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Phosphatidylinositol 3-Kinase Activation Is Required To Form the NKG2D Immunological Synapse

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri 63110,¹ Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo 4-1, Sendai 980-8575, Japan,² RIKEN Research Center for Allergy and Immunology, Laboratory for Lymphocyte Differentiation, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan,³ Departments of Medicine and Cell Biology, Washington University, St. Louis, Missouri 63110⁴

Received 15 August 2007/ Returned for modification 19 September 2007/ Accepted 25 September 2007

The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP₃), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP₃. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.

Published ahead of print on 8 October 2007.