Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1

The type V TGF-β receptor (TβR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-β in responsive cells. Unexpectedly, TβR-V was recently found to be identical to the LRP-1/α₂M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-β₁, IGFBP-3, and α₂M*). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding α₂ M* at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by α₂M* affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and ¹²⁵I- α₂M*. Cell surface annexin VI is involved in ¹²⁵I-TGF-β₁ and ¹²⁵I-α ₂M* binding to the acidic pH binding sites and ¹²⁵I-α₂M* binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound ¹²⁵-TGF-β₁ and ¹²⁵I-α₂M* at pH 6 and of forming ternary complexes with ¹²⁵I-α₂M* and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound ¹²⁵I-TGF- β₁ or ¹²⁵I-α₂M*. These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.