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The uptake of triglyceride-rich lipoproteins has been described as being mediated by apolipoprotein E and lipoprotein lipase (LpL). Proteoglycans, the LDL-receptor, and the LDL receptor-related protein (LRP) are the cellular acceptors. In addition to LpL, hepatic lipase (HL) has been shown to bind to LRP. In this study, the role of HL in lipoprotein uptake was investigated. Human chylomicrons and rabbit beta-VLDL were used as ligands for human hepatoma cells, primary human hepalocytes, normal and proteoglycan-deficient Chinese hamster ovary (CHO) cells, and normal and LDL receptor-deficient human fibroblasts. We show that HL induces stimulation of the uptake of chylomicrons and beta-VLDL into the different cell lines. HL is known to bind to heparan sulfate, and experiments on normal and proteoglycan-deficient CHO cells showed that cell surface proteoglycans are essential for HL-mediated uptake of lipoproteins. To exclude LDL receptor-mediated uptake. we performed experiments on LDL receptor-deficient fibroblasts that demonstrated that the LDL receptor was not important for the HL-mediated uptake of lipoproteins. Crosslinking experiments confirmed the binding of HL to LRP on the cell surface. To identify the region of HL involved in the interaction with LRP, we used a C-terminal fragment of LpL, known to inhibit LpL-mediated uptake. HL-mediated lipoprotein uptake was suppressed by this fragment. Our experiments indicate that HL, like LpL, can mediate the uptake of lipoproteins into cells, most probably via a C-terminal binding site. The uptake, initiated by proteoglycan binding, is mediated by LRP.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.

Apolipoprotein A-V is a potent modulator of plasma triacylglycerol levels. To investigate the molecular basis for this phenomenon we explored the ability of apolipoprotein A-V, in most experiments complexed to disks of dimyristoylphosphatidylcholine, to interact with two members of the low density lipoprotein receptor family, the low density lipoprotein receptor-related protein and the mosaic type-1 receptor, SorLA. Experiments using surface plasmon resonance showed specific binding of both free and lipid-bound apolipoprotein A-V to both receptors. The binding was calcium dependent and was inhibited by the receptor associated protein, a known ligand for members of the low density lipoprotein receptor family. Preincubation with heparin decreased the receptor binding of apolipoprotein A-V, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double mutant, apolipoprotein A-V (Arg210Glu/Lys211Gln), showed decreased binding to heparin and decreased ability to bind the low density lipoprotein receptor-related protein. Association of apolipoprotein A-V with the low density lipoprotein receptor-related protein or SorLA resulted in enhanced binding of human chylomicrons to receptor-covered sensor chips. Our results indicate that apolipoprotein A-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of triacylglycerol-rich lipoproteins. Udgivelsesdato: 2007-Mar-27

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

The expression of tissue transglutaminase (tTG) was studied during the formation of the normal human placenta and in molar pregnancies and choriocarcinoma, in order to correlate its expression with the functional characteristics of the recognized trophoblast cell types. tTG expression was found to be developmentally regulated. Before 6-7 weeks' gestation, only the chorionic villous cytotrophoblast expresses tTG. Thereafter the overlying syncytiotrophoblast becomes positive. tTG expression is gradually downregulated in the intermediate trophoblast cells emerging from the tips of the chorionic villi invading the uterine tissue. In the decidual wall, the intermediate trophoblast does not express tTG, whereas scattered syncytial cells, the placental bed giant cells, express tTG. Villi from complete hydatidiform mole (CHM) show tTG expression in both the cyto- and the syncytiotrophoblast. The intermediate trophoblast cells from CHM show heterogeneous tTG expression, with a majority of negative cells, whereas extravillous syncytia always express tTG. In choriocarcinoma, the tumour cells show heterogeneous tTG expression, with a majority of positive cells. Analysis of tTG protein and mRNA in placental extracts by Western and Northern blotting did not provide evidence for expression of the truncated form of tTG found in some cell types. The regulated expression of tTG in the normal placenta suggests that the enzyme is involved in important trophoblastic functions and may participate in the control of invasion.

Udgivelsesdato: 28. feb. 1997 Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C-terminal folding domain binds to alpha2MR/LRP, it remains uncertain whether it binds to heparin and to HSPG. To identify segments important for binding to alpha2MR/LRP and to clarify possible binding to heparin, we produced constructs of the human C-terminal folding domain, LpL-(313-448), and of the fragment LpL-(347-448) in Escherichia coli. In addition to binding to alpha2MR/LRP, LpL-(313-448) displayed binding to heparin with an affinity similar to that of the LpL monomer, whereas it bound poorly to lipoprotein particles. Moreover, LpL-(313-448) displayed heparin sensitive binding to normal, but not to HSPG deficient Chinese hamster ovary cells. LpL-(313-448) and LpL-(347-448) showed similar affinities for binding to both purified alpha2MR/LRP and to heparin. Deletion of LpL residues 380-384 abolished the binding to LRP, whereas binding to heparin was unperturbed. The binding to both heparin and alpha2MR/LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448). We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids within residues 404-430. Two segments of the domain are necessary for efficient binding to alpha2MR/LRP, one comprising residues 380-384 and another overlapping the segment important for binding to heparin.

Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity.

The β-amyloid peptide (βA4), derived from a larger amyloid precursor protein, is the principal component of senile plaques in Alzheimer's disease. Here we report that the full-length (1-40) synthetic βA4 peptide, containing one glutamine and two lysine residues, is able to form homopolymers in a transglutaminase-mediated reaction. Moreover, transglutaminase catalysed the formation of heteropolymers in reactions of βA4 with α2M receptor, a constituent of amyloid plaques, and with extracellular matrix proteins. Incorporation of site-specific probes followed by enzymatic digestion and sequencing of tracer-containing fractions demonstrated that both Lys16, Lys28 and Gin15 in βA4 were susceptible to cross-linking by transglutaminase.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Moller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thogersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

Udgivelsesdato: 1990-May-22 Receptors for the 54 kDa plasminogen activator urokinase were characterized in freshly isolated and 5-14 day cultured human monocytes. The half saturation constant was about 55 pM in freshly isolated monocytes at 4 degrees C and 140 pM at 37 degrees C. Diisopropylfluorophosphate-inactivated urokinase was bound with the same affinity as catalytically active urokinase. Binding per cell of 2-5 pM urokinase increased progressively during cell culture with a concomitant decrease in the apparent affinity. By 14 days, binding had increased 5-7-fold and the half-saturation constant had increased to 500 pM at 4 degrees C, indicating a large increase in the binding capacity. Affinity cross-linking of labelled urokinase to receptors showed a 110 kDa complex in both freshly isolated and cultured monocytes. When cells with labelled urokinase (prebound at 4 degrees C) were incubated at 37 degrees C, about 80% of the urokinase dissociated as the intact molecule, whereas about 20% was degraded to iodide and iodotyrosine. Electron microscopic autoradiography of cultured monocytes incubated at 4 degrees C showed a marked heterogeneity between cells with regard to bound urokinase. Autoradiographic grains were mainly seen over the plasma membrane in areas rich in microvilli and invaginations. Transfer of the cells to 37 degrees C caused no major alteration in the distribution of grains. Thus, freshly prepared monocytes have urokinase receptors (approx. 55 kDa) of high affinity. Development to macrophage-like cells in culture causes a decrease in affinity and a large increase in capacity. The receptors are confined mainly to certain areas of the plasma membrane. Internalization and degradation of the ligand occurs only to a minor extent.

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand.

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi-endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (Ψ-Ψ-X-X-∅) defining minimum requirements for GGA binding to cytoplasmic receptor domains. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

The cellular binding and uptake was studied for α 1 -inhibitor 3 , a monomeric 200 kDa proteinase inhibitor present in rat plasma. After intravenous injection in the rat the inhibitor disappeared from the circulation with a half-time of 2.5 min when complexed with chymotrypsin, whereas the half-time for uncomplexed inhibitor was more than 60 min. 6 min after the injection of labelled complex, 83% was in the liver and 2.5% in the spleen. In vitro experiments at 4°C with isolated hepatocytes and peritoneal macrophages showed binding to the previously described receptors which bind and internalize the tetrameric rat and human α 2 -macroglobulin-proteinase complexes. The binding affinities were similar for the two types of complexes and binding was followed by uptake and degradation of the labelled complex when the cells were warmed to 37°C. The binding of uncomplexed α 1 -inhibitor 3 was low and did not increase following treatment with methylamine in spite of cleavage of the internal thiol ester. α 1 -Inhibitor 3 -methylamine was changed to the receptor binding form when treated with chymotrypsin which caused the cleavage of at least one peptide bond in the bait region.