The 39-kDa Receptor-associated Protein Modulates Lipoprotein Catabolism by Binding to LDL Receptors

Jheem D Medh,Glenna L Fry,Susan L Bowen,Marc W Pladet,Dudley K Strickland,David A Chappell

doi:10.1074/jbc.270.2.536

Jheem D Medh, Glenna L Fry + Show 4 more

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https://doi.org/10.1074/jbc.270.2.536

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Abstract

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.

Highlights

From the Wepartment of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242 and the §Biochemistry Laboratory, American Red Cross, Rockville, Maryland 20855
Because low density lipoprotein (LDL) bind via apolipoprotein B100 whereas very low density lipoprotein (VLDL) bind via apoE, our results show that receptor-associated protein (RAP) inhibits LDL receptor interactions with both apoB100 and apoE
It is well established that RAP inhibits the interactions of lipoprotein receptor-related protein (LRP) with all of its known ligands including o2M-proteinase complexes [6, 7], apoE-enriched {:l-migrating very low density lipoproteins ({:lVLDL) [6], lipoprotein lipase [8, 9], complexes of tissue-type and urokinase plasminogen activators with their inhibitor (l014), and Pseudomonas exotoxin A [15], In addition, fibroblasts genetically engineered to overexpress RAP overexpress LRP. 2 it is believed that RAP modulates LRP-ligand interactions and may function in the intracellular transport of LRP as well

Summary

11 To whom reprint requests should be addressed

Dept. of Internal Medicine, E318 GH, University ofIowa College of Medicine, Iowa City, IA 52242. 2 it is believed that RAP modulates LRP-ligand interactions and may function in the intracellular transport of LRP as well. Kounnas et al [3] have shown that RAP binds gp330 with a high affinity and may modulate interactions of gp330 with its ligands [3, 16]. Because RAP binds to LRP [17, 18] and gp330 [3, 19], both members of the LDL receptor family, we speculated that RAP might bind LDL receptors themselves. We demonstrate that RAP binds to LDL receptors both in normal human foreskin fibroblasts and solid-phase binding assays.

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DISCUSSION