression of the APP intracellular domain (AICD). We further show that AICD, together with Fe65 and Tip60, interacts with the LRP1 promoter and suppresses its transcription. Together, our findings support that the γ-secretase cleavage of APP plays a central role in regulating apoE and cholesterol metabolism in the CNS via LRP1 and establish a biological linkage between APP and apoE, the two major genetic determinants of AD.
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macrophage low density Lipoprotein Receptor related protein deficiency enhances atherosclerosis in apoe ldlr double knockout mice
Arteriosclerosis Thrombosis and Vascular Biology, 2006Co-Authors: Joachim Herz, Lianne S M Boesten, Petra May, Niels Bovenschen, Menno V Huisman, J F P Berbee, Louis M Havekes, B J M Van VlijmenAbstract:Objective— In vitro studies implicate that the low-density Lipoprotein Receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of Lipoproteins. Methods and Results— We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, Lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3+ T cells we...
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39 kda protein modulates binding of ligands to low density Lipoprotein Receptor related protein alpha 2 macroglobulin Receptor
Journal of Biological Chemistry, 1991Co-Authors: Joachim Herz, Joseph L. Goldstein, Dudley K Strickland, Michael S. BrownAbstract:A 39-kDa protein of unknown function has previously been reported to copurify with the low density Lipoprotein Receptor-related protein (LRP)/alpha 2-macroglobulin Receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin Receptor. This interaction inhibits the binding and uptake of the Receptor's two known ligands: 1) beta-migrating very low density Lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched Lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.
Michael S. Brown - One of the best experts on this subject based on the ideXlab platform.
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nuclear protein that binds sterol regulatory element of low density Lipoprotein Receptor promoter i identification of the protein and delineation of its target nucleotide sequence
Journal of Biological Chemistry, 1993Co-Authors: Michael R Briggs, Michael S. Brown, Chieko Yokoyama, Xiaodong Wang, Joseph L. GoldsteinAbstract:Abstract The current paper reports the identification of a protein in rat liver nuclei that binds to the sterol regulatory element (SRE-1) in the promoter of the gene for the low density Lipoprotein Receptor. The 10-base pair SRE-1 is embedded within a 16-base pair sequence designated Repeat 2 located immediately upstream of a related sequence designated Repeat 3. To confirm that DNA recognition by the SRE-1 binding protein (SREBP) correlates with sterol-regulated transcription, we synthesized an artificial promoter that contains two copies of wild-type or mutant Repeat 2 + 3 sequences immediately upstream of a TATA box from adenovirus. The synthetic promoters were inserted upstream of a reporter gene and tested for transcriptional activity in the absence and presence of sterols after transient transfection into monkey CV-1 cells. The reporter gene with two copies of the wild-type Repeat 2 + 3 sequence was transcribed actively in sterol-deprived cells and was repressed by more than 80% when sterols were present. Binding of SREBP to the SRE-1 sequence, assessed by gel mobility shift assays, correlated precisely on a nucleotide-by-nucleotide basis with the transcriptional activity of each of 16 synthetic promoters with point mutations in Repeat 2. The SREBP bound to the nine mutant promoters that were positive for sterol-regulated transcription, and it did not bind to any of the nine point mutants that abolished transcription. We conclude that SREBP is a DNA binding protein that mediates sterol-regulated transcription of the low density Lipoprotein Receptor gene.
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39 kda protein modulates binding of ligands to low density Lipoprotein Receptor related protein alpha 2 macroglobulin Receptor
Journal of Biological Chemistry, 1991Co-Authors: Joachim Herz, Joseph L. Goldstein, Dudley K Strickland, Michael S. BrownAbstract:A 39-kDa protein of unknown function has previously been reported to copurify with the low density Lipoprotein Receptor-related protein (LRP)/alpha 2-macroglobulin Receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin Receptor. This interaction inhibits the binding and uptake of the Receptor's two known ligands: 1) beta-migrating very low density Lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched Lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.
Tokuo T. Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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the very low density Lipoprotein vldl Receptor characterization and functions as a peripheral Lipoprotein Receptor
Journal of Atherosclerosis and Thrombosis, 2004Co-Authors: Sadao Takahashi, Yasuo Zenimaru, Jinya Suzuki, Isamu Miyamori, Juro Sakai, Hiroaki Hattori, Takahiro Fujino, Tokuo T. YamamotoAbstract:The very low-density Lipoprotein (VLDL) Receptor is a member of the low-density Lipoprotein (LDL) Receptor family. In vitro and in vivo studies have shown that VLDL Receptor binds triglyceride (TG)-rich Lipoproteins but not LDL, and functions as a peripheral remnant Lipoprotein Receptor. VLDL Receptor is expressed abundantly in fatty acid-active tissues (heart, skeletal muscle and fat), the brain and macrophages. It is likely that VLDL Receptor functions in concert with Lipoprotein lipase (LPL), which hydrolyses TG in VLDL and chylomicron. In contrast to the LDL Receptor, VLDL Receptor binds apoLipoprotein (apo) E2/2 VLDL particles as well as apoE3/3 VLDL, and the expression is not down-regulated by intracellular Lipoproteins. Recently, various functions of the VLDL Receptor have been reported in Lipoprotein metabolism, metabolic syndrome/atherosclerosis, cardiac fatty acid metabolism, neuronal migration and angiogenesis/tumor growth. Gene therapy of VLDL Receptor into the liver showed a benefit effect for Lipoprotein metabolism in both LDL Receptor knockout and apoE mutant mice. Beyond its function as a peripheral Lipoprotein Receptor, possibilities of its physiological function have been extended to include signal transduction, angiogenesis and tumor growth.
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The very low density Lipoprotein (VLDL) Receptor--a peripheral Lipoprotein Receptor for remnant Lipoproteins into fatty acid active tissues.
Molecular and cellular biochemistry, 2003Co-Authors: Sadao Takahashi, Isamu Miyamori, Juro Sakai, Takahiro Fujino, Tokuo T. YamamotoAbstract:The VLDL (very low density Lipoprotein) Receptor is a member of the LDL (low density Lipoprotein) Receptor family. The VLDL Receptor binds apoLipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich Lipoprotein particles to the VLDL Receptor. By the unique ligand specificity, VLDL Receptor practically appeared to function as IDL (intermediate density Lipoprotein) and chylomicron remnant Receptor in peripheral tissues in concert with LPL. In contrast to LDL Receptor, the VLDL Receptor expression is not down regulated by Lipoproteins. Recently several possible functions of the VLDL Receptor have been reported in Lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL Receptor into the LDL Receptor knockout mice liver showed a benefit effect for Lipoprotein metabolism and atherosclerosis. Further researches about the VLDL Receptor function will be needed in the future.
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seven novel sequence variants in the human low density Lipoprotein Receptor related protein 5 lrp5 gene
Human Mutation, 2002Co-Authors: Minoru Okubo, Dongho Kim, Asako Horinishi, Tokuo T. Yamamoto, Toshio MuraseAbstract:We identified seven novel polymorphisms in the human low density Lipoprotein Receptor related protein 5 (LRP5) gene. Two of them are predicted to replace amino acid in LRP5 protein (c.314A>G: Q89R and c.4037T>C: V1330A), whereas three are silent mutations in the coding region (c.2268T>C: N740N, c.3405A>G: V1119V, and c.4137C>T: D1363D) and two are polymorphisms in introns (IVS10+6T>C and IVS17-30G>A). Since LRP5 recognizes apoLipoprotein E and is genetically linked with type 1 diabetes, these novel polymorphisms will be useful in genetic studies of hyperLipoproteinemia and diabetes. To our knowledge, this is the first report in the literature of sequence variants in the human LRP5 gene. ©2002 Wiley-Liss, Inc.
Manfred Huettinger - One of the best experts on this subject based on the ideXlab platform.
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metabolism of activated complement component c3 is mediated by the low density Lipoprotein Receptor related protein alpha 2 macroglobulin Receptor
Journal of Biological Chemistry, 1999Co-Authors: Melinda Meilinger, Christa Gschwentner, Irmgard Burger, Markus Haumer, Markus Wahrmann, Lajos Szollar, Johannes Nimpf, Manfred HuettingerAbstract:Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density Lipoprotein-Receptor-related protein/alpha(2)-macroglobulin Receptor (LRP/alpha(2)MR), a member of the low density Lipoprotein Receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by Receptor-associated protein (the 39-kDa Receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional Receptor. Binding of C3 is sensitive to reduction of the Receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density Lipoprotein Receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of Receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.
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metabolism of activated complement component c3 is mediated by the low density Lipoprotein Receptor related protein α2 macroglobulin Receptor
Journal of Biological Chemistry, 1999Co-Authors: Melinda Meilinger, Christa Gschwentner, Irmgard Burger, Markus Haumer, Markus Wahrmann, Lajos Szollar, Johannes Nimpf, Manfred HuettingerAbstract:Abstract Complement component 3 (C3) and α2-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density Lipoprotein-Receptor-related protein/α2-macroglobulin Receptor (LRP/α2MR), a member of the low density Lipoprotein Receptor family, is responsible for the clearance of α2-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/α2MR. Ligand blot experiments with human 125I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/α2MR by the following criteria: it is competed by Receptor-associated protein (the 39-kDa Receptor-associated protein that impairs binding of all ligands to LRP/α2MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional Receptor. Binding of C3 is sensitive to reduction of the Receptor and is Ca2+-dependent. All these features are typical for cysteine-rich binding repeats of the low density Lipoprotein Receptor family. In LRP/α2MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of Receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/α2MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K Dvalue of 17 nm. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/α2MR to bind and endocytose C3 and provide evidence for an LRP/α2MR-mediated system participating in C3 metabolism.