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Vol. 15, Issue 7, 3210-3223, July 2004

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Exclusion of Lipid Rafts and Decreased Mobility of CD94/NKG2A Receptors at the Inhibitory NK Cell Synapse

Tolib B. Sanni ^*, Madhan Masilamani, Juraj Kabat, John E. Coligan, and Francisco Borrego 

Receptor Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852

Submitted November 3, 2003; Revised April 16, 2004; Accepted April 27, 2004
Monitoring Editor: Jennifer Lippincott-Schwartz

CD94/NKG2A is an inhibitory receptor expressed by most human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes.

Abbreviations used: APC, antigen presenting cells; BCR, B-cell receptor; Ctx-B, cholera toxin B; D_eff, diffusion coefficient; DIC, differential interference contrast; FcR1, Fc receptor for IgE alpha chain; HLA, human leukocyte antigen; ILT, Ig like transcript; IS, immunological synapse; ITIM, immunoreceptor tyrosine-based inhibitory motif; KIR, killer Ig-like receptor; MCD, methyl--cyclodextrin; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; NK, natural killer; NKIS, NK immunological synapse; RBL, rat basophilic leukemia; RMA-SE, HLA-E-transfected RMA-S cells; ROI, region of interest; SHP, SH2 domain-bearing tyrosine phosphatase; TCR, T-cell receptor.

Online version of this article contains supporting material. Online version is available at www.molbiolcell.org.

^* Present address: Department of Cell Biology, Georgetown University, Washington, DC 20007.

Corresponding author. E-mail address: Fborrego{at}niaid.nih.gov.

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