Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts

A dynamic balance of positive and negative signals regulates target cell lysis by natural killer (NK) cells upon engagement of a variety of different activation receptors and of inhibitory receptors that recruit the tyrosine phosphatase SHP-1. However, the step at which activation signals are blocked by SHP-1 is not known. We have been using activation receptor 2134 (CD244) to study the influence of inhibitory receptors on NK cell activation. Engagement of inhibitory receptors by HLA class I on target cells blocks phosphorylation of 2134, placing the inhibitory step at the level, or upstream of 2134 phosphorylation. Here we show that phosphorylated 2134, after engagement with either antibodies or target cells that express the 2134 ligand, is found exclusively in a detergent-resistant membrane fraction that contains lipid rafts. Integrity of lipid rafts was essential for phosphorylation and activating function of 2134. Coengagement of inhibitory receptors blocked 2134 phosphorylation and 2134 association with detergent-resistant membranes, indicating that inhibitory receptors function upstream of raft-dependent signals. Recruitment of 2134 into detergent-resistant membrane fractions and 2134 phosphorylation were dependent on actin polymerization. Blocking actin cytoskeleton-dependent raft recruitment of different receptors may be a general mechanism by which inhibitory receptors control NK cell activation.