Mutation in the DC-SIGN cytoplasmic triacidic cluster motif markedly attenuates receptor activity for phagocytosis and endocytosis of mannose-containing ligands by human myeloid cells.
The transmembrane C-type lectin, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), has three conserved cytoplasmic tail motifs: the tyrosine (Y)-based, dileucine (LL), and triacidic cluster (EEE), which are believed to regulate ligand binding, uptake, and trafficking. We mutated each of these motifs by alanine substitution and tested their roles in phagocytosis and receptor-mediated endocytosis of the highly mannosylated ligands, Mycobacterium tuberculosis mannose-capped lipoarabinomannan (ManLAM) and HIV-1 surface glycoprotein gp120, respectively, in transfected human myeloid K-562 cells. Compared with wild-type and other mutants, the EEE mutant of DC-SIGN showed a reduced cell-surface expression, near abolishment in the phagocytosis of ManLAM-coated beads (90.5+/-0.4%), and a marked reduction in the endocytosis of soluble gp120 (79.3+/-0.7%). Although, the Y mutant of DC-SIGN did not exhibit any effect on phagocytosis and intracellular trafficking to the phagolysosome, the LL mutant caused the majority of the receptor and/or ligands to remain bound to the cell surface, indicating a role for the LL motif as an internalization signal. The majority of the EEE mutant protein was found to be retained by the intracellular trans-Golgi network and not by the late endosomal/lysosomal compartment of transfected K-562 cells. Collectively, our data indicate a dual role for the EEE motif as a sorting signal in the secretory pathway and a lysosomal targeting signal in the endocytic pathway.
Azad
Abul K
AK
The Center for Microbial Interface Biology, Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University, 460 West 12th Avenue, Biomedical Research Tower, Room 1004, Columbus, OH 43210, USA.
Torrelles
Jordi B
JB
Schlesinger
Larry S
LS
D019943:Amino Acid Substitution; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008561:Membrane Fusion; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D022423:Myeloid Cells / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D055571:Secretory Pathway; D014162:Transfection; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D015815:Cell Adhesion Molecules; D015699:HIV Envelope Protein gp120; D037181:Lectins, C-Type; D008070:Lipopolysaccharides; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin; C514793:gp120 protein, Human immunodeficiency virus 1; C050016:lipoarabinomannan
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