Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor.
The GM-CSF receptor (GM-CSFR) is composed of alpha and beta subunits. Surface expression of the alpha chain alone leads to low affinity GM-CSF binding and of both subunits to high affinity binding; the beta 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, KD 50 +/- 15 pM) and a GM-CSF dependent human leukaemic cell line, TF-1 (2000 +/- 450 R/cell KD 15 +/- 5 pM) and 8600 +/- 1150 R/cell KD 1.8 +/- 0.3 nM). The addition of GM-CSF to TF-1 cells (350 pM, 4 h at 37 degrees C) caused a reduction in subsequent binding of 125I-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 degrees 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 degrees 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 4972 +/- 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Khwaja
A
A
Department of Haematology, University College London Medical School.
Carver
J
J
Jones
H M
HM
Linch
D C
DC
D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007942:Leukemia, Experimental / Q000378:metabolism; D008024:Ligands; D009504:Neutrophils / Q000378:metabolism / Q000648:ultrastructure; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000187:drug effects / Q000502:physiology*; D016897:Respiratory Burst / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007377:Interleukin-3; D008024:Ligands; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013755:Tetradecanoylphorbol Acetate