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 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.
Haynes
P A
PA
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston 77555-0647.
Oka
J A
JA
Weigel
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D019000:Phosphotyrosine; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D037102:Lectins; D011956:Receptors, Cell Surface; D019000:Phosphotyrosine; D014443:Tyrosine