BLOCKING INTRACELLULAR DEGRADATION OF THE ERYTHROPOIETIN AND ASIALOGLYCOPROTEIN RECEPTORS BY CALPAIN INHIBITORS DOES NOT RESULT IN THE SAMEINCREASE IN THE LEVELS OF THEIR MEMBRANE AND SECRETED FORMS

The erythropoietin receptor (EPO-R), a type 1 membrane glycoprotein, i s degraded mainly in the lysosomes or endosomes, whereas the asialogly coprotein receptor (ASGP-R) H2a subunit, a type 2 membrane glycoprotei n, is degraded exclusively in the endoplasmic reticulum. The present s tudy describes compounds that inhibit the intracellular degradation of these receptors in an efficient manner, However, the levels of cell-s urface expression and secretion of their soluble exoplasmic domains we re not enhanced to the same extent. The calpain inhibitors N-acetyl-le ucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methional (A LLM) inhibited EPO-R degradation profoundly. After 3 h of chase using Ba/F3 cells and NIH 3T3 fibroblasts expressing the EPO-R, virtually al l of the receptor molecules were degraded, whereas 80%, of the pulse-l abelled receptor remained intact in the presence of the inhibitor. EPO -R cell-surface expression was elevated 1.5-fold after 1 h of incubati on with ALLN. In the absence of protein synthesis, ALLN caused the acc umulation 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 tre atment increased secretion of the soluble exoplasmic domain of the EPO -R 2-5-fold. Similarly, in NIH 3T3 cells singly transfected with the A SGP-R H2a subunit cDNA, ALLN inhibited degradation of the ASGP-R H2a s ubunit precursor, as well as the degradation of the 35 kDa proteolytic fragment corresponding to the receptor ectodomain, by 3-6-fold. Howev er, accumulation of secreted proteolytic fragment in the medium was au gmented 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 th e 35 kDa fragment [Yuk and Lodish (1993) J. Cell Biol. 123, 1735-1749] , degradation of the precursor was inhibited. Overall, our data sugges t the involvement of cysteine proteinases located in the endoplasmic r eticulum, 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 inhibi tory compounds on cell-surface and secreted forms of the EPO-R and ASG P-R H2a subunit illustrates the complexity and the tight regulation of the cellular localization and stability of membrane proteins.