The 39-kDa receptor-associated protein (RAP) is cosynthesized and co-purifies with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and is thought to modulate ligand binding to LRP. In addition to binding LRP, RAP binds two other members of the low density lipoprotein (LDL) receptor family, gp330 and very low density lipoprotein (VLDL) receptors. Here, we show that RAP binds to LDL receptors as well. In normal human foreskin fibroblasts, RAP inhibited LDL receptor-mediated binding and catabolism of LDL and VLDL with Sf 20-60 or 100-400. RAP inhibited 125I-labeled LDL and Sf 100-400 lipoprotein binding at 4 degrees C with KI values of 60 and 45 nM, respectively. The effective concentrations for 50% inhibition (EC50) of cellular degradation of 2.0 nM 125I-labeled LDL, 4.7 nM 125I-labeled Sf 20-60, and 3.6 nM 125I-labeled Sf 100-400 particles were 40, 70, and 51 nM, respectively. Treatment of cells with lovastatin to induce LDL receptors increased cellular binding, internalization, and degradation of RAP by 2.3-, 1.7-, and 2.6-fold, respectively. In solid-phase assays, RAP bound to partially purified LDL receptors in a dose-dependent manner. The dissociation constant (KD) of RAP binding to LDL receptors in the solid-phase assay was 250 nM, which is higher than that for LRP, gp330, or VLDL receptors in similar assays by a factor of 14 to 350. Also, RAP inhibited 125I-labeled LDL and Sf 100-400 VLDL binding to LDL receptors in solid-phase assays with KI values of 140 and 130 nM, respectively. Because LDL bind via apolipoprotein (apo) B100 whereas VLDL bind via apoE, our results show that RAP inhibits LDL receptor interactions with both apoB100 and apoE. These studies establish that RAP is capable of binding to LDL receptors and modulating cellular catabolism of LDL and VLDL by this pathway.
SummaryLow Density Lipoprotein (LDL) is known to sensitize platelets for physiological agonists. To clarify the basis of this sensitization, we investigated the involvement of p38MAP Kinase (p38MAPK). As dual phosphorylation on Thr180 and Tyr182 of p38MAPK is the trigger for activation of the kinase, p38MAPK-activity was measured with an antibody that recognizes the dual-phosphorylated sequence. LDL induced a rapid and dose dependent activation of p38MAPK. The activation was not inhibited by a wide variety of inhibitors of platelet signalling, including TxA2-formation, Phospholipase C-activation, Ca2+-mobilization and ERK 1/2-activation. Only a slight reduction in p38MAPK-activation was observed when protein kinase C was inhibited. Activation of p38MAPK was strongly inhibited by a rise in cAMP. Thus, p38MAPK-activation was upstream of most signalling pathways and close to the LDL-receptor. A number of platelet receptors was screened with the use of antibodies. Integrins αIIbβ3 and α2β1, as well as the FcγRII-receptor, CD36 (platelet glycoprotein IV), CD68 (gp110) and Low Density Lipoprotein-receptor related protein (LRP) were not implicated in LDL-induced p38MAPK-activation. Inhibition of LDL binding by modification of apo B100 lysines reduced p38MAPK-activation by 80 %. Activation of p38MAPK resulted in an increase in release of arachidonic acid, the precursor for thromboxane A2 synthesis. In conclusion, activation of p38MAPK might be the first step in platelet sensitization by LDL, leading to formation of arachidonate metabolites and increased aggregation and secretion responses to physiological agonists.
Abstract-LDL is known to increase the sensitivity of human platelets for agonists and to induce aggregation and secretion independently at high concentrations, but its mechanism of action is largely obscure. To clarify how LDL increases platelet sensitivity, cells were incubated in lipoprotein-poor plasma and treated with collagen at a concentration that induced Ϸ20% secretion of 14 C-serotonin. Preincubation with LDL (30 minutes at 37°C) enhanced secretion in a dose-dependent manner to 60Ϯ14% at a concentration of 2 g LDL protein/L. Similar stimulation by LDL was seen when secretion was induced by the thrombin receptor-activating peptide. This enhancement was strongly reduced (1)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.