Abstract
Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I–specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed that signaling by an HLA-C–specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that KIR binding to HLA-C must override the hindrance that His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C+ target cells than did expression of wild-type KIR2DL1. These results revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.
Introduction
Regulation of many cellular processes depends on inhibitory receptors that carry ITIMs in their cytoplasmic tail (1). NK cell cytotoxicity and cytokine secretion are tightly regulated by ITIM-containing inhibitory receptors (2). Engagement of NK cell inhibitory receptors with their MHC class I ligands on target cells leads to ITIM phosphorylation and recruitment of the protein tyrosine phosphatase (PTPase) SHP-1. SHP-1 bound to phosphorylated ITIMs in inhibitory killer cell Ig-like receptors (KIR) is activated and dephosphorylates the guanine nucleotide exchange factor Vav1, thereby inhibiting actin-dependent signals for NK cell activation (2, 3). Inhibition of activation signals by ITIM-bearing receptors is local and transient and does not globally impair NK cell responses to other stimuli (4, 5). Based on these characteristics, inhibitory receptors have been considered coinhibition receptors (6). According to earlier models, their phosphorylation is dependent on tyrosine kinases associated with signaling by activation receptors. Kinases of the Src family, such as Lyn and Lck, have been implicated in the phosphorylation of ITIMs (7–9).
Signals from inhibitory receptors in NK cells block activation signals at a very early step, prior to the actin-dependent clustering of activation receptors and the full signaling cascade for activation. For example, inhibition by ITIM-containing receptors blocks inside-out signals from coactivation receptors to β2 integrin (10) and β2 integrin–mediated adhesion (11). Photoactivation of HLA-C–peptide complexes induced microclusters of inhibitory KIR within seconds of ligand binding, as well as inhibited NK cell attachment to ICAM-1 and the formation of activation receptor clusters (12). These properties of inhibitory KIR are not easily reconciled with models that place the phosphorylation of ITIMs downstream of signaling by activation receptors.
Recent studies revealed that the binding of inhibitory KIR2DL1 to HLA-C, as well as of inhibitory receptor CD94-NKG2A to HLA-E, results in tyrosine phosphorylation of the small adaptor molecule Crk (13). Because binding of CD94-NKG2A to purified HLA-E is sufficient to trigger Crk phosphorylation, the ITIM-bearing CD94-NKG2A can signal independently of activation receptor signaling (14). We studied the regulation of KIR2DL1 phosphorylation and its association with SHP-1. We identified a gain-of-function, single amino acid mutant of KIR2DL1 that is constitutively phosphorylated. We propose that KIR2DL1, in its basal state, is subjected to a continuous cycle of phosphorylation and dephosphorylation and that KIR2DL1 self-association facilitates phosphorylation by protecting phosphorylated ITIMs from PTPases, thereby shifting the equilibrium in favor of phosphorylation.
Materials and Methods
Cell lines and reagents
The human NK cell line YTS was transfected with wild type (WT), ITIM Tyr mutant (wherein both Tyr residues were mutated to Phe) (2YF), and His36-to-Ala substitution (H36A) mutant of KIR2DL1. Each one of them was tagged with Venus at the cytosolic end. The transfectants were selected in 1 μM puromycin. They are referred to as YTS-2DL1-WT-Venus, YTS-2DL1-2YF-Venus, and YTS-2DL1-H36A-Venus in this article. Expression of KIR2DL1 in these transfectants was comparable to KIR2DL1 in primary NK cells (Supplemental Fig. 1). YTS cells were cultured in RPMI 1640 supplemented with glutamine, 10% FBS, and 50 μM 2-ME (R10 medium). YTS cells express HLA-C*01 and HLA-C*08, two group C1 allotypes that are not ligands for KIR2DL1. The YTS transfectants were cultured in R10 medium supplemented with 1 μM puromycin.
721.221 cell lines (referred to as 221 cells) transfected with HLA-Cw3 and HLA-Cw4 were obtained from J. Gumperz and P. Parham (Stanford University, Stanford, CA). These 221 transfectants were cultured in R10 medium. TAP-deficient 221-HLA-Cw4 cells were generated by transfection of ICP-47-IRES-GFP (15) into 221-HLA-Cw4 cells, followed by selection in 1 μM puromycin. Cells were sorted for high GFP expression and low HLA-C on the cell surface.
Abs
The following Abs were used in this study: anti-GFP (11814460001, Roche; A6455, Invitrogen), anti–phosphotyrosine-biotin (4G10-biotin; Upstate), anti–SHP-1 (610126, BD Transduction Laboratories; 07-419, Upstate), and anti–HLA-C (F4/326 [IgG2a], a gift from S.Y. Yang, Memorial Sloan-Kettering Cancer Center, New York, NY). The HRP-conjugated Abs were from Santa Cruz. Streptavidin-HRP Ab was obtained from GE Healthcare. Allophycocyanin-conjugated anti-KIR2DL1 Ab used in flow cytometer studies was from Beckman Coulter (A22332).
DNA mutagenesis and transfection
A KIR2DL1 cDNA tagged at the C terminus with Venus was cloned into a lentiviral vector pCDH-EF1-MCS-T2A-Puro (CD520A-1; System Biosciences) using XbaI and NotI. The resulting plasmid is referred to as p2DL1-WT-Venus. The ITIM tyrosine mutant Y281,311F (2YF) and H36A mutants were generated by site-directed mutagenesis on p2DL1-WT-Venus. Stable YTS transfectants, expressing 2DL1-WT, 2DL1-2YF, and 2DL1-H36A, each tagged with Venus at the C terminus, were generated by transduction of lenti-pseudoviral particles packaged with the cDNA into YTS cells, followed by selection in 1 μM puromycin (System Biosciences). The construct expressing Cerulean-tagged SHP-1 was made by cloning a SHP-1 cDNA, tagged at the C terminus with a glycine-serine linker of 12 residues followed by Cerulean, into pCDH-EF1-MCS-T2A-Puro (CD520A-1; System Biosciences) using XbaI and NotI. The resulting plasmid is referred to as pSHP-1–Cerulean. The cDNA encoding the N-terminal (1–154; VN155) and C-terminal (155–238 with I152L mutation; VC155) fragments of Venus were PCR amplified from the plasmids pBiFC-VN155I152L [pBiFC-VN155(I152L) was a gift from Chang-Deng Hu (Addgene plasmid # 27097)] and pBiFC-VC155 (pBiFC-VC155 was a gift from Chang-Deng Hu [Addgene plasmid # 22011]), respectively. The GFP portion of the plasmid 2DL1-TG (16) was replaced with VN155 cDNA or VC155 cDNA. These resulting plasmids are referred to as p2DL1-WT-VN155 and p2DL1-WT-VC155, and they express KIR2DL1 tagged at the C terminus (cytosolic end) with VN155 and VC155, respectively. The H36A and H36-to-phenylalanine substitution (H36F) mutants of KIR2DL1 tagged with VN155 or VC155 were generated by site-directed mutagenesis on p2DL1-WT-VN155 or pDL1-WT-VC155. To make the construct expressing VN155-tagged SHP-1, the GFP coding region of pEGFP expression vector (Clontech Laboratories) was replaced with a SHP-1 cDNA, tagged at C terminus with a glycine-serine linker of 12 residues followed by VN155 using XhoI and NotI sites. The resulting plasmid is referred to as pSHP-1-VN155. Transfection for transient protein expression in YTS cells was achieved using nucleofection (VCA-1003; Amaxa Nucleofection Kit V). Typically, two million cells and 2–4 μg plasmid DNA were used for each transfection. The plasmids encoding Venus tagged with a K-Ras membrane-targeting sequence (V-K-Ras) were described previously (17). In the construct encoding Venus dimer, the two Venus molecules are attached in tandem with a 5-aa linker. Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The following primers were used for site-directed mutagenesis: H36A, 5′-GTCATGTTTGAAGCCTTCCTTCTGCACAGAGAGGGG-3′ and its complement and H36F, 5′-GTCATGTTTGAATTCTTCCTTCTGCACAGAGAGGGG-3′ and its complement.
Bimolecular fluorescence complementation and flow cytometry
YTS cells were transfected with combinations of VN155 and VC155 constructs using nucleofection (VCA-1003; Amaxa Nucleofection Kit V). After 4 h of incubation at 37°C, cells were washed once with PBS supplemented with 1% FBS. Transfected cells were stained with allophycocyanin-conjugated anti-KIR2DL1 Ab (A22332; Beckman Coulter) for 30 min on ice to probe the receptor surface expression. Fluorescence signals arising from bimolecular fluorescence complementation (BiFC) and anti-KIR2DL1–allophycocyanin were measured using a flow cytometer (BD FACSCalibur). The data were analyzed using FlowJo software. The BiFC signals from different samples were compared within gates that had the same expression level of the receptor, as determined by surface staining with the anti-KIR2DL1–allophycocyanin Ab.
Confocal microscopy
Confocal microscopy was performed using a Zeiss LSM-710 confocal microscope with a 63×/1.4 oil-immersion objective. Differential interference contrast images were acquired simultaneously with the fluorescence images of cells. The measurements of fluorescence intensities were performed using ImageJ.
Förster resonance energy transfer
YTS cells stably expressing 2DL1-WT-Venus, 2DL1-2YF-Venus, or 2DL1-H36A-Venus were transfected with pSHP-1–Cerulean. After 4 h at 37°C, cells were washed and resuspended in RPMI 1640 supplemented with 10% FBS (R10) medium. The cells were applied to poly-l-lysine–coated chambers, fixed with 4% paraformaldehyde, and mounted with ProLong Gold (Life Technologies). Förster resonance energy transfer (FRET) measurements were performed using a confocal microscope (Zeiss LSM-710). Cerulean molecules (FRET donor) were excited using a 440-nm laser. The fluorescence intensity of Cerulean at the cell surface of Venus molecules was monitored before and after photobleaching (using a 514-nm laser). The samples were recorded at two time points before and two time points after the photobleaching of Venus. The percentage FRET signal was calculated as the percentage change in the fluorescence intensity of Cerulean after photobleaching of Venus. The values for fluorescence intensity were determined using ImageJ. A small amount of spontaneous photobleaching was observed during acquisition; therefore, a few cells had a negative FRET signal.
Steady-state anisotropy
A total of 4 × 106 cells in phenol red–free RPMI 1640 solution supplemented with 2% FBS (R2 medium) was plated onto a 35-mm glass-bottom dish coated with poly-l-lysine. The cells were washed twice with the R2 medium before imaging. Images were acquired on a DCS-120 Confocal Scanning FLIM System (Becker & Hickl). The system was equipped with a Ti-Sapphire–pulsed laser (Chameleon, Coherent), an Observer.Z1 (Zeiss) microscope with a 40×/1.2 W objective, and MH-100-40 hybrid detectors (Hamamatsu). The images were acquired for 2 min in Time Correlated Single Photon Counting mode on two detectors, detecting parallel and perpendicular emissions. The parallel and perpendicular images were recorded simultaneously and were processed later for anisotropy and intensity images, using Igor Pro software, version- 6.34A (WaveMetrics).
The following equations were used to calculate Intensity (Itotal) and Anisotropy (r)
(1)
(2)In Eqs. 1 and 2, III is the count from the parallel detector, I
is the count from the perpendicular detector, and G is the instrumentation factor (calculated to be 0.99). The intensity and anisotropy images were color coded to create a representative image. The mean and SEM were calculated from the nonzero pixel values of the image.
Immunoprecipitation and Western blotting
A total of 20 × 106 cells was lysed in 1 ml lysis buffer (0.5% Triton X-100, 150 mM NaCl, 50 mM Tris [pH 7.4]), protease inhibitor mixture (11836170001; Roche), 2 mM EGTA, 1 mM NaVO3) for 30 min on ice. Lysate was centrifuged, and supernatants were incubated with Ab-coated protein G Sepharose beads for 2.5 h. Beads were washed thrice in lysis buffer and resuspended in 1× NuPage LDS sample buffer (Invitrogen). After the addition of 10 mM DTT, the samples were heated at 70°C for 10 min. The samples were run on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore) for 1.5 h at 40 V. Membrane was blocked in PBS containing 5% BSA and 0.5% Tween 20 for 1 h and then incubated with primary Ab for 1 h. After three washes (each wash of 5 min) with PBS containing 350 mM NaCl and 1% Tween 20, the membrane was incubated with secondary Ab for 1 h. Then, the membrane was washed three times (each wash of 5 min) with PBS containing 1% Tween 20. The membrane was developed with Super Signal West Dura (Pierce). The intensities of bands were calculated using ImageJ.
Expression and purification of KIR2DL1 (1–224)
For bacterial expression of KIR2DL1, a cDNA encoding the extracellular portion (1–224) of KIR2DL1 was cloned into pET30a (+) using the restriction sites NdeI and XhoI. The resulting plasmid is referred to as p2DL1 (1–224). The H36A mutant variant was generated by site-directed mutagenesis on p2DL1 (1–224). The plasmids were expressed in Escherichia coli strain BL21-Gold (DE3) (Agilent Technologies). The protein was purified as described (18). The concentration of protein was determined by measuring absorbance at 280 nm, using ε280 nm = 52921 M−1 cm−1 (18).
Fluorescence spectroscopy
The intrinsic fluorescence spectra were collected on a FluoroMax-3 spectrofluorometer (Jobin Yvon). The protein was dissolved in 10 mM Tris (pH 7.4) with 50 mM NaCl at a concentration of 1.5 μM. The intrinsic tryptophan residues in the protein were excited at 295 nm, and emission at different wavelengths was monitored. The excitation and emission bandwidths were set at 1 and 10 nm, respectively.
Circular dichroism
The far-UV circular dichroism spectra were collected on a Jasco J-815 spectropolarimeter. The protein was dissolved in 10 mM Tris (pH 7.4) with 50 mM NaCl at a concentration of 5 μM. The spectra were acquired using a 1-mm path-length cuvette. Each spectrum was averaged over 10 scans.
Cytotoxicity assay
Propidium iodide–based cytotoxicity assays were performed. The TAP-deficient 221-Cw4 target cells were incubated with increasing amounts of HLA-Cw4–specific peptide QYDDAVYKL (Biosource) (19) for 16 h at 26°C. The surface expression of HLA-C at different peptide concentrations was determined using an anti–HLA-C Ab, F4/326. YTS-2DL1-WT-Venus and YTS-2DL1-H36A-Venus cells were labeled with Vybrant DiD Cell-Labeling Solution (V-22887; Invitrogen) and mixed with TAP-deficient 221-HLA-Cw4 target cells, loaded with different amounts of the peptide, at an E:T ratio of 6:1 in a V-bottom 96-well plate. The plate was centrifuged at 48 × g for 3 min and incubated for 5 h at 37°C. The cells were analyzed on a flow cytometer (BD FACSCalibur). The percentage of target cell killing was determined as the percentage of target cells showing propidium iodide fluorescence minus the percentage of spontaneous killing of target cells in the absence of NK cells.
Statistical analysis
Statistical analysis was performed by the nonparametric t test using Prism software (GraphPad).
Results
His-36 of KIR2DL1 is a negative regulator of SHP-1 recruitment
To test the role of histidine residues in the D1 domain of KIR2DL1 in the inhibitory function of the receptor, we used BiFC to measure KIR2DL1 association with the phosphatase SHP-1 using WT and single His-to-Ala mutants of the receptor. The BiFC method is based on complementation of two nonfluorescent fragments of Venus, which refold into a fluorescent Venus when the proteins fused with these fragments are in close proximity (20, 21). Once refolded, the reconstituted Venus is stable. Therefore, the BiFC signal is cumulative and is well suited for detection of transient interactions. However, this method has inherent noise arising from random encounters, particularly at higher expression levels.
The WT and the indicated His-to-Ala mutants of KIR2DL1, tagged with the C-terminal fragment of Venus (155–238; VC155), and SHP-1, tagged with the N-terminal fragment of Venus [1–154 with I152L mutation (22); VN155], were cotransfected into YTS cells (Fig. 1A). The association of SHP-1 with KIR2DL1 was monitored by the fluorescence of reconstituted Venus using flow cytometry (Fig. 1B, 1C). With WT KIR2DL1 (2DL1-WT), as well as H1A, H5A, H40A, H55A, and H85A mutants, a basal BiFC signal was observed only at high receptor expression (Fig. 1B). To test whether this BiFC signal at high receptor expression originated from the specific association of SHP-1 with phosphorylated ITIMs of KIR2DL1 or from random interactions due to high expression, we tested the ITIM tyrosine mutant variant, in which both Tyr residues were mutated to Phe (2DL1-2YF). The observation that 2DL1-2YF showed the same profile (Fig. 1C) indicated that the BiFC signal at high receptor expression originated from nonspecific interactions. To avoid the contribution of this nonspecific BiFC signal, we gated and performed analysis on cells with lower expression levels at which the WT receptor showed no or negligible BiFC signal (Fig. 1B). The values of median fluorescence intensities determined from these gates were compared (Fig. 1C). A BiFC signal for 2DL1-H36A was evident at low receptor expression and was higher in magnitude than that for 2DL1-WT and other His-to-Ala mutants (Fig. 1B, 1C). This suggested that the H36A mutant of KIR2DL1 has higher constitutive association with SHP-1. Given the absence of KIR2DL1 engagement by HLA-C or by Ab, this was a surprising result.
His-36 of KIR2DL1 is a negative regulator of SHP-1 recruitment. (A) Diagram of KIR2DL1 and SHP-1 tagged at the C terminus with the Venus fragments VC155 and VN155, respectively. (B) Constitutive SHP-1 association with KIR2DL1 in transfected YTS cells monitored by BiFC of reconstituted Venus using flow cytometry. BiFC signal of KIR2DL1 WT (red) and the indicated histidine-to-alanine mutants (blue) are plotted against surface expression, as detected by anti-2DL1–allophycocyanin binding. (C) Median fluorescence intensity of the BiFC signal in the gates shown in (B). The error bars represent spread in the values of median BiFC signal determined from two independent experiments. *p = 0.015. (D) Confocal microscopy images of the BiFC signal in YTS cells transfected with SHP-1−VN and either 2DL1-WT (upper panel) or 2DL1-H36A (lower panel). Insets show enlarged views of the indicated cells.
We performed confocal fluorescence microscopy to visualize the subcellular localization of the BiFC signal emanating from the association of SHP-1 with KIR2DL1. The localization of the BiFC signal was similar in the cells expressing the WT or H36A mutant of KIR2DL1. In both cases, the signal was evenly distributed around the cell periphery, and some signal was in a discrete intracellular region, consistent with an association of SHP-1 with KIR2DL1 at the plasma membrane and on the intracellular membrane (Fig. 1D).
We validated the role of His-36 of KIR2DL1 in controlling the constitutive recruitment of SHP-1 to the receptor using FRET measurements. Although BiFC is cumulative and only produces a signal after a time delay needed for the folding and maturation of the fluorophore, FRET enables instantaneous detection of protein interactions (23). YTS cells stably expressing 2DL1-WT (YTS-2DL1-WT-Venus), 2DL1-2YF (YTS-2DL1-2YF-Venus), or 2DL1-H36A (YTS-2DL1-H36A-Venus) (Supplemental Fig. 1), each tagged with Venus at the end of the cytosolic tail, were transfected for transient expression of SHP-1 that was tagged at the C terminus with Cerulean (Fig. 2A). The FRET signal was measured as the increase in donor (Cerulean) fluorescence upon acceptor (Venus) photobleaching (Fig. 2B). In accordance with the BiFC measurements (Fig. 1), the FRET signals arising from the association of SHP-1 with 2DL1-WT (1.3 ± 0.6% [mean ± SEM]) and 2DL1-2YF (0.5 ± 1%) were similar, whereas 2DL1-H36A (3.7 ± 0.4%) gave a stronger signal (p < 0.002). The FRET signals were not dependent on the strength of the donor (Cerulean) or the acceptor (Venus) fluorescence signals (Fig. 2B). We conclude that the increased association of SHP-1 with 2DL1-H36A was not due to high expression level. The detection of association by FRET confirmed the result obtained by BiFC and showed that 2DL1-H36A−SHP-1 association is detectable at a single time point.
H36A mutation of KIR2DL1 results in constitutive SHP-1 recruitment. (A) Diagram of KIR2DL1 and SHP-1 tagged at their C terminus with Venus and Cerulean, respectively. (B) SHP-1 proximity to KIR2DL1 monitored by FRET. SHP-1−Cerulean was transiently expressed in YTS cells stably transfected with 2DL1-WT-Venus, 2DL1-2YF-Venus, or 2DL1-H36A-Venus. FRET signal at the plasma membrane was calculated as the percentage increase in donor (Cerulean) fluorescence after acceptor (Venus) photobleaching. The negative values for the FRET signal are due to unintentional photobleaching of donor fluorescence during acquisition. Data points from three independent experiments are combined (left panel). Horizontal and vertical lines represent the mean and SE, respectively. **p = 0.001. FRET signals of SHP-1 with 2DL1-H36A relative to the expression of FRET donor (SHP-1-Cerulean; middle panel) and FRET acceptor (2DL1-H36A-Venus; right panel). (C) Endogenous SHP-1 was immunoprecipitated from the indicated YTS-2DL1-Venus cells, and its association with 2DL1 was probed with an anti-GFP immunoblot (left panel). Band intensities were obtained by scanning immunoblots and calculated using ImageJ (right panel). The error bars represent spread in the mean value obtained from two independent experiments. ***p = 0.0004.
Next, we tested the recruitment of SHP-1 to KIR2DL1 by immunoprecipitation and Western blotting. Endogenous SHP-1 was immunoprecipitated from YTS-2DL1-WT-Venus, YTS-2DL1-2YF-Venus, or YTS-2DL1-H36A-Venus cells, and its association with the receptor was monitored with an Ab to Venus (Fig. 2C). As expected, 2DL1-2YF was not associated with SHP-1. In contrast, and in accordance with the BiFC and FRET measurements, 2DL1-H36A showed much higher constitutive association with SHP-1 than did 2DL1-WT (∼4-fold) (Fig. 2C).
Recruitment of SHP-1 to ITIM-bearing receptors is usually dependent on tyrosine phosphorylation of the ITIMs. To test whether increased recruitment of SHP-1 by 2DL1-H36A correlated with tyrosine phosphorylation of the receptor, we assessed the phosphorylation of 2DL1-WT, 2DL1-2YF, and 2DL1-H36A in these cell lines by immunoprecipitation of the receptor, followed by immunoblotting with Ab to p-Tyr. 2DL1-WT and 2DL1-2YF displayed no or very low phosphorylation, whereas 2DL1-H36A displayed much higher constitutive phosphorylation (Fig. 3A). Because ITIM phosphorylation is sufficient for SHP-1 binding (7, 24, 25), this result suggested that SHP-1 association with 2DL1-H36A was a consequence of receptor phosphorylation.
His-36 of KIR2DL1 prevents constitutive ITIM phosphorylation. (A) Constitutive tyrosine phosphorylation of KIR2DL1 monitored by immunoblotting. KIR2DL1 was immunoprecipitated from YTS-2DL1-WT-Venus, YTS-2DL1-2YF-Venus, and YTS-2DL1-H36A-Venus cells with an anti-GFP Ab, and tyrosine phosphorylation was detected by immunoblotting with mAb 4G10 (left panel). The intensity of bands in immunoblots, relative to YTS-2DL1-2YF-Venus cells (set to 1) (right panel). The error bars represent spread in the mean value determined from two independent experiments. **p = 0.001. (B) KIR2DL1 tyrosine phosphorylation, measured as in (A), after treatment of transfected YTS cells with 1 mM pervanadate for 20 min.
The constitutive phosphorylation of 2DL1-H36A could be due to selective recruitment of a tyrosine kinase. Alternatively, because KIR2DL1 may be subjected to a continuous cycle of phosphorylation and dephosphorylation, the self-association of 2DL1-H36A may result in enhanced phosphorylation through the protection of phosphorylated ITIMs from PTPases. We assessed the effect of inhibition of tyrosine phosphatases on receptor phosphorylation. In the case of selective kinase recruitment by 2DL1-H36A, the difference in phosphorylation of 2DL1-WT and 2DL1-H36A may persist after treatment with pervanadate, a PTPase inhibitor. Conversely, protection of 2DL1-H36A from PTPase would be expected to result in equal phosphorylation of 2DL1-WT and 2DL1-H36A after treatment with pervanadate. 2DL1-WT and 2DL1-H36A were phosphorylated to the same extent after pervanadate treatment (Fig. 3B). This suggested that the constitutive phosphorylation of 2DL1-H36A was due to selective protection from PTPase activity, rather than selective recruitment of tyrosine kinase.
H36A mutation does not perturb the overall structure of KIR2DL1
It was conceivable that the H36A mutation in KIR2DL1 caused a conformational change of the receptor, which could protect it from ITIM dephosphorylation and enhance constitutive SHP-1 recruitment. To test this possibility, we compared the spectroscopic characteristics, using intrinsic tryptophan fluorescence and circular dichroism spectroscopy, of the purified extracellular portions (aa 1–224) of 2DL1-WT and 2DL1-H36A, which had been expressed in E. coli, refolded, and purified (Fig. 4A).
H36A mutation does not perturb the overall structure of KIR2DL1. (A) Ribbon diagram structure of KIR2DL1. Tryptophan residues 29, 188, and His-36 are shown as sticks. Tryptophan residue 207 is not visible in the KIR2DL1 structure, probably as the result of a disordered stem (201–224) structure. The diagram was generated from the PDB file 1NKR using PyMOL. (B) Intrinsic tryptophan fluorescence of purified, soluble KIR2DL1. The spectra of the WT (2DL1-WT; red) and the H36A mutant (2DL1-H36A; blue) of KIR2DL1 (1–224) (left panel). Maximum wavelengths of emission (middle panel) and fluorescence (right panel) intensities at 348 nm (I348 nm). The error bars represent spread in the mean values determined from two independent experiments. (C) Far-UV circular dichroism of purified, soluble KIR2DL1. Far-UV circular dichroism spectra of 2DL1-WT (red) and 2DL1-H36A (blue) (left panel). Positions of minimum ellipticity (middle panel) and the ellipticity values at 213 nm (right panel). The error bars represent spread in the mean values determined from two independent experiments.
Intrinsic tryptophan fluorescence spectroscopy probes the environment of tryptophan residues in a protein. The emission maximum, as well as the quantum yields of intrinsic tryptophan fluorescence spectra, is sensitive to the solvent exposure of the tryptophan residues (26, 27). A tryptophan residue buried in the hydrophobic core of a protein shows the emission maximum at ∼330 nm, upon excitation at 295 nm. A solvent-exposed tryptophan residue has the emission maximum at 355–360 nm and has a lower quantum yield of fluorescence. KIR2DL1 (1–224) has three tryptophan residues: at positions 29 in the D1 domain, 188 in the D2 domain, and 207 in the stem region (Fig. 4A). A difference in the conformation of 2DL1-WT and 2DL1-H36A may lead to a change in the environment of tryptophan residue(s) and, thus, a difference in the tryptophan fluorescence spectrum. 2DL1-WT (1–224) showed the emission maximum at ∼348 nm, indicative of tryptophan residue(s) residing in an environment that is exposed, in part, to the solvent (Fig. 4B). The wavelength of the emission maximum, as well as the quantum yield of fluorescence, was the same in 2DL1-H36A (1–224), showing that the mutation did not affect the overall environment of the tryptophan residues in the receptor (Fig. 4B).
Far-UV circular dichroism measures secondary structure (α-helix and β-strands) in proteins and peptides (28). The far-UV circular dichroism spectrum of 2DL1-WT (1–224) had a peak at 213 nm, indicative of a protein rich in β-strands (Fig. 4C), consistent with the known X-ray structure of the receptor (Fig. 4A). The spectrum of 2DL1-H36A (1–224) was similar (Fig. 4C), which indicated that the mutation did not have a major effect on the secondary structure of the receptor.
His-36 is pointing outward in the KIR2DL1 crystal structure and is positioned away from the HLA-C binding site (29). His-36 is not required for binding to HLA-C (30). Because His-36 resides in a solvent-accessible region of the protein, it is more likely to control intermolecular interactions compared with the receptor structure.
Phe mimics His at position 36 in KIR2DL1
The side-chain of a histidine residue has several physicochemical properties that could affect protein interactions, such as zinc-binding ability and the ability to create steric hindrance through its bulky imidazole ring. KIR2DL1 is known to bind zinc (31), and zinc contributes to inhibitory signaling through the receptor (30). To test whether constitutive SHP-1 recruitment to 2DL1-H36A results from an altered zinc binding, we assessed constitutive association of SHP-1 with 2DL1-WT and 2DL1-H36A by coimmunoprecipitation and BiFC, in the presence of 200 μM supplemental ZnCl2 or 1 mM EDTA. The constitutive association of SHP-1 with 2DL1-H36A, as well as its low basal association with 2DL1-WT, did not change upon treatment with either 200 μM ZnCl2 or 1 mM EDTA (Supplemental Fig. 2). Therefore, the prevention of constitutive SHP-1 recruitment, as well as constitutive ITIM phosphorylation by His-36, is not dependent on zinc.
To test whether His-36 prevents KIR2DL1 phosphorylation by steric hindrance we replaced it with another bulky amino acid, phenylalanine. We monitored the constitutive recruitment of SHP-1 to 2DL1-WT, 2DL1-H36A, or 2DL1-H36F with BiFC assays using flow cytometry (Fig. 5A, 5B). As seen earlier, 2DL1-H36A showed greater association with SHP-1 than did 2DL1-WT. In contrast, constitutive SHP-1 association with 2DL1-H36F was similar to that with 2DL1-WT (Fig. 5A, 5B). We monitored the constitutive association of SHP-1 with KIR2DL1 at the plasma membrane using confocal fluorescence microscopy. In accordance with BiFC, SHP-1 association was higher with 2DL1-H36A than with 2DL1-WT, and association with 2DL1-H36F was similar to that with 2DL1-WT (Fig. 5C).
A large side-chain at aa 36 of KIR2DL1 prevents constitutive SHP-1 recruitment. (A) Constitutive SHP-1 association with KIR2DL1 in transfected YTS cells monitored by BiFC of reconstituted Venus using flow cytometry. BiFC signal of 2DL1-WT and 2DL1-H36A (upper panel) or WT and 2DL1-H36F (lower panel) is plotted against surface expression, as detected by anti-2DL1–allophycocyanin binding. (B) Median fluorescence intensity of BiFC signal in the gates shown in (A). The error bars represent spread in the values of median BiFC signal determined from two independent experiments. *p = 0.037. (C) Constitutive SHP-1 association with KIR2DL1 in transfected YTS cells monitored by BiFC of reconstituted Venus using confocal microscopy. BiFC signal of 2DL1-WT, 2DL1-H36A, and 2DL1-H36F, determined at the plasma membrane. The horizontal and vertical lines represent mean and SE, respectively. ***p = 0.0007.
A residue at position 36 with similar bulk as His (Phe) prevented SHP-1 recruitment to the receptor, suggesting that steric hindrance by the imidazole ring of His-36 of KIR2DL1 may prevent interactions that result in constitutive SHP-1 recruitment to the receptor.
His-36 prevents KIR2DL1 self-association
His-36 or Phe-36 may prevent interaction of KIR2DL1 with itself (self-association) or with something else. We first tested the role of His-36 in regulating KIR2DL1 self-association. Constitutive self-association of 2DL1-WT, 2DL1-H36A, or 2DL1-H36F was assessed by a BiFC assay with YTS cells coexpressing KIR2DL1 tagged at the cytosolic tail with either VN155 or VC155 (Fig. 6A). Self-association of 2DL1-WT was evident only at higher levels of receptor expression (Fig. 6B), probably as the result of random encounters on the two-dimensional surface of the plasma membrane. However, the self-association propensity of 2DL1-H36A was clearly higher than that of 2DL1-WT, and it occurred at much lower expression levels (Fig. 6B, 6C). The propensity of self-association of 2DL1-H36F was similar to that of 2DL1-WT. As observed with the KIR2DL1−SHP-1 association assay (Fig. 5), H36F had the same property as 2DL1-WT and prevented KIR2DL1 self-association (Fig. 6B, 6C).
A bulky side-chain at aa 36 of KIR2DL1 prevents constitutive receptor self-association. (A) Diagram of KIR2DL1 tagged at the C terminus with Venus fragments VN155 and VC155. (B) Constitutive KIR2DL1 self-association in transfected YTS cells monitored by BiFC. BiFC signal of 2DL1-WT and 2DL1-H36A (upper panel) or 2DL1-WT and 2DL1-H36F (lower panel) is plotted against surface expression, as detected by anti-2DL1–allophycocyanin binding. (C) Median fluorescence intensity of BiFC signal in the gates shown (B). The error bars represent spread in the values of median BiFC signal determined from two independent experiments. **p = 0.006.
To validate the constitutive self-association of 2DL1-H36A, steady-state anisotropy imaging was performed using YTS-2DL1-WT-Venus and YTS-2DL1-H36A-Venus cells (Fig. 7). Fluorescence anisotropy measures how well the orientation (polarization) of emitted light correlates with the orientation of the linearly polarized light used for excitation (Fig. 7A). When a high m.w. fluorophore, such as Venus, is excited, the orientation of emitted light is typically highly correlated with the orientation of the electric field of the light used for excitation, resulting in a high anisotropy value (32). This arises primarily because large fluorophores, such as GFP and its derivatives, have slow rotational motion relative to their fluorescence lifetime. However, if two such fluorophores are in close proximity, energy absorbed by one fluorophore can be transferred to the second fluorophore by a phenomenon called homo-FRET. Homo-FRET, per se, does not alter the emission intensity or spectrum. However, because the emission arises, in part, from a molecule other than the one that was photo-selected, which potentially has a different orientation, the orientation of emitted light tends to be less well correlated with the excitation light source. Thus, homo-FRET may occur when two fluorophores are in close proximity; typically, this can be observed as a decrease in anisotropy.
Self-association of 2DL1-WT and 2DL1-H36A monitored by anisotropy measurements. (A) Diagram depicting how receptor self-association generates homo-FRET and a reduced fluorescence anisotropy. In the absence of homo-FRET, the emission of Venus is detected mostly in the parallel plane (||) of polarization. If homo-FRET occurs between two Venus molecules in close proximity, the emission becomes more evenly distributed between both parallel and perpendicular (
) planes of polarization, and anisotropy will decrease. (B) Anisotropy (upper panels) and fluorescence intensity (lower panels) images of YTS-2DL1-WT-Venus and YTS-2DL1-H36A-Venus cells. Arrows show YTS-2DL1-WT-Venus and YTS-2DL1-H36A-Venus cells with matched fluorescence intensity (expression level) but distinct anisotropy values. (C) Quantitation of anisotropy and fluorescence intensity of YTS-2DL1-WT-Venus (red) and YTS-2DL1-H36A-Venus (blue) cells shown in (B). Individual pixel anisotropy values are plotted parametrically as a function of fluorescence intensity. Quantitation was performed using IGOR Pro software (WaveMetrics) (left panel). Same data potted separately for YTS-2DL1-WT-Venus cells (red; upper right panel) and YTS-2DL1-H36A-Venus cells (blue; lower right panel). (D) Median pixel anisotropy values (n = 350) are plotted as a function of fluorescence intensity. YTS-2DL1-WT-Venus (red) and YTS-2DL1-H36A-Venus (blue) cells (left panel). Anisotropy of membrane tethered Venus monomer (V-K-Ras) and Venus dimer (VV-K-Ras) (right panel). The median pixel anisotropy values were determined from three independent images.
To control for the homo-FRET signals resulting from high expression rather than receptor self-association, anisotropy and fluorescence intensity (receptor expression) were acquired simultaneously. We performed simultaneous two-photon anisotropy and fluorescence intensity imaging using YTS-2DL1-WT-Venus or 2DL1-H36A-Venus cells (Fig. 7B, Supplemental Fig. 3). Images were color coded to highlight changes in anisotropy and intensity values. Overall, the anisotropy values of 2DL1-H36A were lower than those of 2DL1-WT (Fig. 7B, upper panels). Further, cells expressing 2DL1-WT or 2DL1-H36A with matched fluorescence intensity (Fig. 7B, arrows) showed lower anisotropy values for the H36A mutant. A more quantitative comparison was obtained by plotting the individual pixel anisotropy values as a function of fluorescence intensity (Fig. 7C, 7D). As expected from Poisson counting statistics (33), variance in the anisotropy values was higher at low fluorescence intensity. In this low-intensity region, the anisotropy values were independent of receptor expression level; thus, they indicated the true aggregation state of the receptor. Importantly, at these low-intensity values, the anisotropy for 2DL1-H36A were lower than for 2DL1-WT. At higher fluorescence intensity values, the anisotropy values of both samples decreased, presumably as a result of overcrowding of the fluorophores. However, throughout the intensity range, 2DL1-H36A (0.261 ± 0.004 [mean ± SEM]) had consistently lower anisotropy values than 2DL1-WT (0.284 ± 0.004), indicating higher homo-FRET and, thus, self-association of 2DL1-H36A.
We reasoned that if 2DL1-WT-Venus is a monomer, it should have an anisotropy value similar to that of a membrane-tethered monomeric Venus tagged at the N terminus with a membrane-targeting sequence from K-Ras. We performed the measurements on YTS cells expressing monomeric Venus or dimeric Venus (two Venus molecules joined by a linker of 5 aa) tagged with the membrane-targeting sequence of K-Ras; they are referred as V-K-Ras and VV-K-Ras. The anisotropy values of 2DL1-WT were similar to those of V-K-Ras (Fig. 7D), suggesting that 2DL1-WT exists as a monomer at the cell surface. 2DL1-H36A had lower anisotropy values than V-K-Ras, indicating that H36A mutation facilitates KIR2DL1 self-association and, conversely, that His-36 prevents KIR2DL1 self-association. As expected for a covalent dimer, the anisotropy of the membrane-targeted dimeric Venus (VV-K-Ras) was lower (i.e., higher homo-FRET) than that of 2DL1-H36A. The proportion of 2DL1-H36A receptors that is self-associated at any one time is not known. In addition, it is possible that the homo-FRET efficiency is greater between the covalently joined Venus molecules in VV-K-Ras than between Venus molecules at the C-terminal end of self-associated 2DL1-H36A. Finally, FRET and anisotropy also depend on the relative orientation of Venus pairs.
Overall, our results from BiFC and anisotropy measurements indicated that 2DL1-WT exists as a monomer at the cell surface and that the H36A mutation facilitates KIR2DL1 self-association.
Inhibition of lysis of HLA-C+ target cells is stronger with YTS-2DL1-H36A cells than with YTS-2DL1-WT cells
An important question is how the mutation H36A, which results in constitutive KIR2DL1 self-association, phosphorylation, and recruitment of SHP-1, affects the inhibitory function of KIR2DL1 during interaction of NK cells with sensitive target cells that express HLA-C ligands. We first tested the killing of 221 target cells expressing HLA-Cw3, an HLA-C allotype that does not bind KIR2DL1, or HLA-Cw4, a ligand for KIR2DL1, by YTS-2DL1-WT-Venus and YTS-2DL1-H36A-Venus cells (Supplemental Fig. 4). YTS-2DL1-WT-Venus and YTS-2DL1-H36A-Venus had a similar ability to kill 221-HLA-Cw3 target cells, indicating that the constitutive self-association and phosphorylation of 2DL1-H36A did not affect NK cell cytotoxicity. Inhibition of killing of 221 cells expressing HLA-Cw4 was efficient and similar for WT and the H36A mutant of KIR2DL1 (Supplemental Fig. 4). Therefore, the inhibitory function of KIR2DL1 was not affected by the mutation at His-36.
To expose the role of His-36, if any, in regulating inhibitory KIR2DL1 function, we developed a quantitative assay for the strength of inhibition. We generated a 221-HLA-Cw4 cell line stably expressing ICP47, an inhibitor of TAP that is expressed by HSV (34). HLA-Cw4 at the surface of TAP-deficient cells can be stabilized by the addition of exogenous HLA-Cw4–specific peptide, providing an opportunity to titrate the amount of peptide-loaded HLA-Cw4 available to KIR2DL1 for inhibition of NK cell activation (Fig. 8A). 2DL1-WT and 2DL1-H36A YTS cells had a similar ability to kill 221-Cw4-ICP47 cells in the absence of peptide, indicating that, as seen with normal (TAP-sufficient) 221-HLA-Cw3 cells, the H36A mutation did not affect the cytotoxicity activity of the NK cell (Fig. 8B). Expression of 2DL1-WT or 2DL1-H36A inhibited the cytotoxicity in a peptide concentration–dependent manner. However, 2DL1-H36A inhibited NK cell activation at much lower peptide concentrations (Fig. 8B, 8C). This showed that 2DL1-H36A inhibits NK cell cytotoxicity more efficiently than does the WT receptor (i.e., His-36 of KIR2DL1 impedes inhibitory signaling through KIR2DL1 upon HLA-C binding). Consistent with the observation in TAP-sufficient 221 target cells with higher HLA-C expression, where there is comparable inhibition of killing by 2DL1-WT and 2DL1-H36A (Supplemental Fig. 4), at higher levels of peptide-loaded HLA-Cw4 (5–10 μM of the peptide), the ability to inhibit killing by NK cells was similar for 2DL1-WT and 2DL1-H36A (Fig. 8B).
Inhibition of lysis of HLA-C+ target cells is stronger with YTS-2DL1-H36A cells than with YTS-2DL1-WT cells. (A) Surface expression of HLA-C on TAP-deficient 221-HLA-Cw4 target cells that were loaded with an exogenous HLA-Cw4–specific peptide was determined with anti–HLA-C Ab F4/326. (B) Lysis of TAP-deficient 221-Cw4 cells by YTS-2DL1-WT-Venus (●) and YTS-2DL1-H36A-Venus (▴) cells after loading with peptides at different concentrations. The NK/target ratio was 6. Error bars represent spread in the mean values determined from duplicate samples. (C) Relative lysis of TAP-deficient 221-HLA-Cw4 cells by YTS-2DL1-WT and YTS-2DL1-H36A after loading with 0.5 μM peptide. To compare different experiments, lysis of 221-HLA-Cw4 cells in the absence of exogenous peptide was set to 1. Bars represent the mean and error bars represent SDs obtained from three independent experiments. *p = 0.028.
Discussion
The very proximal block of NK activation signals by inhibitory receptors (10–12) is not easily reconciled with models that place the phosphorylation of ITIMs downstream of signaling by activation receptors. The question that we addressed in this study is how phosphorylation of ITIMs in KIR is regulated. Because ITIMs in inhibitory receptors are readily phosphorylated in cells treated with pervanadate (a general inhibitor of cysteine-dependent PTPase), ITIMs must be subjected to a constitutive cycle of phosphorylation and dephosphorylation, with an equilibrium that favors dephosphorylation. If so, how could this cycle be regulated to control ITIM phosphorylation? The PTPase SHP-1 is the key effector in the inhibitory function of ITIM-bearing KIR (2). SHP-1 is maintained in a self-inhibited conformation in the cytosol, and SHP-1 recruitment by phosphorylated ITIMs results in its activation and ability to dephosphorylate components of activation pathways. Binding of phosphorylated ITIMs to SH2 domains of SHP-1 protects them from tyrosine phosphatases. Therefore, SHP-1 may not be the primary PTPase responsible for the maintenance of ITIMs in a dephosphorylated state.
In this study, we identified a gain-of-function mutant of KIR2DL1, which is phosphorylated in its basal, ligand-free state. In a screen of histidine residues in the D1 domain of KIR2DL1 for their role in receptor function, we noticed that H36A resulted in constitutive association of KIR2DL1 with SHP-1, constitutive ITIM phosphorylation, and KIR2DL1 self-association. These unusual properties of 2DL1-H36A are independent of HLA-C ligands, given that KIR2DL1 exhibits exclusive specificity for group 2 HLA-C allotypes and that YTS cells express two group 1 HLA-C allotypes. The constitutive phosphorylation of 2DL1-H36A implies a change in the balance between phosphorylation and dephosphorylation of this mutant KIR. Our results suggest that KIR2DL1 phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that this access is denied by 2DL1-H36A self-association.
The constitutive tyrosine phosphorylation of 2DL1-H36A measured in biochemical experiments, in which immunoprecipitated receptor was immunoblotted for phosphotyrosine, was much greater than that of KIR2DL1. Constitutive tyrosine phosphorylation of KIR2DL1 was very low, similar to the negative control of KIR2DL1 lacking tyrosines in both ITIMs. However, in cells treated with pervanadate, tyrosine phosphorylation of KIR2DL1 and of 2DL1-H36A was the same, indicating that 2DL1-H36A has acquired some level of protection from the constitutive dephosphorylation the WT receptor is subjected to in the absence of pervanadate. Constitutive association of SHP-1 with 2DL1-H36A was evident in similar biochemical experiments. This association was also detected using both BiFC and FRET between appropriately tagged KIR2DL1 and SHP-1 molecules. The constitutive association of 2DL1-H36A with SHP-1 is not due to the specific loss of histidine at position 36, because the similarly bulky amino acid phenylalanine at position 36 behaved like WT receptor. The folding of a purified, soluble H36A mutant of KIR2DL1 was tested by intrinsic tryptophan fluorescence and far-UV circular dichroism. By both methods, 2DL1-H36A was indistinguishable from purified, soluble WT KIR2DL1. Those results, and the fact that 2DL1-H36A is a gain-of-function mutant, do not support the possibility that 2DL1-H36A is a misfolded receptor.
The constitutive tyrosine phosphorylation of 2DL1-H36A could be due to the loss of a His-36-dependent binding to a PTPase or to a protein complex that includes a PTPase. The maintenance of WT KIR2DL1 in a dephosphorylated state due to this His-36-dependent association would have to be overcome when KIR2DL1 binds to HLA-C. An alternative is that His-36 may prevent an interaction with a protein or a protein complex that favors tyrosine phosphorylation of KIR2DL1. In support of this interpretation, substitution of His-36 by a Phe, which has an equally bulky side chain, resulted in a receptor that was not constitutively associated with SHP-1, just as WT KIR2DL1. Using BiFC assays with YTS cells we found that 2DL1-H36A was associated with itself, whereas WT 2DL1 and 2DL1-H36F did not undergo self-association. For those experiments, cells were transfected with an equal amount of two plasmids, each encoding KIR2DL1 tagged with a different half of unfolded Venus. The refolding of Venus due to KIR2DL1 self-association was monitored at low KIR2DL1 expression levels, to avoid the crowding effect at high receptor densities.
To confirm the propensity of 2DL1-H36A to self-associate, we performed anisotropy measurements on KIR2DL1 tagged at the C terminus with an intact Venus molecule. Anisotropy measures the amount of fluorophore emission that has a polarization (or orientation) similar to the polarized light used for excitation. Close proximity (<10 nm) of two Venus molecules results in homo-FRET, which can lead to depolarization of fluorescence emission and can be detected by a reduction in anisotropy. This technique is different from standard FRET experiments, which measure changes in the wavelength or in the lifetime of emission, and eliminates complications associated with the use of two distinct fluorophores in standard FRET measurements. Emission that has become unpolarized, as the result of homo-FRET from one Venus molecule to another Venus molecule with a different orientation, reduces anisotropy. 2DL1-H36A had reduced mean anisotropy values over a wide range of fluorescence intensity, which was below that of WT 2DL1 at any fluorescence intensity, clearly demonstrating that 2DL1-H36A has the propensity to self-associate.
Our results clearly show that His-36 prevents self-association of the WT 2DL1 receptor. Mutation of His-36 to an alanine resulted in constitutive receptor self-association and tyrosine phosphorylation. We propose that receptor self-association is a mechanism that protects ITIMs from dephosphorylation by PTPases. In this model, WT KIR2DL1 is continuously exposed to tyrosine kinases and PTPases, with an equilibrium that favors dephosphorylation and, thus, disfavors SHP-1 recruitment. The negative effect of His-36 on receptor self-association must be overcome upon binding to HLA-C on target cells. Whether KIR2DL1 bound to HLA-C forms complexes similar to the constitutive self-association of 2DL1-H36A is not known. However, the striking ability of mutant 2DL1-H36A to inhibit YTS cells at a much lower HLA-C–peptide density on target cells than at the HLA-C–peptide density required to inhibit through WT KIR2DL1 clearly demonstrates that the constitutive 2DL1-H36A receptor complexes have strong inhibitory potential. The steric hindrance imposed by His-36 on receptor self-association may serve as a gatekeeper to prevent unregulated inhibition of NK cell activation. Activation of signaling receptors by the relief of structural constraints upon ligand binding has precedents, notably in growth factor receptors (35).
In the kinetic partitioning model proposed to explain TCR phosphorylation, regulation of phosphorylation occurs at the level of the distribution of the trans-membrane phosphatase CD45 and its exclusion from the center of the immunological synapse, and specific recruitment of a tyrosine kinase to the immunoreceptor is not required (36). Our model of KIR phosphorylation is similar, in that ligand-induced receptor phosphorylation is achieved by protection of phosphorylated tyrosines from phosphatase. However, in our model for KIR phosphorylation, the receptor itself switches into a state that protects its cytosolic tyrosine residues from local tyrosine phosphatases.
His-36 is positioned away from the HLA-C–binding region of the receptor, and it is not involved in KIR binding to HLA-C (29, 30). This conserved histidine is present in the D1 domain of 11 of 12 D1-containing KIR family members. Its role in inhibitory signaling by other ITIM-bearing KIRs or in signaling by activating KIRs remains to be tested. A precise understanding of how the hindrance of KIR2DL1 self-association by His-36 is overcome, or bypassed, upon binding to HLA-C ligands on target cells awaits future investigation. Solving the structure of a 2DL1-H36A self-associated complex could shed light on this question.
The observation made in this study that 2DL1-H36A has a greater ability to inhibit NK cell activation than does the WT receptor is remarkable, considering that ITIM-bearing KIRs are potent inhibitors of NK cell cytotoxicity. It shows that KIRs have evolved to achieve an optimal degree of inhibition rather than the maximum possible. Proper tuning of KIR inhibitory signals may also be relevant to their role in maintaining NK cells in a state of high responsiveness (37, 38).
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank David Narum and Karine Reiter for assistance with circular dichroism, Joseph Brzostowski for advice on FRET measurements, Rajat Varma for advice on nucleofection, Emmanuel Wiertz for the plasmid pLZRS-ICP47-IRES-eGFP, and Mary Carrington for HLA typing of YTS cells.
Footnotes
This work was supported by the Intramural Research Program at the National Institutes of Health, National Institute of Allergy and Infectious Diseases, and National Institute on Alcohol Abuse and Alcoholism. M.J.W.S. is supported by a National Institutes of Health–Wellcome Trust studentship.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- BiFC
- bimolecular fluorescence complementation
- FRET
- Förster resonance energy transfer
- H36A
- His-36–to-alanine substitution
- H36F
- His-36–to-phenylalanine substitution
- KIR
- killer cell Ig-like receptor
- PTPase
- protein tyrosine phosphatase
- WT
- wild-type.
- Received July 18, 2014.
- Accepted November 11, 2014.
References
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