title: Perturbation of C6 glial cells by acetate leads to modulation of [125I]IGF-II binding to the IGF-II/M6P receptor: implications for ligand internalization studies.
abstract: Acid washes are used as an experimental tool to differentiate between cell-surface bound and internalized radioligands. We have observed that washes with acid buffers containing 100 mM acetate can modulate [125I]IGF-II binding to rat C6 glial cells in an unexpected manner: when cells in monolayer culture were prewashed with phosphate buffered saline (pH 7.3) (PBS), [125I]IGF-II binding was characteristic of the IGF-II/mannose-6-phosphate (M6P) receptor. Importantly, IgG 3637, which is purified from an antiserum directed against the rat IGF-II/M6P receptor, blocked binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity crosslinking studies using DSS as the crosslinking agent and Western blotting experiments using antiserum 3637 confirmed the presence of the IGF-II/M6P receptor in C6 glial cells. Prewashes of C6 cell monolayers with acid buffers (pH 4-4.5) which contained 100 mM sodium acetate and which have been used in internalization studies reduced [125I]IGF-II binding by 40-60%. Affinity crosslinking studies using C6 cells showed that the formation of the 250 kDa radioligand-receptor complex was not prohibited by IgG 3637 after acid washes with buffers containing high acetate concentrations, while acid washes with buffers containing no acetate did not cause a loss in the blocking ability of IgG 3637. However, acid washes with 100 mM acetate did not alter the recognition of IGF-II/M6P receptors by IgG 3637 in Western blotting experiments. In addition, in a subset of experiments acid prewashes with acetate also decreased binding of [125I]IGF-I to the IGF-I receptor by 20%. We conclude that acid washes with acetate buffers lead to decreased [125I]IGF-I and [125I]IGF-II binding. In addition, the capability of anti-receptor IgG to block radioligand binding to the IGF-II/M6P receptor also declines. We hypothesize that alteration of ligand binding might be partially caused by perturbation of the cell membrane and hence a conformational change in IGF receptors. These data imply that the use of acetate buffers in acid wash experiments in ligand internalization studies--particularly in studies involving the IGF-II/M6P receptor--should be avoided.
authors.affiliation: Children's Hospital, Laboratory of Cell Biology, University of Munich, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Kiess
authors.forename: U
authors.initials: U
authors.lastname: Kessler
authors.forename: B
authors.initials: B
authors.lastname: Funk
authors.forename: A
authors.initials: A
authors.lastname: Hoeflich
mesh_terms: D000085:Acetates / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007457:Iodine Radioisotopes; D009457:Neuroglia / Q000187:drug effects*; D017527:Receptor, IGF Type 2 / Q000378:metabolism*
chemical_list: D000085:Acetates; D007457:Iodine Radioisotopes; D017527:Receptor, IGF Type 2; D007335:Insulin-Like Growth Factor II