Haynes P A, Oka J A, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston 77555-0647.
J Biol Chem. 1994 Dec 30;269(52):33146-51.
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.
大鼠去唾液酸糖蛋白受体（ASGPR）是一种完整的跨膜糖蛋白，由三个多肽亚基组成，分别命名为大鼠肝凝集素（RHL）1、2和3。每个亚基在其胞质结构域中含有一个或多个丝氨酸（Ser）和苏氨酸（Thr）残基，这些残基是潜在的磷酸化位点；此外，RHL1还含有一个胞质酪氨酸（Tyr）。基于[32P]PO4代谢放射性标记实验，高桥等人（高桥，T.，中田，H.，冈村，T.，泽村，T.，和田代，Y.（1985年）生物化学与生物物理研究通讯126，1054 - 1060）得出结论，RHL2和RHL3是磷蛋白，但RHL1不是。我们在此报告，活性ASGPR中的RHL1实际上是一种磷蛋白。使用抗磷酸酪氨酸（Tyr(P)）抗体的蛋白质印迹分析在亲和纯化的ASGPRs的RHL1中鉴定出了磷酸酪氨酸。RHL2和RHL3在其胞质结构域中不含有酪氨酸，不与该抗体反应。当分离的肝细胞用[32P]PO4进行代谢放射性标记时，RHL1、RHL2和RHL3都被放射性标记。在有或没有配体去唾液酸-血清类黏蛋白存在的情况下，每个ASGPR亚基的放射性标记程度相似，这表明ASGPR的功能不会改变其稳态的32P放射性标记。对放射性标记的ASGPR亚基进行磷酸氨基酸分析，确定丝氨酸磷酸（Ser(P)）是每个多肽中的主要（约95%）磷酸氨基酸，苏氨酸磷酸（Thr(P)）是次要（约5%）磷酸氨基酸，并证实了RHL1中存在磷酸酪氨酸（约1%）。此外，在37℃用3 mM钒酸盐处理肝细胞30分钟，使RHL1中磷酸酪氨酸的稳态水平增加了一倍。
