39 – 41). This prevented an interaction with the phosphatase SHP-2
(10, 39, 41, 42) and facilitated the recruitment of the phosphatase
SHIP (10, 43) or the Src family kinases FynT (43). Although this
issue has not been addressed directly in this study, it is unlikely
that this ITSM serves as a docking site for SAP-family adaptor
molecules. Indeed Siglec-10, which contains two membrane-prox-
imal ITIMs and one membrane-distal ITSM-like motif, does not
bind to the SAP molecule in a three-hybrid system (44).
Finally, we have shown here that mutation of the membrane-
proximal ITIM of Siglecs-7 and -9 leads to an increase in the sialic
acid-dependent binding of RBC, whereas mutation of the mem-
brane-distal motif has no effect. Similar results have been reported
previously with Siglec-3/CD33 using transiently transfected COS
cells (22) and may be a general feature of human CD33-related
Siglecs. The fact that SHP-2 was only recruited with Y2 mutants,
which displayed low levels of RBC binding similar to the WT
forms of Siglecs-7 and -9, raised the possibility that interactions of
this phosphatase could negatively regulate the ligand binding ac-
tivity of Siglecs-7 and -9. Binding of sialic acid residues by mo-
nomeric Siglecs is generally of very low afﬁnity and therefore
clustering of receptors within the plasma membrane is essential for
high-avidity interactions with ligands on other cells. Although we
failed to see gross changes in distribution of Siglecs-7 or -9 com-
paring WT and mutant forms of the proteins expressed on trans-
fected RBL cells by confocal light microscopy, there could be
subtle changes that would require higher resolution imaging tech-
niques for their visualization.
In conclusion, we demonstrate that Siglec-9 is a new member
the inhibitory receptor superfamily and that the membrane-proxi-
mal ITIM is essential for the inhibitory function of both Siglecs-7
and -9 molecules.
Note added in proof. A recent publication demonstrated the in-
hibitory activity of Siglec-7 and Siglec-9 in transfected Jurkat cells
(Ikehara, Y., S. K. Ikehara, and J. C. Paulson. 2004. Negative
regulation of T cell receptor signaling by Siglec-7 (p70/AIRM)
and Siglec-9. J. Biol. Chem. 279:43117.).
We thank Benjamin Neel for the kind gift of SHP-1 and SHP-2 cDNA,
Lars Nitshke for providing the anti-phosphotyrosine mAb, Mathias Lucas
for the mouse IgE Abs, Claire Jones for the sheep anti-Siglec-9 Abs,
Gavin Nicoll and Kevin Lock for the anti-Siglec-7 Abs, Jiquan Zhang for
the anti-Siglec-9 mAb, and Andrew Ferenbach and Faye Wesley for the
SHP-1 and SHP-2-GFP constructs.
1. Crocker, P. R., and A. Varki. 2001. Siglecs, sialic acids and innate immunity.
Trends Immunol. 22:337.
2. Crocker, P. R. 2002. Siglecs: Sialic-acid-binding immunoglobulin-like lectins in
cell-cell interactions and signalling. Curr. Opin. Struct. Biol. 12:609.
3. Cornish, A. L., S. Freeman, G. Forbes, J. Ni, M. Zhang, M. Cepeda, R. Gentz,
M. Augustus, K. C. Carter, and P. R. Crocker. 1998. Characterization of siglec-5,
a novel glycoprotein expressed on myeloid cells related to CD33. Blood 92:2123.
4. Floyd, H., J. Ni, A. L. Cornish, Z. Zeng, D. Liu, K. D. Carter, J. Steel, and
P. R. Crocker. 2000. Siglec-8. A novel eosinophil-speciﬁc member of the im-
munoglobulin superfamily. J. Biol. Chem. 275:861.
5. Vivier, E., and M. Daeron. 1997. Immunoreceptor tyrosine-based inhibition mo-
tifs. Immunol. Today 18:286.
6. Ravetch, J. V., and L. L. Lanier. 2000. Immune inhibitory receptors. Science
7. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.
8. Long, E. O. 1999. Regulation of immune responses through inhibitory receptors.
Annu. Rev. Immunol. 17:875.
9. Staub, E., A. Rosenthal, and B. Hinzmann. 2004. Systematic identiﬁcation of
immunoreceptor tyrosine-based inhibitory motifs in the human proteome. Cell
10. Shlapatska, L. M., S. V. Mikhalap, A. G. Berdova, O. M. Zelensky, T. J. Yun,
K. E. Nichols, E. A. Clark, and S. P. Sidorenko. 2001. CD150 association with
either the SH2-containing inositol phosphatase or the SH2-containing protein
tyrosine phosphatase is regulated by the adaptor protein SH2D1A. J. Immunol.
11. Sidorenko, S. P., and E. A. Clark. 2003. The dual-function CD150 receptor sub-
family: The viral attraction. Nat. Immunol. 4:19.
12. Meyaard, L., G. L. Adema, C. Chang, E. Woollatt, G. R. Sutherland, L. L. Lanier,
and J. H. Phillips. 1997. LAIR-1, a novel inhibitory receptor expressed on human
mononuclear leukocytes. Immunity 7:283.
13. Zhang, J. Q., G. Nicoll, C. Jones, and P. R. Crocker. 2000. Siglec-9, a novel sialic
acid binding member of the immunoglobulin superfamily expressed broadly on
human blood leukocytes. J. Biol. Chem. 275:22121.
14. Falco, M., R. Biassoni, C. Bottino, M. Vitale, S. Sivori, R. Augugliaro,
L. Moretta, and A. Moretta. 1999. Identiﬁcation and molecular cloning of p75/
AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory
receptor in human natural killer cells. J. Exp. Med. 190:793.
15. Nicoll, G., J. Ni, D. Liu, P. Klenerman, J. Munday, S. Dubock, M. G. Mattei, and
P. R. Crocker. 1999. Identiﬁcation and characterization of a novel siglec, siglec-7,
expressed by human natural killer cells and monocytes. J. Biol. Chem.
16. Blixt, O., B. E. Collins, I. M. van den Nieuwenhof, P. R. Crocker, and
J. C. Paulson. 2003. Sialoside speciﬁcity of the siglec family assessed using novel
multivalent probes: Identiﬁcation of potent inhibitors of myelin-associated gly-
coprotein. J. Biol. Chem. 278:31007.
17. Yamaji, T., T. Teranishi, M. S. Alphey, P. R. Crocker, and Y. Hashimoto. 2002.
A small region of the natural killer cell receptor, Siglec-7, is responsible for its
preferred binding to
2,8-disialyl and branched
2,6-sialyl residues. A compar-
ison with Siglec-9. J. Biol. Chem. 277:6324.
18. Nicoll, G., T. Avril, K. Lock, K. Furukawa, N. Bovin, and P. R. Crocker. 2003.
Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity
via siglec-7-dependent and -independent mechanisms. Eur. J. Immunol. 33:1642.
19. Vitale, C., C. Romagnani, M. Falco, M. Ponte, M. Vitale, A. Moretta,
A. Bacigalupo, L. Moretta, and M. C. Mingari. 1999. Engagement of p75/AIRM1
or CD33 inhibits the proliferation of normal or leukemic myeloid cells. Proc.
Natl. Acad. Sci. USA 96:15091.
20. Blery, M., J. Delon, A. Trautmann, A. Cambiaggi, L. Olcese, R. Biassoni,
L. Moretta, P. Chavrier, A. Moretta, M. Daeron, and E. Vivier. 1997. Reconsti-
tuted killer cell inhibitory receptors for major histocompatibility complex class I
molecules control mast cell activation induced via immunoreceptor tyrosine-
based activation motifs. J. Biol. Chem. 272:8989.
21. Angata, T., S. C. Kerr, D. R. Greaves, N. M. Varki, P. R. Crocker, and A. Varki.
2002. Cloning and characterization of human Siglec-11. A recently evolved sig-
naling that can interact with SHP-1 and SHP-2 and is expressed by tissue mac-
rophages, including brain microglia. J. Biol. Chem. 277:24466.
22. Taylor, V. C., C. D. Buckley, M. Douglas, A. J. Cody, D. L. Simmons, and
S. D. Freeman. 1999. The myeloid-speciﬁc sialic acid-binding receptor, CD33,
associates with the protein-tyrosine phosphatases, SHP-1 and SHP-2. J. Biol.
23. Ulyanova, T., J. Blasioli, T. A. Woodford-Thomas, and M. L. Thomas. 1999. The
sialoadhesin CD33 is a myeloid-speciﬁc inhibitory receptor. Eur. J. Immunol.
24. Paul, S. P., L. S. Taylor, E. K. Stansbury, and D. W. McVicar. 2000. Myeloid
speciﬁc human CD33 is an inhibitory receptor with differential ITIM function in
recruiting the phosphatases SHP-1 and SHP-2. Blood 96:483.
25. Ulyanova, T., D. D. Shah, and M. L. Thomas. 2001. Molecular cloning of MIS,
a myeloid inhibitory siglec, that binds protein-tyrosine phosphatases SHP-1 and
SHP-2. J. Biol. Chem. 276:14451.
26. Yu, Z., M. Maoui, L. Wu, D. Banville, and S. Shen. 2001. mSiglec-E, a novel
mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that
recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases
SHP-1 and SHP-2. Biochem. J. 353:483.
27. Fry, A. M., L. L. Lanier, and A. Weiss. 1996. Phosphotyrosines in the killer cell
inhibitory receptor motif of NKB1 are required for negative signaling and for
association with protein tyrosine phosphatase 1C. J. Exp. Med. 184:295.
28. Bruhns, P., P. Marchetti, W. H. Fridman, E. Vivier, and M. Daeron. 1999. Dif-
ferential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition
motifs during inhibition of cell activation by killer cell inhibitory receptors. J. Im-
29. Burshtyn, D. N., A. S. Lam, M. Weston, N. Gupta, P. A. Warmerdam, and
E. O. Long. 1999. Conserved residues amino-terminal of cytoplasmic tyrosines
contribute to the SHP-1-mediated inhibitory function of killer cell Ig-like recep-
tors. J. Immunol. 162:897.
30. Yusa, S., and K. S. Campbell. 2003. Src homology region 2-containing protein
tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function
of killer cell Ig-like receptors in human NK cells. J. Immunol. 170:4539.
31. Yusa, S., T. L. Catina, and K. S. Campbell. 2004. KIR2DL5 can inhibit human
NK cell activation via recruitment of Src homology region 2-containing protein
tyrosine phosphatase-2 (SHP-2). J. Immunol. 172:7385.
32. Verbrugge, A., T. T. Ruiter, H. Clevers, and L. Meyaard. 2003. Differential
contribution of the immunoreceptor tyrosine-based inhibitory motifs of human
leukocyte-associated Ig-like receptor-1 to inhibitory function and phosphatase
recruitment. Int. Immunol. 15:1349.
33. Kabat, J., F. Borrego, A. Brooks, and J. E. Coligan. 2002. Role that each NKG2A
immunoreceptor tyrosine-based inhibitory motif plays in mediating the human
CD94/NKG2A inhibitory signal. J. Immunol. 169:1948.
34. Yusa, S., T. L. Catina, and K. S. Campbell. 2002. SHP-1- and phosphotyrosine-
independent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic do-
main in human NK cells. J. Immunol. 168:5047.
35. Burshtyn, D. N., A. M. Scharenberg, N. Wagtmann, S. Rajagopalan, K. Berrada,
T. Yi, J. P. Kinet, and E. O. Long. 1996. Recruitment of tyrosine phosphatase
HCP by the killer cell inhibitor receptor. Immunity 4:77.
6848 MUTATIONAL ANALYSIS OF THE TYROSINE-BASED MOTIFS IN SIGLECS-7 AND -9