INHIBITION OF TYROSINE PHOSPHORYLATION IN THE RAT HEPATIC LECTIN-1 SUBUNIT OF THE RAT ASIALOGLYCOPROTEIN RECEPTOR PREVENTS ATP-DEPENDENT RECEPTOR INACTIVATION IN PERMEABILIZED HEPATOCYTES

We previously reconstituted the ATP dependent inactivation of asialogl ycoprotein 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 subu nit that becomes radiolabeled when permeabilized washed hepatocytes ar e incubated at 4 degrees C in the presence of [gamma-P-32]ATP; RHL2 an d RHL3 are not radiolabeled. Phosphorylation of RHL1 was rapid (t1/2 a pproximate to 4 min) and complete within 30 min. Inclusion of 20 mM ED TA inhibited phosphorylation of RHL1 completely. Phosphoamino acid ana lysis identified Tyr(P) as the predominant (>90%) radiolabeled phospho amino acid. Addition of vanadate enhanced phosphorylation of Tyr in RH L1 4-fold. Phosphorylation of RHL1 occurred to the same extent in hepa tocytes permeabilized with either 0.006% (w/v) or 0.055% digitonin and in the presence or the absence of ligand (50 mu g/ml asialo-orosomuco id; ASOR) and/or 10 mM CaCl2. Sequential purification of active ASGPRs (using ASOR-Sepharose) and inactive ASGPRs from the ASOR-Sepharose fl ow-through (using anti-ASGPR antibody-Sepharose) demonstrated that rad iolabeled RHL1 was present almost exclusively in active ASGPR oligomer s. When permeabilized hepatocytes radiolabeled with [gamma-P-32]ATP at 4 degrees C were warmed to 37 degrees C, a temperature at which ATP-d ependent ASGPR inactivation occurs, RHL1 was dephosphorylated rapidly (t1/2 approximate to 4 min) and completely within similar to 30 min. W estern blot analysis using a monoclonal anti-Tyr(P) antibody showed th at the steady-state level of endogenous Tyr(P) in RHL1 doubled as a re sult of ATP treatment at 4 degrees C and then decreased to undetectabl e levels upon warming to 37 degrees C. The protein-tyrosine kinase inh ibitor tyrphostin 51 inhibited phosphorylation of RHL1 at 4 degrees C and also prevented ATP-dependent ASGPR inactivation at 37 degrees C. W e conclude that phosphorylation of Tyr in RHL1 of active ASGPRs is a p rerequisite for ATP-dependent ASGPR inactivation.