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Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of the CD16 complex (3), and the pp36 molecule (7) that may
be the recently cloned LAT (8). The activity of the catalytic domain of
SHP-1 is regulated by its tandem Src homology 2 (SH2) domains (9, 10).
Phosphopeptides that bind to the N-terminal SH2 domain induce the
phosphatase activity, suggesting that recruitment of SHP-1 through its
SH2 domains would lead to localized activation of SHP-1. Therefore, the
specificity of the SH2 domains is a critical determinant of where and
when SHP-1 is active.
The consensus motif I/VxYxxL/V was deduced from the sequences of
several receptors known to bind to the C-terminal SH2 domain of SHP-1
(1, 11). This ITIM occurs in NK receptors such as KIR, Ly49, NKG2A, and
gp49B; in B cell receptors such as CD22, FcRIIb, and PIR; and in
receptors expressed in monocytes and dendritic cells such as
ILT/MIR/LIR (reviewed in Refs. 12 and 13). The tyrosines in the
cytoplasmic tail of KIR, PIR, and Ly49A are required for inhibition
(14, 15, 16, 17). The involvement of upstream residues in a binding motif for
an SH2 domain was unprecedented. In vitro analysis with phosphopeptides
has established that the conserved residues both upstream and
downstream of the motif are important for binding to the C-terminal SH2
domain of SHP-1 (1, 18). Furthermore, a longer peptide with two
phosphorylated ITIMs produced greater activation of SHP-1 than the
combination of two shorter peptides, each carrying a separate ITIM
(10). We have performed mutagenesis of the ITIMs in KIR to assess the
relative importance of each ITIM and of the conserved I/V amino acid
N-terminal of the tyrosine in the functional context of MHC class
I-mediated inhibition of NK cells.
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Materials and Methods |
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The cell line 721.221 (19) was provided by R. DeMars, and the transfected 721.221 cell line .221-Cw3 (20) was provided by J. Gumperz and P. Parham. HEK 293T/17 cells were obtained from American Type Culture Collection (Manassas, VA) (21). Mouse NK cells were isolated from 4–6-month-old C57BL/6 mice as described (22). Briefly, nylon wool nonadherent cells were stimulated with 800 U/ml human rIL-2. On day 3 the plastic adherent cells were retained and restimulated with rIL-2. Purity was assessed by flow cytometry on day 6 with anti-NK1.1 and phycoerythrin-coupled anti-mouse CD3 (PharMingen, San Diego, CA). Cells were >99% NK1.1+ and ranged from 10 to 33% CD3+. Cytotoxicity assays were performed on day 7 or 8. Biochemical analysis of receptors was done on day 11. The mAb GL183 (Immunotech, Westbrook, ME) is an IgG1 reactive with the ectodomain of KIR-2DL3, and CH-L (23) is an IgG2b with similar specificity (a kind gift of S. Ferrini). MOPC21 is an IgG1 used as a control for immunoprecipitation and flow cytometric analysis (Sigma, St. Louis, MO). Western blotting was performed with polyclonal anti-SH-PTP1 reactive with human and mouse SHP-1- and biotin-conjugated 4G10 specific for phosphotyrosine (Upstate Biotechnology, Lake Placid, NY). Western blots were developed with horseradish peroxidase-goat anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) or horseradish peroxidase-streptavidin and the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).
Mutagenesis of KIR2DL3
The double-tyrosine mutant Y2F of KIR2DL3 (formerly known as KIR-6) has been described (15). Mutagenesis was performed using the Transformer Mutagenesis Kit (Clontech, Palo Alto, CA) in the plasmid RSV.5 (24). The fragment was subcloned into the vector pSC65 modified to include SalI–NotI cloning sites and used for recombination with vaccinia strain WR (25). The single tyrosine-to-phenylalanine mutant Y312F was generated as the first step for making Y2F, and the SalI–StuI fragment was subcloned into the modified pSC65. All other mutagenesis was done with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer’s guidelines. All mutagenic primers contained 10–15 flanking residues. The remainder of the mutagenesis was performed on the KIR2DL3 cDNA sequence in the vector pSPORT (26). Y282F was generated with the same forward mutagenic primer as described for Y2F (15) and the complementary reverse primer. The mutants 280A (Val280 to Ala) and 310A (Ile310 to Ala) were generated by the codon substitutions of GTG to GCG and ATC to GCC, respectively. To create the mutant -2A2, the 310A mutation was introduced into the 280A mutant. 310A/282F was created by introducing the 282F mutation onto the 310A in pSPORT. The SalI–NotI fragment of all mutants generated in pSPORT was subcloned into the modified pSC65. Introducing the 280A mutation into the 312F mutant in pSC65 created 280A/312F. The mutations were confirmed by sequence analysis. A silent mutation arose in the 282F in the region of the mutagenic primer. The entire coding sequence of each final product was verified to ensure that no other mutation was introduced into the receptors.
Infection with recombinant vaccinia viruses
Generation of recombinant vaccinia virus was as described (25). All vaccinia viruses were purified, and the titer in plaque-forming units (pfu) was determined in TK- cells. Mouse NK cells were used between days 7 and 9 of culture for cytotoxicity assays. For each condition, 750,000 cells were infected in 0.5 ml of Iscove’s medium supplemented with 0.1% BSA, nonessential amino acids, 2 mM glutamine, and 100 U/ml recombinant IL-2. The duration of the infection was 1.5–2 h at 37°C with 5% C02. The cells were washed once in 10 ml of warm assay medium (Iscove’s medium with 5% FBS, glutamine, and gentamicin). The cells were counted and diluted into assay medium with 100 U/ml rIL-2 at the appropriate concentration for E:T ratios of 9 and 3. NIH-3T3 cells were infected with crude preparations of recombinant vaccinia as described (1).
Immunoprecipitation
Infected cells were washed once with Dulbecco’s PBS and resuspended in Dulbecco’s PBS with 0.03% H2O2 and 0.2 µM NaV03 and incubated at 37°C for 10 min. The cells were pelleted and lysed in 0.5 ml of 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 µg/ml aprotinin, 1 mM pepstatin A, 1 mM NaVO3, 1 mM NaF, 5 mM iodoacetamide, 1 mM PMSF, and 20 mM Tris-HCl, pH 8. Debris was removed from the lysates by a 20-min centrifugation at 4°C. Receptors were immunoprecipitated by a 30-min incubation with 5 µg of GL183 and collected for 20 min with protein G bound to agarose beads (Life Technologies, Grand Island, NY). The pellets were washed three times with 1 ml of lysis buffer and analyzed by 10.5% SDS-PAGE under nonreducing conditions and immunoblotting.
Transient transfection
lck505F in the vector pSXSR
(27) was a gift from L. Samelson.
Wild-type and mutant KIR2DL3 were expressed from the plasmid RSV.5
(24). One day before transfection, HEK 293T/17 cells were plated at
5 x 105 cells per well in six-well plates in DMEM
supplemented with 10% FBS and 2 mM L-glutamine. Cells were
transfected with 2 µg of each plasmid using the CellPhect
transfection kit calcium phosphate reagents prepared essentially
according to the manufacturer’s instructions (Pharmacia Biotech,
Piscataway, NJ). The DNA mixture was added to the wells in 1 ml of
culture medium with 40 µM of chloroquine and incubated for 5 h.
The monolayer was washed twice with medium and cultured for 48 h
before analysis. Cells were removed from the plastic with PBS
containing 1 mM EDTA, and 5% of the recovered cells were used for flow
cytometric analysis. The remaining cells were lysed and
immunoprecipitated with GL813 as described, except that the samples
were precleared overnight in the presence of 5 µg of MOPC21 and
protein G-agarose beads.
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Results |
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KIR2DL3, a member of the KIR family, is specific for HLA-Cw3. The
two tyrosine residues in the cytoplasmic tail of KIR2DL3 were mutated
to phenylalanine independently and in combination (Fig. 1
). Receptor function was evaluated in
mouse NK cells that were mixed with human target cells coated with Abs
to induce Ab-dependent cellular cytotoxicity (ADCC) through the
FcRIII receptor. Mutated receptors expressed by recombinant vaccinia
viruses were compared at a multiplicity of infection of 20 pfu/NK cell,
which produced comparable levels of surface expression for all of the
receptors. Mutation of the membrane-distal tyrosine (312F) had little
to no effect on the ability of the receptor to inhibit an activation
signal delivered by the Fc receptor (Fig. 2). The data in Fig. 2 represent
activation with the highest dose of Ab and suggest that 312F is still
able to deliver an inhibitory signal under strong NK activation
conditions. In contrast, the receptor in which the membrane-proximal
tyrosine was mutated (282F) was unable to provide inhibition under the
same conditions. The inhibitory activity of mutant 282F was similar to
that of KIR2DL3 without a cytoplasmic tyrosine (Y2F).
Similar results were obtained at a 10-fold lower dose of Ab (data not
shown).
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A mutant of KIR2DL3 was constructed with Ala residues substituted
for the Val280 and Ile310 upstream of the
tyrosines and named -2A2 (Fig. 1
). The role of position -2
was also tested in the context of the first Tyr alone by substituting
Ala for Val280 in conjunction with the mutation of
Tyr312 to Phe (280A/312F) (Fig. 1). Recombinant vaccinia
viruses were employed to obtain expression of these mutant receptors in
mouse NK cells, and the strength of the inhibitory signal was tested in
a cytotoxicity assay (Fig. 3). A similar
level of surface expression for 280A/312F and -2A2 required
10-fold higher doses of virus than for the wild-type or the
Tyr-to-Phe mutants of KIR2DL3 (Fig. 3A and Table I). Flow cytometry was used to determine
the dose necessary to obtain matched levels of receptor. Two doses of
280A/312F are included in Fig. 3 for comparison. The mouse cells
displayed a low level of natural cytotoxicity toward .221 and .221-Cw3
cells in the absence of Abs. This low killing activity was inhibited by
all mutant receptors except Y2F. However, the ability of
the mutant receptors to prevent killing of .221-Cw3 cells decreased as
activation through ADCC was increased by addition of Ab specific for
target cells. The strength of inhibition by 280A/312F and
-2A2 correlates with the relative affinities for binding to
SHP-1 of phosphopeptides corresponding to the various mutant receptors
(10).
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To test the ability of the various receptors to coprecipitate
SHP-1, mouse NK cells were infected with vaccinia viruses encoding the
receptors and treated with pervanadate to induce tyrosine
phosphorylation (28). Mutation of either tyrosine alone decreased the
amount of tyrosine phosphorylation detectable by Western blotting to
less than half (Fig. 4,
middle). This low level of phosphorylation may be due to a
different efficiency of phosphorylation by the relevant kinases or to a
diminished ability of the Ab 4G10 to bind to a monophosphorylated
protein. In addition, despite treatment of the cells with pervanadate,
protection from phosphatases by stronger binding of SH2 domains to a
biphosphorylated tail than to a monophosphorylated one cannot be ruled
out. Similar results were observed in mouse 3T3 cells that lack SHP-1
(data not shown). However, phosphorylation of -2A2 was very
similar to that of the wild-type receptor, indicating that mutation of
residues at position -2 did not affect overall phosphorylation or
dephosphorylation. On the other hand, mutation of these residues
reduced the amount of SHP-1 immunoprecipitated, with the receptor below
the limit of detection. Even long exposures did not reveal SHP-1
associated with -2A2, 282F, or 312F. A similar amount of
receptor in each sample was detected with the mAb CH-L (Fig. 4), which
binds to the extracellular domain of KIR2DL3.
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). lck505 produced much higher
phosphorylation of the wild-type receptor compared with the 312F
mutant, in agreement with the data obtained in pervanadate-treated
cells.
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). The
amount of SHP-1 associated with -2A2 was much smaller than
that associated with the wild-type receptor (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results indicate that inhibition of ADCC requires the membrane-proximal tyrosine but not the membrane-distal tyrosine. A similar analysis of a chimeric molecule consisting of the extracellular domain of CD8 and a KIR cytoplasmic tail concluded that the membrane-proximal tyrosine was sufficient for inhibition of TCR activation of NFAT in Jurkat T cells (14). However, the CD8 chimeric receptor lacking the membrane-proximal tyrosine (corresponding to our 282F receptor) retained partial inhibitory activity in Jurkat cells (14). The difference with our results may relate to the nature or the efficiency of the inhibitory signal required to block cytotoxicity by NK cells as compared with transcriptional activation in T cells. An alternative explanation is that the single membrane-distal tyrosine is sufficient to provide an inhibitory signal when included in a receptor expressed as a homodimer, such as CD8, but not when present in the tail of a monomeric receptor such as KIR. Despite this difference, both studies show that the membrane-proximal ITIM is more potent than the membrane-distal ITIM. This conclusion is supported by the observation that a synthetic phosphopeptide corresponding to the membrane-proximal tyrosine binds SHP-1 with greater affinity than a peptide corresponding to the membrane-distal tyrosine (10, 18). It is noteworthy that the membrane-proximal tyrosine is in the context of the sequence QE(I/V)TYAQL that is conserved in a subset of the receptor families that bind SHP-1 such as PIR, ILT/MIR/LIR, Ly-49, and NKG2 (reviewed in 13 .
The mutant receptors carrying a single tyrosine were weakly phosphorylated in pervanadate-treated NK cells, as compared with the wild-type receptor, and did not coimmunoprecipitate SHP-1. Phosphorylation of receptors with a single tyrosine was also weak in cotransfection experiments with an active form of lck, the kinase believed to be responsible for phosphorylation of KIR (3). Despite weak tyrosine phosphorylation, the 312F mutant receptor was fully inhibitory when expressed in NK cells. These results reveal limitations in extrapolation of function from detection of protein-protein complexes by coimmunoprecipitation and emphasize the necessity to test mutant receptors in functional assays. A similar requirement for two tyrosines in the biliary glycoprotein (CD66a) for interaction with SHP-1 has been reported (29).
The residues at position -2 from the tyrosine contribute to the strength of the signal delivered by KIR but are not an absolute requirement for inhibition. The degree of inhibition delivered by mutants -2A2 and 280A/312F parallel the ability of phosphopeptides corresponding to these sequences to bind and activate SHP-1 in vitro (10). The I/V at position -2 is important for maintaining the association of KIR and SHP-1 in detergent lysates. The -2A2 mutant receptor was phosphorylated as well as the wild-type receptor, yet we failed to detect SHP-1 in association with -2A2 in NK cells. However, an association of SHP-1 with -2A2 was observed after overexpression of the molecules in heterologous cells. These results suggest that SHP-1 mediates the inhibition by -2A2 but do not rule out the possibility that another molecule binding to the mutants can substitute for SHP-1. However, we have not observed association of the mutants with likely candidates such as SHP-2 or SHIP (18, 30) (data not shown). The importance of position -2 for SHP-1 association may depend on the particular ITIM and may be compensated by the presence of more than one SHP-1 binding motif in the receptor. The overall avidity of SHP-1 for a peptide carrying two phosphorylated ITIMs is greater than the sum of the affinities for each individual phosphorylated ITIM (10). Consistent with this in vitro finding, the mutant -2A2 with tyrosines was a stronger inhibitor than either mutant 282F or mutant 280A/312F that have a single tyrosine.
An unexpected observation was that mutation of the I/V at position -2 affects the surface expression of KIR. The reduced surface expression occurred when either Val280 or Ile310 was mutated to Ala, suggesting that these mutations exposed a cryptic determinant in the sequence that perturbs surface expression levels. Matched surface expression levels required higher doses of recombinant vaccinia virus for the receptors mutated at position -2. Despite greater doses of recombinant vaccinia virus, the total cellular pool of KIR detected by immunoprecipitation and Western blotting was not greater. This observation would be consistent with a faster turnover of the mutated KIR. Further analysis is required to determine the underlying mechanism and the implications for receptors with similar sequences.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric Long, LIG-NIAID-NIH Twinbrook II, 12441 Parklawn Drive, Rockville, MD 20852-1727. E-mail address:
3 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; ITIM, immunoreceptor tyrosine-based inhibitory motif; SH2, Src homology 2; pfu, plaque-forming unit; ADCC, antibody-dependent cellular cytotoxicity.
Received for publication May 26, 1998. Accepted for publication October 2, 1998.
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signaling complex in human natural killer (NK) cells. J. Exp. Med. 184:2243.RIIB. Nature 383:263.[Medline]
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) or
infected at 20 pfu per cell with vaccinia virus expressing wild type
(
), Y282F (
), Y312F (
), or Y2F (•). Cells were
infected for 1.5 h and assayed for the ability to lyse .221 and
.221-Cw3 cells coated with L243 mAb at 0.1 µg/ml in a 4-h assay of
51Cr release. The level of receptor expressed at the cell
surface at the beginning of the lysis assay was assessed with the mAb
GL183. The mean fluorescence intensity in this experiment was 9 for
uninfected cells; 240 for cells infected with wild type; and 257 for
Y282F, 263 for Y312F, and 258 for Y2F cells. Each condition
was tested in triplicate, and the experiment presented is
representative of three independent experiments.
), 280A/312F at 48 pfu/cell
(
), -2A2 (
), and Y2F (•), as well as
the uninfected cells (
