title: Blocking intracellular degradation of the erythropoietin and asialoglycoprotein receptors by calpain inhibitors does not result in the same increase in the levels of their membrane and secreted forms.
abstract: The erythropoietin receptor (EPO-R), a type 1 membrane glycoprotein, is degraded mainly in the lysosomes or endosomes, whereas the asialoglycoprotein receptor (ASGP-R) H2a subunit, a type 2 membrane glycoprotein, is degraded exclusively in the endoplasmic reticulum. The present study describes compounds that inhibit the intracellular degradation of these receptors in an efficient manner. However, the levels of cell-surface expression and secretion of their soluble exoplasmic domains were not enhanced to the same extent. The calpain inhibitors N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methional (ALLM) inhibited EPO-R degradation profoundly. After 3 h of chase using Ba/F3 cells and NIH 3T3 fibroblasts expressing the EPO-R, virtually all of the receptor molecules were degraded, whereas 80% of the pulse-labelled receptor remained intact in the presence of the inhibitor. EPO-R cell-surface expression was elevated 1.5-fold after 1 h of incubation with ALLN. In the absence of protein synthesis, ALLN caused the accumulation of non-degraded EPO-R molecules in endosomes and lysosomes, as determined by double immunofluorescence labelling of NIH 3T3 cells expressing EPO-Rs. In Ba/F3 cells expressing a soluble EPO-R, ALLN treatment increased secretion of the soluble exoplasmic domain of the EPO-R 2-5-fold. Similarly, in NIH 3T3 cells singly transfected with the ASGP-R H2a subunit cDNA, ALLN inhibited degradation of the ASGP-R H2a subunit precursor, as well as the degradation of the 35 kDa proteolytic fragment corresponding to the receptor ectodomain, by 3-6-fold. However, accumulation of secreted proteolytic fragment in the medium was augmented in the presence of ALLN by only 1.75-fold. In cells expressing the G78R mutant of the ASGP-R H2a subunit, which is not cleaved to the 35 kDa fragment [Yuk and Lodish (1993) J. Cell Biol. 123, 1735-1749], degradation of the precursor was inhibited. Overall, our data suggest the involvement of cysteine proteinases located in the endoplasmic reticulum, as well as in post-Golgi compartments, in degradation of the EPO-R and the ASGP-R H2a subunit. The much lower effect of the inhibitory compounds on cell-surface and secreted forms of the EPO-R and ASGP-R H2a subunit illustrates the complexity and the tight regulation of the cellular localization and stability of membrane proteins.
authors.affiliation: Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Neumann
authors.forename: M H
authors.initials: MH
authors.lastname: Yuk
authors.forename: H F
authors.initials: HF
authors.lastname: Lodish
authors.forename: G Z
authors.initials: GZ
authors.lastname: Lederkremer
mesh_terms: D016475:3T3 Cells; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006868:Hydrolysis; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000187:drug effects* / Q000378:metabolism
chemical_list: D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; C089487:acetyl-leucinyl-leucinyl-methional; C063261:acetylleucyl-leucyl-norleucinal; D002154:Calpain