Abstract
We previously reconstituted the ATP-dependent inactivation of asialoglycoprotein receptors (ASGPRs) in digitonin-permeabilized hepatocytes (Medh, J. D., and Weigel, P. H. (1991) J. Biol. Chem. 266, 8771-8778). Here we report that rat hepatic lectin 1 (RHL1) is the only ASGPR subunit that becomes radiolabeled when permeabilized washed hepatocytes are incubated at 4 degrees C in the presence of [gamma-32P]ATP; RHL2 and RHL3 are not radiolabeled. Phosphorylation of RHL1 was rapid (t1/2 appoximately 4 min) and complete within 30 min. Inclusion of 20 mM EDTA inhibited phosphorylation of RHL1 completely. Phosphoamino acid analysis identified Tyr(P) as the predominant (> 90%) radiolabeled phosphoamino acid. Addition of vanadate enhanced phosphorylation of Tyr in RHL1 4-fold. Phosphorylation of RHL1 occurred to the same extent in hepatocytes permeabilized with either 0.006% (w/v) or 0.055% digitonin and in the presence or the absence of ligand (50 micrograms/ml asialo-orosomucoid; ASOR) and/or 10 mM CaCl2. Sequential purification of active ASGPRs (using ASOR-Sepharose) and inactive ASGPRs from the ASOR-Sepharose flow-through (using anti-ASGPR antibody-Sepharose) demonstrated that radiolabeled RHL1 was present almost exclusively in active ASGPR oligomers. When permeabilized hepatocytes radiolabeled with [gamma-32P]ATP at 4 degrees C were warmed to 37 degrees C, a temperature at which ATP-dependent ASGPR inactivation occurs, RHL1 was dephosphorylated rapidly (t1/2 approximately 4 min) and completely within approximately 30 min. Western blot analysis using a monoclonal anti-Tyr(P) antibody showed that the steady-state level of endogenous Tyr(P) in RHL1 doubled as a result of ATP treatment at 4 degrees C and then decreased to undetectable levels upon warming to 37 degrees C. The protein-tyrosine kinase inhibitor tyrphostin 51 inhibited phosphorylation of RHL1 at 4 degrees C and also prevented ATP-dependent ASGPR inactivation at 37 degrees C. We conclude that phosphorylation of Tyr in RHL1 of active ASGPRs is a prerequisite for ATP-dependent ASGPR inactivation.
Highlights
- abilized washedhepatocytesare incubated at 4 "C in the for degradation, and the receptors return to the cell surface presence of [ys2P]ATP;RHL2 and RHL3 are not radiola- where they bind more ligand
ASGPRs from the ASOR-Sepharose flow-through(using phosphatase inhibitors suchas vanadate (18,19). These agents anti-ASGPR antibody-Sepharose)demonstrated that ra- induce inactivation andor redistribution of State 2 ASGPRs diolabeled RHLl was present almost exclusively in active ASGPR oligomers
ASGPR internalization via coated pits is a prerequisite for ASGPR inactivation (17)
Summary
Dept. of Biochemistry and Molecular Biology, BMSB 860, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190. The abbreviations used are: ASGPR, asialoglycoprotein receptor; ASOR, asialo-orosomucoid; RHL,rat hepatic lectin; a-"yr(Pm) Ab, antiTyr(P) monoclonalantibody;PMSF,phenylmethanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis. Cells were pelletedby centrifugation at 350 x g, resuspended in 500 pl of medium l/BSA containing 1.5 pg/ml '251-ASOR(50-100 c p d fmol ASOR) and 0.055%(w/v) digitonin and incubated at4 "C for 1h. We Treatment with 0.055% (w/v) digitoninpermeabilizes the cells comused digitonin-permeabilized rat hepatocytes to examine the pletely and allows determination of total (surface and internaAl)SGPR relationship between phosphorylation and inactivation of the activity (32). Balanced salt solution and resuspended i5n00 pl of Hanks', transferred to y counter tubes, and total bound lZ5I was determined.
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