Phosphorylation-dependent interaction of the asialoglycoprotein receptor with molecular chaperones.
A membrane protein trafficking mutant (Trf1) of HuH-7 alters the asialoglycoprotein (ASGPR) and transferrin receptor subcellular distribution. Expression cloning of a cDNA complementing the trf1 mutation led to the discovery of a novel casein Kinase 2 catalytic subunit (CK2alpha"). To purify potential CK2alpha" phosphorylation-dependent sorting proteins from cytosol, the ASGPR cytoplasmic domain was expressed as a GST fusion protein and immobilized on glutathione-agarose. In the absence of phosphorylation, only trace amounts of cytosol protein were bound and eluted. When the fusion protein was phosphorylated, a heterocomplex of potential sorting proteins was recovered. Mass spectrometer and immunoblot analysis identified five of these proteins as gp96, HSP70, HSP90, cyclophilin-A, and FKBP18. Treatment of HuH-7 with rapamycin to disrupt the heterocomplex reduced surface ASGPR binding activity by 65 +/- 5.7%. In Trf1 cells, surface-binding activity was 48 +/- 7% of that in HuH-7 and was not further reduced by rapamycin treatment. Immunoanalysis showed significantly fewer surface receptors on rapamycin-treated HuH7 cells than on nontreated cells, with no affect on the level of surface receptors in Trf1 cells. The data presented provide evidence that phosphorylation of the ASGPR cytoplasmic domain is required for the binding of specific molecular chaperones with the potential to regulate receptor trafficking.
Huang
Tianmin
T
Marion Bessin Liver Research Center and the Departments of Medicine and Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Deng
Haiteng
H
Wolkoff
Allan W
AW
Stockert
Richard J
RJ
D000595:Amino Acid Sequence; D037263:Asialoglycoprotein Receptor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D035321:Telomeric Repeat Binding Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D037263:Asialoglycoprotein Receptor; D018076:DNA, Complementary; D018832:Molecular Chaperones; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D035321:Telomeric Repeat Binding Protein 1; D020123:Sirolimus