title: Mannose-6-phosphate enhances cross-linking efficiency between insulin-like growth factor-II (IGF-II) and IGF-II/mannose-6-phosphate receptors in membranes.
abstract: Endogenous and exogenous phosphomannosyl ligands inhibit binding of insulin-like growth factor-II (IGF-II) to the IGF-II/mannose-6-phosphate receptor (IGF-II/Man-6-P receptor). In the present study, the mechanism of this antagonism was examined using a [125I]IGF-II cross-linking assay with disuccinimidyl suberate in cell membranes. Treatment with 5 mM Man-6-P enhanced [125I]IGF-II cross-linking to the receptor. The magnitude of the Man-6-P enhancement differed depending on the source of the membranes, ranging from a 30% increase in JEG-3 human choriocarcinoma up to a 560% increase in B16-F1 mouse melanoma. Man-6-P stimulated [125I]IGF-II-receptor cross-linking in H-35 hepatoma membranes by about 80%, even at concentrations of labeled IGF-II (greater than or equal to 10 nM) that nearly saturated the receptors. Thus, in addition to its effect on IGF-II-binding affinity, Man-6-P caused a 1.5- to 2-fold increase in cross-linking efficiency within the IGF-II-receptor complex. Furthermore, Man-6-P enhanced [125I]IGF-II cross-linking to the H-35 receptor by a constant (approximately 80%) increment 1) when the cross-linking reaction was conducted in buffers of different pH over the range 6.8-8.0, or 2) using cross-linking agents differing in spacer arm length from 6.4-16.1 A. Washing membranes before assay with either Man-6-P (pH 7.4) or 0.5 M NaCl (pH 4.5) reduced the subsequent Man-6-P enhancement of [125I]IGF-II-receptor cross-linking, suggesting that this phenomenon was actually due to displacement of inhibitory phosphomannosyl ligands bound endogenously to the Man-6-P sites of the receptor. In support of this hypothesis, Man-6-P produced a minimal (8-14%) enhancement of [125I]IGF-II-receptor cross-linking in membranes from I-cell fibroblasts lacking such phosphomannosyl ligands. Thus, phosphomannosyl ligands bound to the IGF-II/Man-6-P receptor decrease both IGF-II-binding affinity and IGF-II-receptor cross-linking efficiency. Membrane-associated receptors appear to exist in experimentally and perhaps functionally distinct populations, depending on occupancy of the Man-6-P-binding sites.
authors.affiliation: Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198-4525.
authors.forename: R G
authors.initials: RG
authors.lastname: MacDonald
mesh_terms: D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism / Q000494:pharmacology*; D008970:Molecular Weight; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017451:Receptors, Somatomedin
chemical_list: D003432:Cross-Linking Reagents; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; C027693:mannose-6-phosphate; D007335:Insulin-Like Growth Factor II