Abstract
The binding of 125I-labeled nerve growth factor (NGF) to human melanoma cell (A875) membranes, detergent-soluble membrane extracts, and membrane extracts reconstituted into phospholipid vesicles was significantly increased when binding was carried out in the presence of wheat germ agglutinin (WGA). In the absence of WGA, all 125I-NGF binding was rapidly eliminated by trypsin treatment or rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, in the presence of WGA, up to 75% of 125I-NGF bound was resistant to trypsin digestion and was only slowly dissociated by a high concentration of unlabeled NGF. The effects of WGA can be blocked or reversed by N-acetylglucosamine. Both WGA and NGF rapidly associate with soluble extracts and reconstituted vesicles and, at the concentrations used here, reach binding equilibrium within 2 min. The conversion to slowly dissociating, trypsin-resistant binding, however, was not complete for at least 10 min. Both WGA and NGF are required for maximum accumulation of trypsin-resistant, slowly dissociating binding. The order of addition of NGF and WGA has no effect on the rate of conversion of NGF-receptor, and the conversion occurs after both NGF and WGA are present. The amount of conversion is dependent on the incubation temperature, and significantly greater conversion occurs at 37 than at 0 degrees C. The generation of the trypsin-resistant, slowly dissociating state of NGF-receptor is consistent with a time- and temperature-dependent conformational change in NGF-receptor which occurs after interaction of both NGF and WGA with the receptor or closely associated structures.
Highlights
The binding of ‘261-labelednerve growth factor (NGF) is binding of the hormone to specific receptors. This initial to human melanoma cell (A875)membranes, detergent- interaction of NGF with its receptor hasbeen studied extensoluble membrane extracts, and membrane extracts sively (5-8), and two hypotheses have been proposed to exreconstituted into phospholipidvesicles was signifi- plain the kinetic and equilibrium binding data obtained
The datawere consistent with a model inwhich hormone ever, was not complete for at least 10 min. Both wheat germ agglutinin (WGA) binding triggered conversionof a low affinity state of receptor and NGF are required for maximum accumulation of from which NGF rapidly dissociated to a high affinity state trypsin-resistant, slowly dissociating binding
Schechter and der of addition of NGF and WGA has no effect on the Bothwell ( l l ), on the other hand, describedtwoclasses of rate of conversion of NGF-receptor, and the conversion NGF-receptors on PC12 cells with different kinetic and biooccurs after both NGF and WGA are present
Summary
The datawere consistent with a model inwhich hormone ever, was not complete for at least 10 min Both WGA binding triggered conversionof a low affinity state of receptor and NGF are required for maximum accumulation of from which NGF rapidly dissociated to a high affinity state trypsin-resistant, slowly dissociating binding. ‘”I-NGF dissociatedvery slowly from one class of receptor which was closely associated with the cytoskeletal matrix, resistant to trypsindigestion, and insoluble in low concentrations of Triton X-100 detergent. This slowly dissociating form of receptor hasalso been correlated with trypsin resistanceof the NGF’ is a peptide hormone which promotes morphological receptor (10, 11). it is not clear how the different and biochemical differentiation of sensory and sympathetic states of NGF-receptor originate, evaluation of receptor or neurons (I).Regulation of growth and differentiationby NGF hormone-receptor complex states is of interest in examining in other cell types of neural crest origin including PC12 rat early eventsin hormone-mediated stimulation, pheochromocytoma (2) and human melanomas(3,4) has Slowly reversible binding states for several other peptide been reported.
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