Abstract
The rat asialoglycoprotein receptor (ASGPR) is an integral transmembrane 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 sites of phosphorylation; in addition, RHL1 also contains one cytoplasmic Tyr. Based on [32P]PO4 metabolic radiolabeling experiments, Takahashi et al. (Takahashi, T., Nakada, H., Okumura, T., Sawamura, T., and Tashiro, Y. (1985) Biochem. Biophys. Res. Commun. 126, 1054-1060) concluded that RHL2 and RHL3 are phosphoproteins but that RHL1 is not. We report here that RHL1 in active ASGPR is, in fact, a phosphoprotein. Western 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 isolated hepatocytes were radiolabeled metabolically with [32P]PO4, RHL1, RHL2, and RHL3 became radiolabeled. Each ASGPR subunit was radiolabeled to a similar extent in the presence or absence of the ligand asialo-orosomucoid, indicating that functioning of the ASGPR does not change its steady-state 23P-radiolabeling. Phosphoamino acid analysis of radiolabeled ASGPR subunits identified Ser(P) as the predominant (approximately 95%) and Thr(P) as a minor (approximately 5%) phosphoamino acid in each polypeptide and confirmed the presence of Tyr(P) (approximately 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 RHL1.
Highlights
The rat asialoglycoproteinreceptor (ASGPR)is an in- H1, contains a cytoplasmic Tyr residue at position 5, we sustegral transmembrane glycoprotein composed of three pected that Tyr-5 in RHLl mightbe phosphorylated
We report here that RHLl in active ASGPR is, a phosphoprotein
Each ASGPR subunit was radiolabeled to a similar extent in the presence or absence of the ligand asialo-orosomucoid, indicating that functioning of the ASGPR does not change its steady-state=P-radiolabeling.Phosrecognize and bind these determinants and stimulate the assembly of a clathrin coat [5]
Summary
ASGPR Sepharose from total IgG fractions isolfartoemd egg yolks by a phoresis buffer and spotted ontEoM Science 20 x 20-cm plastic-backed modification [29] of the procedure describedby Polson et al [30]A. lka- cellulose thin layer plates. Phosphoamino acids were separabteydelecline phosphatase-conjugated rabbit anti-chickIegnG was obtained from trophoresis in pH 1.9 electrophoresis bufafetr1500 Vfor 30 min in the Jackson ImmunoResearch Laboratories. Phosphate-free Williams' medium E was prepared from indi- Western Blot Analysis-Subunits of affinity-purified ASGPR were vidual components[31].Buffer 1contains 142m~ NaC1,6.7 mM KCI, 10 separated by SDS-PAGE and electroblotted ontoImmobilon P. Ge~teral-~~rPadioactivity was determined by Cerenkov counting or cells/ml in medium 1BSA and incubatedat 37 "C for 1h in a gyratory by scintillation counting using Bio-Safe I1 scintillation mixture in a water bath in order to increase atnodstabilize the numbeorf ASGPRs Beckman LS 6000SE liquid scintillation spectrometer. Metabolic Radiolabeling a n d Purification ofActiue ASGPR-Isolated rat hepatocytes (3 x lo6celld35-mm plate) were culturedat 37 "C for 1
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