Ligation of Low-Density Lipoprotein Receptor-Related Protein with Antibodies Elevates Intracellular Calcium and Inositol 1,4,5-Trisphosphate in Macrophages

We have probed the signaling characteristics of the macrophage low-density lipoprotein receptor-related protein (LRP) with monoclonal antibody 8G1, its Fab and F(ab′)2 fragments directed against the ligand binding heavy chain, and monoclonal antibody 5A6 directed against the membrane-spanning light chain of LRP. Ligation of LRP with 8G1, its Fab and F(ab′)2 fragments, or 5A6 increased intracellular Ca2+ levels two- to threefold. Prior ligation of LRP with 8G1 did not affect the increase in [Ca2+]i observed on subsequent ligation of LRP with lactoferrin, P. exotoxin A, or lipoprotein lipase. Binding to LRP by 8G1, its Fab and F(ab′)2 fragments, or 5A6 increased inositol 1,4,5-trisphosphate (IP3) levels by 50 to 100%. Incubation of macrophages with guanosine 5′,3′-O(thio)-triphosphate (GTP-γ-S) before treatment with antibody potentiated and sustained the 8G1-induced increase in IP3 levels. Treatment of macrophages with guanyl-5′-yl thiophosphate prior to GTP-γ-S treatment abolished the GTP-γ-S-potentiated increase in IP3 levels in 8G1-treated macrophages. Antibody-induced increases in IP3 and [Ca2+]i in macrophages on ligation of LRP were pertussis toxin sensitive. Binding of 8G1 or its Fab or F(ab′)2 fragments to LRP stimulated macrophage protein kinase C (PKC) activity as evaluated by histone IIIs phosphorylation by about two- to sevenfold. Staurosporin inhibited the anti-LRP antibody-induced increase in PKC activity. Ligation of LRP with 8G1 increased cellular cAMP levels about twofold. Preincubation of macrophage with the LRP-binding protein receptor-associated protein suppressed the 8G1-induced increase in cAMP levels. Thus, binding of antibodies directed against either chain of LRP triggers complex signaling cascades.