39-kD protein inhibits tissue-type plasminogen activator clearance in vivo.

Background: Pericytes are a major cerebrovasculature component, producing abundant apolipoprotein E (apoE). Results: Deletion of apoE accelerates pericyte migration and adhesion by increasing RhoA activity. The effects are reversed by exogenous apoE3 but not apoE4. Conclusion: ApoE suppresses pericyte mobility in a RhoA-mediated manner. Significance: Pericyte-derived apoE regulates cell mobility and can be explored as a target for neurovascular disease therapy.

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“…5, C and D). Similar results were obtained when LRP1 function was blocked by an LRP1 antagonist, RAP (23,27) (Fig. 5, E and F).…”

Section: Low-density Lipoprotein Receptor-related Protein 1 (Lrp1) Issupporting

confidence: 86%

“…In some experiments, apoE3 and apoE4 particles (5 ng/ml), an LRP1 antagonist, RAP (500 nM), and a RhoA inhibitor, Rhodblock 1a (22) (Sigma, 1 M), were used. Recombinant RAP was produced and purified as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

Casey

Atagi

Yamazaki

et al. 2015

Journal of Biological Chemistry

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“…Human recombinant RAP was expressed in a glutathione S-transferase expression vector and isolated as described previously (22). Human ␣ 2 -macroglobulin (␣ 2 M) was purified from plasma and activated with methylamine (to yield receptor-binding form, ␣ 2 M*) as described before (23). Polyclonal rabbit anti-human apoER2 antibodies were kindly provided by Dr. James S. Owen (University College London, London, UK; see Ref.…”

Section: Methodsmentioning

confidence: 99%

Differential Functions of Members of the Low Density Lipoprotein Receptor Family Suggested by Their Distinct Endocytosis Rates

Lü

Marzolo

et al. 2001

Journal of Biological Chemistry

136

134

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The low density lipoprotein receptor (LDLR) family is composed of a class of cell surface endocytic receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2), and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its chimeric receptor constructs, and full-length VLDLR and apoER2 stably expressed in LRP-null Chinese hamster ovary cells. We find that the initial endocytosis rates mediated by different cytoplasmic tails are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR and apoER2 exhibit least endocytosis function. Compared with the tail of LRP, the tails of the LDLR and megalin display significantly lower levels of endocytosis rates. Ligand degradation analyses strongly support differential endocytosis rates initiated by these receptors. Interestingly apoER2, which has recently been shown to mediate intracellular signal transduction, exhibited the lowest level of ligand degradation efficiency. These results thus suggest that the endocytic functions of members of the LDLR family are distinct and that certain receptors in this family may play their main roles in areas other than receptor-mediated endocytosis.

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“…Bovine lipoprotein lipase was a gift from Dr. Ira Goldberg (Columbia University, New York, NY, U.S.A.). Recombinant human full-length glutathione S-transferase 39 kDa protein was produced in and purified from E. coli as described previously [13]. The murine monoclonal 2H8 anti-TFPI antibody, which recognizes an epitope within the first Kunitz domain of TFPI, was produced as described previously [5].…”

Section: Proteinsmentioning

confidence: 99%

Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor

Mast

Higuchi

Huang

et al. 1997

Biochemical Journal

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Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

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39-kD protein inhibits tissue-type plasminogen activator clearance in vivo.

Cited by 95 publications

References 49 publications

Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

Differential Functions of Members of the Low Density Lipoprotein Receptor Family Suggested by Their Distinct Endocytosis Rates

Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor

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