Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits.
The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.
Fuhrer
C
C
Department of Biochemistry, University of Basel, Switzerland.
Geffen
I
I
Spiess
M
M
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001483:Base Sequence; D002460:Cell Line; D002966:Clathrin / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry*
D037263:Asialoglycoprotein Receptor; D002966:Clathrin; D011971:Receptors, Immunologic; D014443:Tyrosine; D004247:DNA