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RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 degrees C, whereas temperature change from 22 degrees C to 37 degrees C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.

The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3-59 nm) and most that contain only two CR domains, although binding energies to different (CR)(3) fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.

Analysis of genetic susceptibility factors for Alzheimer's disease (AD) in populations with different genetic and environmental background may be useful to understand AD etiology. There are few genetic association studies of AD in Latin America. In the present work, we analyzed polymorphisms in 3 candidate genes; the LDL receptor related protein-1, the microtubule-associated protein Tau and the serotonin transporter genes in a sample of 106 Colombian AD patients and 97 control subjects. We did not find a significant allelic or genotypic association with any of the three polymorphisms analyzed using different statistical analysis, including a neural network model or different sample stratifications. To date, APOE polymorphisms are the only genetic risk factors identified for AD in the Colombian population. It may be factible that future combination of high-throughput genotyping platforms and multivariate analysis models may lead to the identification of other genetic susceptibility factors for AD in the Colombian population. © Springer-Verlag 2005.

Udgivelsesdato: 2004-Dec-24 The low density lipoprotein receptor-related protein (LRP) mediates the endocytic clearance of various proteinases and proteinase.inhibitor complexes, including thrombospondin (TSP)-dependent endocytosis of matrix metalloproteinase (MMP)-2 (or gelatinase A), a key effector of extracellular matrix remodeling and cancer progression. However, the zymogen of MMP-2 (pro-MMP-2) mostly occurs in tissues as a complex with the tissue inhibitor of MMPs (TIMP-2). Here we show that clearance of the pro-MMP-2.TIMP-2 complex is also mediated by LRP, because addition of receptor-associated protein (RAP), a natural LRP ligand antagonist, inhibited endocytosis and lysosomal degradation of (125)I-pro-MMP-2.TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of (125)I-pro-MMP-2.TIMP-2 complex. Surface plasmon resonance studies indicated that pro-MMP-2, TIMP-2, and pro-MMP-2.TIMP-2 directly interact with LRP in the absence of TSP. LRP-mediated endocytic clearance of (125)I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on (125)I-pro-MMP-2.TIMP-2 uptake. This implies that mechanisms of clearance by LRP of pro-MMP-2 and pro-MMP-2.TIMP-2 complex are different. Interestingly, RAP did not inhibit binding of (125)I-pro-MMP-2.TIMP-2 to the cell surface. We conclude that clearance of pro-MMP-2.TIMP-2 complex is a TSP-independent two-step process, involving (i) initial binding to the cell membrane in a RAP-insensitive manner and (ii) subsequent LRP-dependent (RAP-sensitive) internalization and degradation.

Background. Human mesothelial cells (HMCs) have an important role in maintaining an adequately functioning fibrinolytic system in the peritoneal cavity by secreting the fibrinolytic enzymes tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), as well as a specific PA inhibitor, PA inhibitor type 1 (PAI-1). In this study, we investigated whether the fibrinolytic capacity of HMCs is further counterbalanced by rapid uptake of t-PA and u-PA from the medium. Methods. Cultured HMCs were used to study the uptake and degradation of radiolabeled t-PA and u-PA in the absence or presence of an inhibitor of cellular protein degradation, chloroquine, and of specific receptor antagonists. Northern blotting and ligand-blotting techniques were applied to demonstrate the presence of specific receptors for binding of t-PA and u-PA. Results. At 37°C, HMCs rapidly internalized and degraded 125I-t-PA and 125I-u-PA, which could be inhibited by an excess of unlabeled t-PA anti u-PA, respectively, and by the lysosomotropic agent chloroquine. Northern blot analysis showed the expression of low-density lipoprotein (LDL) receptor-related protein (LRP), very low density lipoprotein (VLDL) receptor, and u-PA receptor. The addition of recombinant 39 kDa receptor-associated protein (RAP; an inhibitor of LRP and VLDL receptor) almost completely blocked the degradation of t-PA and partly that of u-PA. RAP ligand blotting demonstrated predominantly the presence of LRP, suggesting a major role for the LRP in mediating uptake and degradation of t- PA in HMCs. Endocytosis of u-PA occurs via two different pathways. After binding to u-PA receptor, a RAP-inhibitable and a non-RAP-inhibitable route for u-PA degradation was demonstrated. Tumor necrosis factor α (TNFα) diminished the fibrinolytic activity of HMCs by decreasing t-PA and increasing PAI-1 synthesis. The fall in t-PA levels could be counteracted by inhibiting t-PA degradation by either RAP or chloroquine. Interestingly, chloroquine also quenched the TNFα-induced changes in t-PA and PAI-1 mRNA levels. Using TNFα mutants and agonistic or blocking monoclonal antibodies specific for the TNF receptors p55 and p75, we found evidence that chloroquine interfered with the activation of the TNF receptor p55 and/or its intracellular signaling route. Conclusions. Receptor-mediated endocytosis plays a crucial role in regulating the fibrinolytic capacity of HMCs by its participation in the degradation of t-PA and u-PA, and in the TNFα-induced decrease in t-PA and the increase in PAI-1 expression. Chemicals/CAS: Antibodies; Carrier Proteins; Chloroquine, 54-05-7; Culture Media, Conditioned; Glutathione Transferase, EC 2.5.1.18; GST-RAP protein, recombinant; Iodine Radioisotopes; LDL-Receptor Related Protein 1; Lectins, C-Type; mannose receptor; Mannose-Binding Lectins; Plasminogen Activator Inhibitor 1; plasminogen activator, urokinase receptors; Receptors, Cell Surface; Receptors, Immunologic; Receptors, LDL; Recombinant Fusion Proteins; RNA, Messenger; Tissue Plasminogen Activator, EC 3.4.21.68; Tumor Necrosis Factor-alpha; Urinary Plasminogen Activator, EC 3.4.21.73; VLDL receptor

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)- independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor- associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre+LRP(flox/flox)LDLR(-/-)) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDL-sized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre+LRP(flox/flox)LDLR(-/-) mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K(d) = 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation. Chemicals/CAS: Cholesterol, 57-88-5; Chylomicrons; Detergents; Heymann Nephritis Antigenic Complex; LDL-Receptor Related Protein 1; Lipase, EC 3.1.1.3; Lipoprotein Lipase, EC 3.1.1.34; Membrane Glycoproteins; Polyethylene Glycols; Receptors, Immunologic; Receptors, LDL; Triglycerides; tyloxapol, 25301-02-4

Udgivelsesdato: 1987-Oct-1 125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.