Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II.
The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Mathieu
M
M
Unité Hormones and Cancer (U 148) INSERM, Montpellier, France.
Rochefort
H
H
Barenton
B
B
Prebois
C
C
Vignon
F
F
D001667:Binding, Competitive; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002402:Cathepsin D / Q000378:metabolism* / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007335:Insulin-Like Growth Factor II / Q000378:metabolism* / Q000494:pharmacology; D008360:Mannosephosphates / Q000378:metabolism; D008970:Molecular Weight; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013388:Succinimides; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D013388:Succinimides; C027693:mannose-6-phosphate; D007335:Insulin-Like Growth Factor II; D002402:Cathepsin D; C019358:disuccinimidyl suberate