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National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
The human NKG2A chain of the CD94/NKG2A receptor contains two
immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic
tail. To determine the relative importance of membrane-distal (residues
6–11) and membrane-proximal (residues 38–43) ITIMs in mediating the
inhibitory signal, we made site-directed mutants of NKG2A at the Y
(Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2)
of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated
NKG2A were then cotransfected with CD94 into rat basophilic leukemia
2H3 cells. Immunochemical analyses after pervanadate treatment showed
that each of the mutant molecules could be phosphorylated to expected
levels relative to wt NKG2A and that all the mutations significantly
reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for
NKG2A. Confocal microscopy was used to determine whether SH2
domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized
intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F
mutant NKG2A molecules failed to show a dramatic colocalization. In
agreement with this result, the Y8F/Y40F mutant was unable to inhibit
Fc
RI-mediated serotonin release and the Y8F mutant was relatively
ineffective compared with wt NKG2A. In contrast, the Y40F mutant was
70% as effective as wt in mediating inhibition, and the Y-2 mutations
did not remarkably affect inhibitory function. These results show that,
like KIR, both NKG2A ITIMs are required for mediating the maximal
inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of
primary importance rather than the membrane-proximal ITIM. This
probably reflects the opposite orientation of the ITIMs in type II vs
type I proteins.
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