Intermediates in degradation of the erythropoietin receptor accumulate and are degraded in lysosomes.
The erythropoietin receptor (EPO-R) is synthesized in transfected Ba/F3 cells as a major 64-kDa endoglycosidase H (Endo H)-sensitive species, with a single N-linked oligosaccharide, and a minor 62-kDa unglycosylated form. Approximately half of the newly made EPO-R is processed to a mature 66-kDa form with a Golgi-processed Endo H-resistant oligosaccharide, of which only a minor fraction is expressed at the cell surface. Both the Endo H-sensitive and the Endo H-resistant forms of the receptor have a half-life of 45-60 min (Yoshimura, A., D'Andrea, A. D., and Lodish, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4139-4143). The mature, Endo H-resistant form of the EPO-R appears to be degraded in lysosomes or in other acidic organelles, since receptor degradation is blocked by treatment with NH4Cl, chloroquine, or leupeptin. A fraction of the Endo H-resistant EPO-R molecules is cleaved, generating two fragments of 46 and 39 kDa. The sizes of these fragments and their reactivities with carboxyl-terminal-specific antibodies indicate that the receptor is cleaved at two sites in the exoplasmic domain, 7 kDa apart, and carboxyl-terminal to the N-glycosylation site. Both fragments are membrane anchored and are probably formed in a late or post-Golgi compartment, since their formation is blocked by incubation of cells at 20 degrees C or by incubation with brefeldin A. These membrane-anchored COOH-terminal fragments are probably degraded in lysosomes or in other acidic vesicles as cell fractionation demonstrates that they colocalize with lysosomes, and similar to the intact EPO-R, their degradation is inhibited by NH4Cl. Finally, double labeling immunofluorescence experiments demonstrate that in NH4Cl-treated cells both intact mature EPO-R and the 46- and 39-kDa fragments accumulate in lysosomes and presumably are normally degraded there. The sensitivity of the EPO-R to endoproteolytic cleavages in its exoplasmic domain may relate to its low surface expression and to its extreme metabolic instability.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
D
D
Neumann
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L
Wikström
S S
SS
Watowich
H F
HF
Lodish
D016475:3T3 Cells; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006596:Hexosaminidases / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis; D017467:Receptors, Erythropoietin / Q000187:drug effects / Q000378:metabolism*
D007976:Leupeptins; D010446:Peptide Fragments; D017467:Receptors, Erythropoietin; D000643:Ammonium Chloride; D002738:Chloroquine; D003513:Cycloheximide; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C032854:leupeptin