Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors.
The binding of erythropoietin (Epo) to its receptor leads to the transient phosphorylation of the Epo receptor (EpoR) and the activation of intracellular signaling pathways. Inactivation mechanisms are simultaneously turned on, and Epo-induced signaling pathways return to nearly basal levels after 30-60 min of stimulation. We show that proteasomes control these inactivation mechanisms. In cells treated with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal (LLnL) or lactacystin, EpoR tyrosine phosphorylation and activation of intracellular signaling pathways (Jak2, STAT5, phosphatidylinositol 3-kinase) were sustained for at least 2 h. We show that this effect was due to the continuous replenishment of the cell surface pool of EpoRs in cells treated with proteasome inhibitors. Proteasome inhibitors did not modify the internalization and degradation of Epo.EpoR complexes, but they allowed the continuous replacement of the internalized receptors by newly synthesized receptors. Proteasome inhibitors did not modify the synthesis of EpoRs, but they allowed their transport to the cell surface. N-Ac-Leu-Leu-norleucinal, but not lactacystin, also inhibited the degradation of internalized Epo.EpoR complexes, most probably through cathepsin inhibition. The internalized EpoRs were not tyrosine-phosphorylated, and they did not activate intracellular signaling pathways. Our results show that the proteasome controls the down-regulation of EpoRs in Epo-stimulated cells by inhibiting the cell surface replacement of internalized EpoRs.
Verdier
F
F
Institut Cochin de Génétique Moléculaire, INSERM U363 and the Service d'Hématologie, Hôpital Cochin, Université René Descartes, 27 Rue du Faubourg Saint Jacques, F75014 Paris, France.
Walrafen
P
P
Hubert
N
N
Chretien
S
S
Gisselbrecht
S
S
Lacombe
C
C
Mayeux
P
P
D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004921:Erythropoietin / Q000502:physiology; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D014443:Tyrosine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex