We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB.mg cell protein-1.h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; beta-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100-containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48-transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.

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The low density receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) binds to several ligands involved in lipoprotein and protease clearance. The receptor-associated protein (RAP) inhibits the binding of all known ligands. We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha 2-MR. Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha 2-macroglobulin (125I-alpha 2-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells. Ni2+ also inhibited the binding of trypsin-activated alpha 2-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low-density lipoproteins. The inhibition of alpha 2-M* binding by Ni2+ was not due to its interaction with alpha 2-M*. Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha 2-M* but does not affect RAP binding. Most of the binding of alpha 2-M* to SMC was due to its binding to the LRP/alpha 2-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP. Moreover, the inhibition of alpha 2-M* binding to the LRP/alpha 2-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates. Two to three molecules of 63Ni2+ bound to the immobilized receptor with equal affinity but not to alpha 2-M*. The specific binding of alpha 2-M* to the immobilized receptor was inhibited in the presence of nickel.(ABSTRACT TRUNCATED AT 250 WORDS)