Role of the proteasome in modulating native G-CSFR expression.
The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis, but the mechanisms controlling its surface expression are poorly understood. Recent studies using transfected cell lines have suggested the activated G-CSFR is routed to the lysosome and not the proteasome. Here, we examined the role of the ubiquitin/proteasome system in regulating G-CSFR surface expression in both ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme and in primary human neutrophils. We show that the G-CSFR is constitutively ubiquitinated, which increases following ligand binding. In the absence of a functional E1 enzyme, ligand-induced internalization of the receptor is inhibited. Pre-treatment of ts20 transfectants with either chloroquine or MG132 inhibited ligand-induced G-CSFR degradation, suggesting a role for both lysosomes and proteasomes in regulating G-CSFR surface expression in this cell line. In neutrophils, inhibition of the proteasome but not the lysosome was found to inhibit internalization/degradation of the activated G-CSFR. Collectively, these data demonstrate the requirement for a functional ubiquitin/proteasome system in G-CSFR internalization and degradation. Our results suggest a prominent role for the proteasome in physiologic modulation of the G-CSFR, and provide further evidence for the importance of the ubiquitin/proteasome system in the initiation of negative signaling by cytokine receptors.
Kindwall-Keller
Tamila L
TL
Department of Medicine, Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA.
Druhan
Lawrence J
LJ
Ai
Jing
J
Hunter
Melissa G
MG
Massullo
Pam
P
Loveland
Megan
M
Avalos
Belinda R
BR
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011485:Protein Binding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D008024:Ligands; D061988:Proteasome Inhibitors; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D046988:Proteasome Endopeptidase Complex