THE RAT HEPATIC LECTIN-1 SUBUNIT OF THE RAT ASIALOGLYCOPROTEIN RECEPTOR IS A PHOSPHOPROTEIN AND CONTAINS PHOSPHOTYROSINE

The rat asialoglycoprotein receptor (ASGPR) is an integral transmembra ne glycoprotein composed of three polypeptide subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Each subunit contains one or more Ser and Thr residues in its cytoplasmic domain that are potential site s of phosphorylation; in addition, RHL1 also contains one cytoplasmic Tyr. Based on [P-32]PO4 metabolic radiolabeling experiments, Takahashi et al. (Takahashi, T., Nakada, H., Okumura, T., Sawamura, T., and Tas hiro, Y. (1985) Biochem. Biophys. Res. Commun. 126, 1054-1060) conclud ed that RHL2 and RHL3 are phosphoproteins but that RHL1 is not. We rep ort here that RHL1 in active ASGPR is, in fact, a phosphoprotein. West ern blot analysis using anti-Tyr(P) antibody identified Tyr(P) in RHL1 of affinity-purified ASGPRs. RHL2 and RHL3, which do not contain Tyr in their cytoplasmic domains, did not react with this antibody. When i solated hepatocytes were radiolabeled metabolically with [P-32]PO4, RH L1, RHL2, and RHL3 became radiolabeled. Each ASGPR subunit was radiola beled to a similar extent in the presence or absence of the ligand asi alo-orosomucoid, indicating that functioning of the ASGPR does not cha nge its steady-state P-23-radiolabeling. Phosphoamino acid analysis of radiolabeled ASGPR subunits identified Ser(P) as the predominant (sim ilar to 95%) and Thr(P) as a minor (similar to 5%) phosphoamino acid i n each polypeptide and confirmed the presence of Tyr(P) (similar to 1% ) in RHL1. Furthermore, treatment of hepatocytes with 3 mM vandate at 37 degrees C for 30 min doubled the steady-state level of Tyr(P) in RH L1.