The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure - PubMed (original) (raw)

Comparative Study

. 2005 May 24;102(21):7641-6.

doi: 10.1073/pnas.0502439102. Epub 2005 May 13.

Affiliations

Comparative Study

The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure

David Garrity et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The activating NKG2D receptor plays a critical role in innate and adaptive immune responses by natural killer cells and subpopulations of T cells. The human receptor assembles with the DAP10 signaling dimer, and it is thought that one NKG2D homodimer pairs with a single DAP10 dimer by formation of two salt bridges between charged transmembrane (TM) residues. However, direct stoichiometry measurements demonstrated that one NKG2D homodimer assembles with four DAP10 chains. Selective mutation of one of the basic TM residues of NKG2D resulted in loss of two DAP10 chains, indicating that each TM arginine serves as an interaction site for a DAP10 dimer. Assembly of the hexameric structure was cooperative because this mutation also significantly reduced NKG2D dimerization. A monomeric NKG2D TM peptide was sufficient for assembly with a DAP10 dimer, indicating that the interaction between these proteins occurs in the membrane environment. Formation of a three-helix interface among the TM domains involved ionizable residues from all three chains, the TM arginine of NKG2D and both TM aspartic acids of the DAP10 dimer. The organization of the TM domains thus shows similarities to the T cell antigen receptor-CD3 complex, in particular to the six-chain assembly intermediate between T cell antigen receptor and the CD3delta epsilon and CD3gamma epsilon dimers. Binding of a single ligand can thus result in phosphorylation of four DAP10 chains, which may be relevant for the sensitivity of NKG2D receptor signaling, in particular in situations of low ligand density.

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Figures

Fig. 1.

Fig. 1.

Stoichiometry of the NKG2D–DAP10 complex. (A) An in vitro translation system with ER microsomes was used to study the assembly of NKG2D and DAP10. After assembly, membranes were solubilized with 0.5% digitonin, and 35S-labeled complexes were isolated in a two-step snIP that targeted the PC and HA epitope tags attached to the two DAP10 chains. In the mixing controls (*, lanes 2 and 6), DAP10 and NKG2D chains were translated separately, and the reactions were combined immediately before solubilization. In the antibody controls (C, lanes 3, 4, 7, and 8), the PC antibody (lanes 3 and 7) or the HA antibody (lanes 4 and 8) used in the first and second IP step, respectively, were replaced with species- and isotype-matched antibodies of irrelevant specificity. Assembly was abrogated by mutation of the TM arginine of NKG2D to alanine (NKG2D R→ A, lane 5). The ratio of NKG2D and DAP10 dimer was determined and expressed as the percentage relative to WT (lane 1). Data are representative of four experiments. †, A faint background band appeared at the indicated position in this and other experiments; this band was nonspecific because it was also observed in mixing controls. (B) The stoichiometry of the complex was directly determined by quantification of 35S-labeled NKG2D and DAP10 with a phosphorimager. The NKG2D dimer was isolated with a two-step snIP that targeted the SBP and HA tags attached to the two NKG2D chains, and the molar ratio between NKG2D and associated DAP10 was determined based on the known methionine content of the chains. Multiple parallel reactions were analyzed in each of the three experiments, and the average and standard deviation were calculated for all reactions. *, In each experiment, a mixing control was included to verify specificity. These experiments demonstrated that four DAP10 chains are present for each NKG2D dimer.

Fig. 2.

Fig. 2.

Cooperative assembly of the hexameric NKG2D–DAP10 complex. (A) NKG2D dimers composed of two WT chains (lanes 1–6) or of one WT and one mutant chain (substitution of TM arginine by alanine, R→ A) (lanes 7–12) were isolated by a two-step snIP that targeted the SBP and HA tags attached to the two NKG2D chains. The DAP10 construct used for this experiment had two additional methionine residues (total of three methionine residues, compared with one for WT DAP10 used in Fig. 1) to increase the signal. The stoichiometry measurements are presented here for NKG2D dimers and DAP10 dimers, rather than individual chains as in Fig. 1. Mutation of one of the TM arginine residues in the NKG2D dimer reduced the number of associated DAP10 dimers from 1.86 for the WT NKG2D dimer (lanes 1–3) to 0.8 (lanes 7–9), indicating that only a single DAP10 dimer was bound to the mixed NKG2D dimer. Furthermore, the yield of the mixed NKG2D dimer was reduced to 37% (lanes 7–9) relative to WT (100%, lanes 1–3). In reactions without DAP10 (lanes 4–6 and 10–12), the yield of NKG2D dimer was reduced to a level similar to the NKG2D dimer with one mutated chain. Data are representative of three experiments. (B) Graphical representation of the TM domains (as simplified helical wheels) for the six-chain complex formed by WT NKG2D dimer, compared with the four-chain complex formed by the mixed NKG2D dimer in which one of the TM arginine residues (R) has been mutated to alanine (A). The aspartic acid (D) residues in the TM domains of the DAP10 dimer are indicated in red and positioned at the interface with the NKG2D TM domains. (C) Predicted TM sequences and flanking segments for NKG2D and DAP10.

Fig. 3.

Fig. 3.

The TM domain of NKG2D is sufficient for assembly of a three-chain complex. A construct representing only the NKG2D TM domain and flanking residues (NKG2D.TM) was used to define the minimal interaction site. (A) The NKG2D.TM protein was monomeric, as shown by two-step PC→ SBP snIP analysis of translation reactions with SBP- and PC-tagged NKG2D.TM domains (lane 3). Data are representative of two experiments. (B) The monomeric NKG2D.TM domain formed a three-chain complex with the DAP10 dimer, as shown by two-step PC→ HA snIP targeting the epitope tags attached to the two DAP10 chains (lane 4). The interaction was disrupted by mutation of the TM aspartic acid of DAP10 (D→ A, lane 6). The low-molecular-weight TM peptides shown here and in Fig. 4 were resolved on 12% NuPAGE [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane gels under reducing conditions. Data are representative of six experiments.

Fig. 4.

Fig. 4.

Interaction of DAP10 TM peptides with NKG2D. (A) DAP10 TM peptides formed a dimer, based on two-step PC→ SBP snIP of translation reactions with PC- and SBP-tagged DAP10.TM chains (lane 3). Data are representative of three experiments. (B) The DAP10.TM protein assembled with full-length NKG2D because the complex could be isolated by two-step snIP for the PC and SBP tags attached to DAP10.TM and NKG2D, respectively. *, Mixing controls demonstrated that assembly occurred in ER membranes. Data are representative of two experiments.

Fig. 5.

Fig. 5.

Evidence for the localization of the aspartic acid pair at or near the DAP10 dimer interface. DAP10 dimers in which one or both TM aspartic acids (D) were mutated to asparagine (N), serine (S), or alanine (A) were isolated by two-step snIP that targeted the PC and HA tags attached to WT or mutant chains. Covalent and noncovalent DAP10 dimers (CD and ND, respectively) are indicated, and the total amount of DAP10 dimer was compared for each combination relative to WT (100%, lane 1). Mutation of one or both TM aspartic acids by asparagine (DN and NN combinations in lanes 2 and 3, respectively) increased the yield of DAP10 dimers, whereas substitution of one aspartic acid by alanine (DA combination, lane 6) yielded the lowest level of DAP10 dimers. *, Lane 8 represents the mixing control for the WT combination. The upper gel was run under nonreducing conditions, and the lower gel was run under reducing conditions. Data are representative of four experiments.

Fig. 6.

Fig. 6.

Both TM aspartic acids of the DAP10 dimer contribute to assembly with NKG2D. (A) Assembly reactions were performed with NKG2D and with PC- and HA-tagged WT and mutant DAP10 chains, and DAP10 dimers were targeted by two-step PC→ HA snIP. Radiolabeled NKG2D and DAP10 chains were quantitated and expressed as the ratio of NKG2D/DAP10 relative to WT (lane 1, 100%). Analysis of an aliquot of the reaction not subjected to IP demonstrated equal amounts of NKG2D in each reaction. Substitution of one of the aspartic acid residues by asparagine (DN combination, lane 3) or serine (DS combination, lane 5) substantially reduced the amount of associated NKG2D. The individual DAP10 chains with mutation of the TM aspartic acid migrated differently from the WT chain. *, Alternate lanes represent mixing controls in which DAP10 and NKG2D chains were translated separately. Data are representative of three experiments.

Fig. 7.

Fig. 7.

The assembly process that leads to the formation of a hexameric NKG2D–DAP10 receptor complex. (A) The data demonstrate that each TM domain of human NKG2D assembles with one DAP10 dimer and that this interaction involves both TM aspartic acids of DAP10 (red circles) and the TM arginine (blue circles). (B and C) The arrangement of the TM domains in the hexameric NKG2D–DAP10 structure (B) thus shows similarities to an assembly intermediate of the TCR–CD3 complex (C) that lacks the ζ–ζ dimer.

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