Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor

Summary. The GM-CSF receptor (GM-CSFR) is composed of α and β subunits. Surface expression of the α chain alone leads to low affinity GM-CSF binding and of both subunits to high affinity binding: the β chain is required for transducing a proliferative signal. Studies of GM-CSFR expression have concentrated largely on static events occurring under conditions of binding equilibrium. We have examined the dynamic regulation of high and low affinity GM-CSFR expression in neutrophils (1100 ± 200 R/cell, K_D 50 ± 15 pm) and a GM-CSF dependent human leukaemic cell line, TF-l (2000 ± 450 R/cell K_D 15 ± 5 PM) and 8600 ± 150 R/cell K_D 1.8 ± 0.3 nm). The addition of GM-CSF to TF-1 cells (350 PM, 4 h at 37°C) caused a reduction in subsequent binding of ¹²⁵ I-GM-CSF at low ligand concentration (100 pm) (following a low pH wash to remove surface bound ligand) to 16 ± 4% and a reduction in binding at high ligand concentration (2 nm ¹²⁵I-GM-CSF) to 36 ± 9% of control. Scatchard analysis showed complete down-regulation of high affinity GM-CSFR and a significant reduction in low affinity GM-CSFR. In neutrophils, concentration-response curves of ligand induced receptor down-regulation at 37°C showed that observed down-modulation was more than 10-fold greater than predicted by static equilibrium binding data and correlated closely with GM-CSF priming of the neutrophil respiratory burst. The addition of IL-3 to TF-1 cells at 37°C reduced 100 PM ¹²⁵I-GM-CSF binding to 18 ± 4% and 2 nm ¹²⁵I-GM-CSF binding to 46 ± 5% of control. TF-1 cells, but not neutrophils, were able to re-express GM-CSFR following removal of GM-CSF from medium. TF-1 proliferation assays showed that pulsed GM-CSF (0.35–3.5 nm) for up to 4 h did not cause a significant increase in ³H-thymidine incorporation which required the continued presence of GM-CSF (control 2875 ± 208 cpm, pulsed GM-CSF 5 ng/ml 49 72 ± 1344. continuous GM-CSF 5 ng/ml 17249 ± 2982). Therefore, proliferation of TF-1 cells required the continued presence of GM-CSF at a time when there was no detectable surface high affinity GM-CSFR. This shows that signal transduction can take place via the GM-CSFR at a time when there is no detectable high affinity GM-CSF binding and introduces a further layer of complexity in the analysis of GM-CSF receptor function.