ITIM-dependent endocytosis of CD33-related Siglecs: role of intracellular domain, tyrosine phosphorylation, and the tyrosine phosphatases, Shp1 and Shp2.
The leukocyte CD33-related sialic acid-binding Ig-like lectins (Siglecs) are implicated in glycan recognition and host defense against and pathogenicity of sialylated pathogens. Recent studies have shown endocytosis by CD33-related Siglecs, which is implicated in clearance of sialylated antigens and antigen presentation and makes targeted immunotherapy possible. Using CD33 as a paradigm, we have now investigated the reasons underlying the comparatively slow rate of endocytosis of these receptors. We show that endocytosis is largely limited and determined by the intracellular domain while the extracellular and transmembrane domains play a minor role. Tyrosine phosphorylation, most likely through Src family kinases, increases uptake of CD33 depending on the integrity of the two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Simultaneous depletion of the protein tyrosine phosphatases, Src homology-2-containing tyrosine phosphatase 1 (Shp1) and Shp2, which bind to phosphorylated CD33, increases internalization of CD33 slightly in some cell lines, whereas depletion of spleen tyrosine kinase (Syk) has no effect, implying that Shp1 and Shp2 can dephosphorylate the ITIMs or mask binding of the phosphorylated ITIMs to an endocytic adaptor. Our studies show that restraint of CD33 internalization through the intracellular domain is relieved partly when the ITIMs are phosphorylated and show that Shp1 and Shp2 can modulate this process.
Walter
Roland B
RB
Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., D2-373, Seattle, WA 98109-1024, USA. rwalter@fhcrc.org
Raden
Brian W
BW
Zeng
Rong
R
Häusermann
Peter
P
Bernstein
Irwin D
ID
Cooper
Jonathan A
JA
D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000276:immunology; D004705:Endocytosis / Q000276:immunology*; D018922:HL-60 Cells; D006801:Humans; D037102:Lectins / Q000502:physiology*; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000502:physiology*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism*; D063268:Sialic Acid Binding Ig-like Lectin 3; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D013154:Spleen / Q000201:enzymology; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000276:immunology
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D037102:Lectins; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D063268:Sialic Acid Binding Ig-like Lectin 3; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6