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.
Department of Medicine, Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA.
Tamila L
TL
Kindwall-Keller
Lawrence J
LJ
Druhan
Jing
J
Ai
Melissa G
MG
Hunter
Pam
P
Massullo
Megan
M
Loveland
Belinda R
BR
Avalos
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