Role of Tyrosine-5 in the Cytoplasmic Tail of the Macrophage Asialoglycoprotein Receptor in the Rapid Internalization of Ligands1

摘要

A macrophage asialoglycoprotein binding protein (M-ASGP-BP), which is an endocytic receptor specific for Gal/GalNAc-terminated glycoproteins, was shown to be functionally active as a homooligomer (hexamer or octamer) of a single polypeptide chain of 42 kDa Ozaki, K., Ii, M., Itoh, N.,Kawasaki, T. (1992)J. BioL Chem.267,9229–9236. In various endocytic receptors, a four-amino-acid sequence, Tyr-X-Y-Phe, in the cytoplasmic domain has been identified as an internalization signal Pearse, B.M.F.Robinson, M.S. (1990) Annu. Rev. Cell BioL 265,151-171. The amino-terminus of the M-ASGP-BP deduced from its cDNA sequence was found to contain the sequence, Tyr5-Glu6-Asn7-Phe8, in its cytoplasmic tail. This was confirmed by the fact that the recombinant M-ASGP-BP isolated from transfected COS-1 cells was found to have the amino-terminal sequence, Thr2-Met3- Ala4-Tyr5-Glu6-Asn7-Phe8. The role of this presumptive internalization signal in the cytoplasmic tail was studied by measuring the endocytic activity of the wild-type and mutant M-ASGP-BPs expressed on COS-1 cells through transfection with the wild-type and mutant cDNAs prepared by oligonucleotide-directed mutagenesis, respectively. On the deletion of Tyr5 or replacement of it with alanine, the internalization of asialoorosomucoid (ASOR) decreased to approximately one-fourth that in the case of the wild-type molecule. On replacement of Tyr5 with phenylalanine, the internalization proceeded at a rate similar to that in the case of the wild-type molecule. On the other hand, the levels of the receptor on the surface of the cells were essentially the same for the wild-type and mutant receptors. These results suggest that Tyr5 in the recombinant M-ASGP-BP molecule is necessary for its rapid internalization. Co-transfection with the wild-type and Tyr5-deleted mutant cDNAs in various ratios resulted in the expression of the surface receptor in essentially the same numbers. However, the internalization rate of the receptor decreased roughly in proportion to the decrease in the amount of the wild-type lectin cDNA. These results suggest that the presence of multiple (three or more) Tyr5 residues on a receptor molecule is required for its rapid internalizatio