Hyperphosphorylation of the asialoglycoprotein receptor in isolated rat hepatocytes following ethanol administration

Ethanol administration leads to altered function and impaired receptor-medi ated endocytosis of the hepatocyte asialoglycoprotein receptor (ASGP-R). Th e purpose of the present study was to examine the effects of ethanol on the phosphorylation of the ASGP-R to determine whether this post-translational modification could contribute mechanistically to the observed ethanol-indu ced alterations. The methodological approach of this work involved the meas urement of the phosphorylation state of the receptor obtained from isolated rat hepatocytes, using a combination of experimental designs from the bios ynthetic incorporation of phosphate to the determination of steady-state ph osphotyrosine levels. We report here that both short-term (1- to 2-week) an d chronic (5- to 7-week) periods of ethanol administration resulted in a si gnificant increase in the steady-state phosphotyrosine protein in the ASGP- R. In addition, in vitro incorporation of [gamma-P-32]ATP using a permeabil ized cell assay system similarly showed an increase in tyrosine-phosphoryla ted receptors. Furthermore, metabolic radiolabeling of hepatocytes with [P- 32]orthophosphate demonstrated hyperphosphorylation of the ASGP-R in cells obtained from chronically ethanol-fed animals. Finally, our results reveale d that dephosphorylation of the ASGP-R was unaffected by ethanol administra tion, indicating that kinase activity rather than impaired phosphatase acti on contributes to the increased phosphorylation state of the receptor. Over all, the results presented in this study demonstrated that the extent of ty rosine phosphorylation of the receptor is significantly higher in hepatocyt es obtained from ethanol-fed animals. We conclude that hyperphosphorylation of the ASGP-R may be a contributing factor to the impaired function of the receptor elicited by ethanol administration. BIOCHEM PHARMACOL 60;3:343-35 1, 2000. (C) 2000 Elsevier Science Inc.