title: Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1.
abstract: The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M 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-beta(1), IGFBP-3, and alpha(2)M(*)). 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 alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) 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)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) 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 (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). 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.
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Thai-Yen
authors.initials: TY
authors.lastname: Ling
authors.forename: Chun-Lin
authors.initials: CL
authors.lastname: Chen
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.forename: I-Hua
authors.initials: IH
authors.lastname: Liu
authors.forename: Shuan Shian
authors.initials: SS
authors.lastname: Huang
authors.forename: Jung San
authors.initials: JS
authors.lastname: Huang
mesh_terms: D000818:Animals; D017317:Annexin A6 / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005476:Fluphenazine / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D007781:Lactoferrin / Q000378:metabolism; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D051379:Mice; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012685:Sepharose / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000378:metabolism; D014268:Trifluoperazine / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D017317:Annexin A6; D007074:Immunoglobulin G; D008024:Ligands; D008077:Lipoproteins, LDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D014168:Transferrin; D000511:alpha-Macroglobulins; D014268:Trifluoperazine; D006493:Heparin; D002809:Chondroitin Sulfates; D012685:Sepharose; D004492:Edetic Acid; D007781:Lactoferrin; D005476:Fluphenazine; D002118:Calcium