Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences.
Rat 6 fibroblast cell lines expressing wild-type chicken liver glycoprotein receptor (CHL) or chimeric receptors with alternate cytoplasmic tails were produced to study the role of the cytoplasmic tail in mediating receptor localization in coated pits and endocytosis of ligand. Cells expressing CHL or cells expressing a hybrid receptor that contains the cytoplasmic tail of the asialoglycoprotein receptor display high-efficiency endocytosis of N-acetylglucosamine-conjugated bovine serum albumin in experiments designed to measure an initial internalization step, as well as in studies of continuous uptake and degradation. Substitution of the cytoplasmic tail by the equivalent domain of rat Na,K-ATPase beta subunit or by a stretch of Xenopus laevis globin beta chain does not abolish endocytosis but decreases the endocytosis rate constant from 15%-16%/min to 2.4% and 6.5%/min, respectively. Electron microscopy was used to visualize the glycoprotein binding sites at the surface of Rat 6 cells transfected with the various receptors. The percentage of receptors found in coated areas ranged from 32% for CHL to 9% for the Na,K-ATPase hybrid, indicating that clustering in coated pits correlates with efficiency of endocytosis. We concluded that replacement of the CHL cytoplasmic tail with unrelated sequences does not prevent, but decreases to varying extents, coated-pit localization and endocytosis efficiency. The construct with NH2-terminal globin tail lacks a signal for high-efficiency localization in coated pits but nevertheless is directed to the pits by an alternative mechanism.
Verrey
F
F
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Gilbert
T
T
Mellow
T
T
Proulx
G
G
Drickamer
K
K
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D002478:Cells, Cultured; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000737:chemistry; D013329:Structure-Activity Relationship; D014162:Transfection
D037263:Asialoglycoprotein Receptor; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic