A Single Amino Acid Change in Inhibitory Killer Cell Ig-like Receptor Results in Constitutive Receptor Self-Association and Phosphorylation

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 immunoreceptor tyrosine-based inhibition motifs (ITIM) results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work has shown 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. Here we show that substitution of histidine 36 (His-36) in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, and recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine (KIR2DL1-H36F), 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 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 expression of wild type KIR2DL1. These results have 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.

Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses

“…KIR2DL1 has three Trp residues, at position 29, 188, and 207. In the absence of zinc, KIR2DL1 had maximum Trp fluorescence at ~ 348 nm (Figure 1A), suggesting that the Trp residues were partially exposed to solvent, consistent with our previous observation (Kumar et al, 2015). Treatment with ZnCl 2 led to a shift in the wavelength of maximum fluorescence to ~ 339 nm, indicating a change in the environment of the Trp residue(s) in the receptor, and a concurrent increase in the fluorescence intensity.…”

“…The median anisotropy value determined in the absence of exogenous zinc was ~ 0.3, which is similar to the value of a monomeric, membrane-tethered GFP (Kumar et al, 2015), suggesting that KIR2DL1 was monomeric. Addition of ZnCl 2 at 50 and 100 μM led to a drop in anisotropy (Figure 2C), which was independent of fluorescence intensity, as shown by plotting the anisotropy value of each pixel as a function of fluorescence intensity (Figure 2D).…”

“…Amyloid fibril formation leads to conformational change into cross-β structures (Chiti and Dobson, 2006), and is typically associated with an increase in β-sheet content. The two Ig domains of native KIR2DL1 are β-strand rich, with far-UV circular dichroism spectra showing a single peak at 212 nm (Fan et al, 1997; Kumar et al, 2015). After treatment with 50 μM ZnCl 2 the far-UV circular dichroism spectrum of KIR2DL1 (1–224) was very similar, showing minimal change in ellipticity at 212 nm (Figure S1J, S1K).…”

“…To test it, we first used steady-state fluorescence anisotropy as a tool to measure KIR2DL1 self-association (Kumar et al, 2015). Homo-FRET between KIR2DL1 molecules fused to Venus fluorescence protein would result in decreased fluorescence anisotropy, indicative of self-association of the receptor (Nguyen et al, 2015).…”

Canonical and cross-reactive binding of NK cell inhibitory receptors to HLA-C allotypes is dictated by peptides bound to HLA-C

“…The 221 and RMA-S cell lines were cultured in RPMI or IMDM (Gibco) supplemented with 10% FCS. YTS cell lines were previously described and cultured in IMDM with 12.5% FCS [51,53]. HLA-C*05:01 was expressed in 221 cells by retroviral transduction.…”