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, it s Fab and F(ab')(2) fragments directed against the Ligand binding heavy cha in, and monoclonal antibody 5A6 directed against the membrane-spanning ligh t 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 Li(i)ga tion of LRP with 8G1 did not affect the increase in [Ca2+](i) observed on s ubsequent ligation of LRP with lactoferrin, P. exotoxin A, or Lipoprotein l ipase, Binding to LRP by 8G1, its Fab and F(ab')(2) fragments, or 5A6 incre ased inositol 1,4,5-trisphosphate (IP3) levels by 50 to 100%. Incubation of macrophages with guanosine 5',3'-O(thio)-triphosphate (GTP-gamma-S) before treatment with antibody potentiated and sustained the 8G1-induced increase in IP3 levels. Treatment of macrophages with guanyl-5'-yl thiophosphate pr ior to GTP-gamma-S treatment abolished the GTP-gamma-S-potentiated increase in IP3 levels in 8G1-treated macrophages. Antibody-induced increases in IP 3 and [Ca2+](i) in macrophages on ligation of LRP were pertussis toxin sens itive. Binding of 8G1 or its Fab or F(ab')(2) fragments to LRP stimulated m acrophage protein kinase C (PKC) activity as evaluated by histone IIIs phos phorylation by about two- to sevenfold, Staurosporin inhibited the anti-LRP antibody-induced increase in PKC activity. Ligation of LRP with 8G1 increa sed cellular cAMP levels about twofold. Preincubation of macrophage with th e LRP-binding protein receptor-associated protein suppressed the 8G1-induce d increase in cAMP levels. Thus, binding of antibodies directed against eit her chain of LRP triggers complex signaling cascades. (C) 1999 Academic Pre ss.