Summary
Activating receptors in cells of hematopoetic origin include members of two unrelated protein families, the immunoglobulin (Ig) and C type lectins, which differ even in the orientation of the transmembrane (TM) domains. We examined assembly of four receptors with diverse function: the NK receptors KIR2DS and NKG2C/CD94, the Fc receptor for IgA, and the GPVI collagen receptor. For each of the four different receptors studied here, assembly results in the formation of a three-helix interface in the membrane involving two acidic TM residues from the signaling dimer and a basic TM residue from the ligand recognition module, an arrangement remarkably similar to the T cell receptor (TCR)-CD3 complex. The fact that the TM domains of Ig family and C type lectins adopt opposite orientations proves that these receptor families independently evolved toward the same structural arrangement of the interacting TM helices. This assembly mechanism is thus widely utilized by receptors in cells of hematopoetic origin.
Introduction
Cells of the hematopoetic system continuously monitor their environment for sudden changes, such as display of viral peptide-MHC complexes (T cells), upregulation of stress-induced ligands (NK cells), circulation of antibody-decorated pathogens (macrophages and other phagocytic cells), and exposure of extracellular matrix proteins at sites of vascular injury (platelets). Each cell type expresses surface receptors that induce a particular activation program, and important examples include the TCR-CD3 complex required for T cell differentiation and function, a family of NK receptors that trigger lysis and cytokine production by NK cells, Fc receptors that induce phagocytosis of antibody-decorated pathogens and immune complexes, and the glycoprotein VI (GPVI) collagen receptor that triggers platelet aggregation. The ligands recognized by these receptors are structurally diverse and include MHC and MHC-like molecules (TCR, several NK receptors), Igs (Fc receptors), and collagen (GPVI receptor) (Garcia et al., 1999; Colonna and Samaridis, 1995; Wagtmann et al., 1995; Braud et al., 1998; Ravetch and Kinet, 1991; Monteiro and Van De Winkel, 2003; Clemetson et al., 1999). A common feature of these receptors is the absence of signaling modules in the cytoplasmic domains of the ligand binding receptor chain(s), which assembles with dimeric-signaling modules with cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) that are phosphorylated after receptor triggering. We refer to this group as “activating immune receptors,” because phosphorylation of ITAMs induces a characteristic activation program in cells of the immune system, including calcium flux. Interestingly, these receptors belong to distinct protein families, the Ig or C type lectin families, which differ not only in their primary sequence and tertiary structure but also in the orientation of the TM domains: the N terminus is located in the extracellular space for Ig family receptors (type I membrane proteins), but in the cytoplasm for C type lectin receptors (type II membrane proteins). The TM domains of many of these receptors carry a basic residue (lysine or arginine) and associate with signaling dimers with a pair of aspartic acid TM residues, raising the question of whether these receptors nevertheless assemble based on the same mechanism.
The majority of these receptors associate with one of four known disulfide-linked signaling dimers: ζ, Fcγ, DAP10, and DAP12. The ζ chain is part of the TCR-CD3 complex (Samelson et al., 1985; Sussman et al., 1988), whereas Fcγ represents a signaling component for several Fc receptors, the GPVI collagen receptor, and other receptors (Kuster et al., 1990; Tsuji et al., 1997). ζ and Fcγ have strong sequence homology, in particular in the TM domains, probably reflecting a gene duplication event. The cysteine residues that form the interchain disulfide bond and the aspartic acid residues are located in the N-terminal segment of the predicted TM domains (positions 2 and 6, respectively), and a helical wheel model of the TM domains indicates that the cysteine and aspartic acid residues are located on the same face of the TM helix (Rutledge et al., 1992). The two aspartic acid residues of the ζ-ζ and Fcγ-Fcγ dimers may thus be positioned at or near the dimer interface. The cytoplasmic domain of ζ has three ITAMs, compared to the single ITAM of Fcγ. DAP10 and DAP12 represent a second group of disulfide-linked signaling dimers. DAP10 assembles with the NKG2D receptor expressed by NK cells and subpopulations of T cells (Wu et al., 1999), whereas DAP12 (also termed KARAP) forms the signaling component of a number of activating receptors, such as the NK receptors KIR (KIR2DS and KIR3DS) and NKG2C/CD94 (Lanier et al., 1998a; Lanier et al., 1998b; Tomasello et al., 1998; Wu et al., 1999; Wu et al., 2000). There is little sequence homology to the ζ/Fcγ pair, and the positions of the cysteine and aspartic acid residues are different: DAP10 and DAP12 have two cysteine residues per chain in the EC rather than the TM domain, and the aspartic acid residue is located close to the center rather than in the N-terminal segment of the predicted TM domain (Figure 1).
Figure 1. Topology of Investigated Receptors.
(A) The assembly of DAP12 with the NK receptors KIR2DS2 and NKG2C/CD94, which belong to distinct families (Ig and C type lectin, respectively) and differ in the topology of the transmembrane (TM) domains (indicated by arrows), was examined. Both molecules have a basic residue within the TM domain (lysine, K) that is required for assembly with DAP12.
(B) The aspartic acid pair (D-D) of the Fcγ-signaling dimer is located in the N-terminal segment of the TM domains, matching the localization of the basic residue (arginine, R) in the TM domains of the Fc receptor for IgA (FcαRI) and the platelet receptor for collagen (GPVI).
(C) The DAP12 and Fcγ-signaling modules have no sequence homology in the extracellular (EC) and TM domains and differ in the position and spacing of the cysteine and aspartic acid (labeled as “C” and “D,” respectively) residues that are required for covalent dimer formation and receptor assembly, respectively.
We recently demonstrated that assembly of the TCR-CD3 complex is organized by interaction of ionizable transmembrane residues. Assembly of each of the three signaling dimers (CD3δε, CD3γε, and ζ-ζ) with TCR requires a basic TCR TM residue and a pair of acidic residues in the TM domains of the respective signaling dimer (Call et al., 2002). Each assembly step thus results in the formation of a three-helix interface with one basic and two acidic TM residues. Both acidic residues in the TM domains of a signaling dimer make similar contributions to assembly, because a conservative substitution from aspartic acid to asparagine in either CD3δ or CD3ε reduced assembly to a similar extent. These results raised three questions. (1) Does formation of such a three-helix interface represent a general mechanism for assembly of activating immune receptors with basic TM residues? (2) Is this mechanism also responsible for the assembly of receptors that represent type II membrane proteins in which the TM helix is in an antiparallel orientation relative to the TM helices of the signaling dimer? (3) Do the acidic TM residues of a signaling dimer represent a functional pair with which the basic TM residue of the receptor interacts? We have examined these questions for four activating receptors that are diverse in their sequence, membrane topology, and function (Figure 1).
Results
The structural requirements for assembly were studied for two human receptors (KIR2DS2 and NKG2C/CD94) that associate with the DAP12-signaling dimer but are members of different protein families, the Ig family (KIR2DS2) and C type lectins (NKG2C/CD94) (Lanier et al., 1998a; Lanier et al., 1998b; Wu et al., 2000). These receptors are expressed by NK cells and subpopulations of T cells, and receptor activation triggers NK cell cytotoxicity and cytokine production (Colonna and Samaridis, 1995; Wagtmann et al., 1995; Braud et al., 1998). This prior work had established that members of these receptor families can assemble with the same signaling modules, indicating convergent evolution. The fact that these receptors are unrelated in sequence and structure and differ even in the orientation of the TM domains thus offered an ideal opportunity to assess whether these receptors had converged on the same structural mechanism for receptor assembly. KIR2DS2 is a classical type I membrane protein, whereas NKG2C/CD94 is a heterodimer of type II membrane proteins whose TM domains run in an antiparallel orientation to those of KIR and the DAP12 dimer (Figure 1A). The difference in topology of the TM domains thus excludes the possibility that potential similarities in the mechanism of receptor assembly can be explained by a gene or exon duplication event: duplication and inversion of the exon encoding the TM sequence of KIR (or NKG2C) would not result in a TM domain but rather a nonsense sequence.
The KIR2DS2 and NKG2C TM domains have no primary sequence homology, and the critical lysine residues are located at positions 9 and 15 from the N terminus of the predicted TM domains in KIR and NKG2C, respectively. The lysines are nevertheless positioned at a similar depth of the plasma membrane due to the type I and type II membrane topology of these proteins: position 9 from the N terminus of the KIR TM domain and position 9 from the C terminus of the NKG2C TM domain (Figure 1A).
Cooperativity in the Assembly of KIR with DAP12
Receptor assembly was examined in two- and three-step sequential nondenaturing immunoprecipitation (snIP) experiments in order to isolate radiolabeled protein complexes with defined composition (Call et al., 2002). The method is based on specialized affinity tags for elution of protein complexes under nondenaturing conditions after the first IP, either by addition of EDTA (calcium-dependent protein C antibody) or competition with biotin (streptavidin binding peptide [SBP]). Assembly experiments were performed in an in vitro translation system in which [35S]-labeled membrane proteins are inserted cotranslationally into ER microsomes. This system faithfully reproduces membrane protein assembly events defined in cells by metabolic labeling (Ribaudo and Margulies, 1992; Huppa and Ploegh, 1997; Hebert et al., 1998). The advantages of this system are that the major fraction of radiolabeled protein is synthesized from input RNAs and that the effect of mutations in any of the receptor components can be assessed by using relatively low quantities of radiolabeled amino acids.
The assembly of KIR with the DAP12 dimer was examined in experiments in which DAP12 chains carried C-terminal protein C (PC) or hemagglutinin (HA) epitope tags so that DAP12 dimers with a mutation in only one of the two chains could be selected. KIR2DS2 specifically assembled with the DAP12 dimer, as shown by two-step PC→HA snIP targeting the epitope tags attached to DAP12 chains (Figure 2A, lane 1). This interaction required the TM lysine of KIR, because its substitution by alanine (KIR K→A) prevented assembly (lane 5). Quantification of [35S]-labeled proteins using a phosphor imager demonstrated substantially larger quantities of the DAP12 covalent dimer (DAP12 CD) in the presence of KIR (lane 1), compared to reactions in which DAP12 and KIR were translated separately (lanes 4 and 8, 11.8% and 10.3% of DAP12 dimer relative to lane 1, respectively). Efficient formation of DAP12 CD required the TM lysine of KIR, because the yield of DAP12 dimer was reduced to 24.1% for the KIR K→A mutant (lane 5). Interaction with the KIR TM domain contributes substantially to efficient DAP12 dimerization, indicating that formation of the three-chain DAP12-KIR complex is a cooperative assembly event.
Figure 2. Cooperative Assembly of KIR and DAP12.
(A) The assembly of KIR (KIR2DS2) with DAP12 was examined in an in vitro translation system with ER microsomes. Membranes were solubilized with digitonin and complexes isolated by sequential nondenaturing immunoprecipitation (snIP) utilizing the protein C (PC) and hemagglutinin (HA) affinity tags on the C terminus of the two DAP12 chains. After the first IP step with the calcium-dependent PC antibody, complexes were released from IP beads with EDTA and reprecipitated with the HA antibody. As controls, the PC (lanes 2 and 6) or HA antibody (lanes 3 and 7) was replaced with isotype controls. The assembly of KIR with the DAP12 dimer occurred cotranslationally in ER microsomes prior to solubilization, because no interaction was detected in mixing controls (asterisk, lanes 4 and 8) in which KIR and DAP12 were assembled in two separate reactions that were mixed prior to solubilization. Mutation of the KIR TM lysine to alanine (KIR K→A, lane 5) abrogated interaction with DAP12. The amount of DAP12 covalent dimer (DAP12 CD) was quantitated by using a phosphor imager and expressed as a percentage relative to wild-type (wt) (lane 1). DAP12 ND: nondisulfide-linked DAP12 dimer isolated with the IP procedure. The experiment shown is representative of four experiments.
(B and C) The KIR-DAP12 complex contains only a single KIR chain. Assembly reactions were performed with DAP12 and two KIR chains (KIR.SBP and KIR.FLAG). Samples were analyzed by single-step IP for either SBP (lane 1) or FLAG (lane 2) tags or by two-step snIP (SBP→FLAG) (lane 3). Lanes 4–6 represent mixing controls. Aliquots of solubilized membranes were analyzed to confirm the presence of equal quantities of radiolabeled proteins (C). The experiment shown is representative of three experiments.
The presence of two acidic residues within the TM domains of the DAP12 dimer raised the question whether one or two KIR chains could assemble with a DAP12 dimer. We addressed this question in assembly experiments in which two KIR chains were used that differed in MW based on the length of the C-terminal affinity tag (KIR.SBP and KIR.FLAG) (Figure 2B). When the SBP tag was targeted in the IP, KIR.FLAG was not coprecipitated (lane 1); the second KIR chain was also not coprecipitated in the reverse experiment in which the FLAG tag was targeted in the IP (lane 2). In addition, no complex containing both KIR.SBP and KIR.FLAG was isolated in a two-step SBP→FLAG snIP (lane 3), even though equal amounts of radiolabeled proteins were present in all reactions (Figure 2C). The results demonstrate that the KIR-DAP12 complex contains only a single KIR chain.
The TM Aspartic Acid Pair Affects DAP12 Dimerization
Experiments in a cellular system have shown that mutation of the DAP12 TM aspartic acid prevents assembly with KIR2DS2 (Lanier et al., 1998a). The two aspartic acid residues are located near the center of the TM domains in the DAP12 dimer, and it would be energetically highly unfavorable to expose these two polar side chains to the hydrophobic interior of the membrane. Because the aspartic acids may be located at or near the DAP12 dimer interface and represent a functional pair that interacts with the TM lysine of KIR, we examined whether substitution of the aspartic acids affects DAP12 dimerization. Assembly reactions were performed with two DAP12 chains in which one or both chains carried a mutation of the TM aspartic acid (D) to asparagine (N), serine (S), or alanine (A). The two-step snIP procedure (PC→HA) allowed us to select DAP12 dimers in which one of the two aspartic acids (DN, DS, and DA combinations) or both (NN, SS, and AA combinations) were mutated (Figure 3A). Of particular interest was the substitution from aspartic acid to asparagine: asparagine is not ionizable but has the same size as aspartic acid and can serve as both hydrogen bond donor and acceptor. Substitution of one or both aspartic acids by asparagine (Figure 3A, lanes 2 and 3, respectively) resulted in substantial increases in the yield of DAP12 dimer (240% and 350% relative to wild-type [wt] for the DN and NN combinations, respectively). Substitution of both aspartic acids by serine, whose side chain is also polar yet shorter than that of asparagine, also increased dimerization (lane 5, 160% relative to wt), but not to the same extent as the DN or NN substitutions. The lowest level of DAP12 dimer was observed when one aspartic acid was substituted by alanine, which has a short hydrophobic side chain (DA combination, lane 6). Analysis of aliquots of the assembly reaction under reducing conditions without IP (Figure 3C) demonstrated that equal quantities of radiolabeled proteins were present in the reactions. DAP12 assembly was thus most efficient with polar side chains at this position in both chains, providing evidence for the localization of the aspartic acid residues at or near the DAP12 dimer interface.
Figure 3. Both TM Aspartic Acid Residues of DAP12 Participate in the Assembly with KIR.
(A–C) Evidence for the localization of the aspartic acid pair at or near the DAP12 dimer interface. Assembly reactions were performed with two DAP12 chains that carried C-terminal PC or HA tags for isolation of DAP12 dimers in which one or both aspartic acids (labeled “D” within the figure) were substituted by asparagine (labeled “N” within the figure), serine (labeled “S” within the figure), or alanine (labeled “A” within the figure). Proteins were separated by SDS-PAGE under nonreducing conditions after PC→HA snIP targeting both DAP12 chains (A) or under nonreducing (B) and reducing conditions (C) without IP. Dimers representing two wt chains are labeled as DD, whereas mixed dimers with a mutation of one of the two aspartic acid residues to N, S, or A are labeled as DN, DS, and DA, respectively. DAP12 covalent dimers are labeled as DAP12 (CD) and DAP12 monomers as DAP12 (M). Representative of three experiments.
(D and E) Critical role of the TM aspartic acid pair of DAP12 for assembly with KIR. Assembly reactions were set up with KIR as well as PC-or HA-tagged DAP12 chains that had either the wt TM sequence or a substitution of the TM aspartic acid. The two DAP12 chains were targeted by PC→HA snIP, and associated KIR was quantitated by using a phosphor imager (expressed as percentage relative to wt, lane 1), both for the experiment shown in this figure and the average of four separate experiments. (E) Schematic of the formation of a three-helix interface between the DAP12 dimer and KIR in the membrane. Chains are depicted as simplified helical wheels, and the critical residues are indicated on the DAP12 (“D,” aspartic acid; “A,” alanine) and KIR chains (“K,” lysine).
(F) All chains of the complex were targeted in a three-step snIP: the KIR chain was targeted via a C-terminal SBP tag and complexes eluted by competition with biotin followed by a PC→HA snIP for the two DAP12 chains. As a control, a two-step PC→HA snIP was performed with the KIR mutant (KIR K→A). The experiment shown is representative of three experiments.
Both TM Aspartic Acids of DAP12 Are Critical for Assembly with KIR
We used the same general approach to examine the contribution of the aspartic acid pair to DAP12 assembly with KIR and quantitated the amount of DAP12-associated KIR after selection of defined DAP12 dimers by two-step snIP (PC→HA) (Figure 3D). Because both DAP12 chains were targeted in the IP, the amount of coprecipitated KIR represented the amount of KIR-DAP12 complex (wt defined as 100% in Figure 3D). Substitution of both DAP12 aspartic acids by polar (NN and SS combinations) or nonpolar residues (AA combination) abrogated assembly with KIR (lanes 5, 9, and 13). Furthermore, mutation of only one of the aspartic acids greatly reduced complex formation: DAP12-associated KIR was reduced to ~4% relative to wt for the DS and to ~1% or less for the DA combination, respectively (lanes 7 and 11). Even the conservative substitution of one aspartic acid to asparagine resulted in a reproducible reduction of assembly with KIR (to 45%, Figure 3D, lane 3), even though DAP12 dimer formation was substantially higher for the DN combination (240% relative to DD, Figure 3A, lane 2). These results were highly reproducible, and the quantification is shown both for the gel in Figure 3D and the average of four independent experiments. The experiment in Figure 2A had demonstrated that assembly of the KIR-DAP12 complex is cooperative, and lower levels of DAP12 were isolated for the DS and DA dimers that did not efficiently assemble with KIR. Inefficient assembly of KIR with these mutants was also reflected by a low KIR/DAP12 ratio (14.8% for the DS dimer and 5.8% for the DA dimer, compared to 100% for the wt DD dimer). We confirmed these results by using a different IP strategy in which all three chains of the complex were directly selected in a three-step snIP (Figure 3F).
The outcome of the DA and DN substitutions was of particular interest because very similar findings had been made for TCR assembly with CD3δε: substitution of either aspartic acid in the TM domains of the CD3δε dimer by alanine reduced TCR association to <10%, whereas substitution of either aspartic acid to asparagine gave an intermediate level of assembly (29%–38%) (Call et al., 2002). These results demonstrate that both aspartic acids of DAP12 are involved in assembly with KIR (Figure 3E) and that this interaction is remarkably similar to assembly of TCR with its signaling dimers, even though the three TM helices are otherwise distinct.
The Three-Helix Assembly Mechanism Is Also Relevant for Receptors with Type II Membrane Topology
These findings raised the question of whether this structural mechanism is also responsible for the assembly of activating immune receptors that do not belong to the Ig superfamily. NKG2C/CD94 is a member of the C type lectin family and assembles with the DAP12 dimer, an interaction that requires the TM lysine of NKG2C (Lanier et al., 1998b). It had previously been shown that NKG2C and CD94 form a heterodimer and that surface expression of NKG2C requires the presence of both CD94 and DAP12 (Lanier et al., 1998b). We examined whether the NKG2C chain, which bears the TM lysine residue, is sufficient for interaction with DAP12, or whether CD94 is also required. Assembly reactions involving PC- and HA-tagged DAP12 chains as well as NKG2C and/or CD94 were analyzed by two-step PC→HA snIP to isolate DAP12-associated proteins (Figure 4A). NKG2C assembled with DAP12 in the presence and absence of CD94 (lanes 1 and 5), and CD94 was only coprecipitated in the presence of NKG2C (lanes 1 and 9). When the TM lysine of NKG2C was mutated to alanine (NKG2C K→A), neither NKG2C nor CD94 were associated with DAP12 (lanes 3 and 7). Assembly experiments with two differentially tagged NKG2C chains demonstrated that only a single NKG2C/CD94 heterodimer assembles with DAP12 (Figure S1 available online with this article).
Figure 4. Structural Requirements for the Assembly of NKG2C/CD94 with the DAP12-Signaling Dimer.
(A) The NKG2C chain is sufficient for assembly with DAP12. Assembly reactions were performed with PC- and HA-tagged DAP12 as well as different combinations of CD94, NKG2C, and a NKG2C (K→A) mutant. Proteins associated with the DAP12 dimer were isolated by PC→HA snIP and resolved by SDS-PAGE. Lanes 2, 4, 6, 8, and 10 represent mixing controls in which the two tagged DAP12 chains were assembled separately from the other protein(s); the corresponding reactions were combined prior to solubilization. The experiment shown is representative of three experiments.
(B) Both aspartic acid residues of the DAP12 TM domains participate in the assembly with NKG2C. Translation/assembly reactions were set up with NKG2C as well as PC- and HA-tagged DAP12 chains in which the TM aspartic acid (“D”) was either present or mutated to asparagine, serine, or alanine (“N,” “S,” and “A,” respectively); reactions were analyzed as described in Figure 3D.
We next examined whether assembly of NKG2C with DAP12 is based on the interaction of ionizable TM residues, as described above for KIR-DAP12. DAP12 dimers with substitution of one or both TM aspartic acid residues were selected by two-step PC→HA snIP as described above in the KIR experiments, and the amount of associated NKG2C was quantitated (Figure 4B). Substitution of only one of the aspartic acid residues of DAP12 by either a polar or nonpolar amino acid (DS and DA combinations, lanes 7 and 11, respectively) greatly reduced the yield of coprecipitated NKG2C (to 7% and 10% relative to wt, respectively). In the presence of NKG2C, these substitutions also reduced the yield of DAP12 dimers (lanes 7 and 11), as described above for KIR. A substantial reduction in the yield of complex was also observed with the DN combination (23% relative to DD). These results were highly reproducible, and the quantification is shown both for the gel in Figure 4B and the average of three independent experiments. The striking similarities in the outcome of this experiment between KIR and NKG2C (Figures 3 and 4) demonstrate that a similar three-helix interface is formed in the membrane based on the contributions of the three ionizable TM residues, even though the TM helix of NKG2C has an orientation opposite to that of KIR.
The TM Domains of NKG2C and KIR Are Sufficient for Assembly with DAP12
The interaction was further defined with a construct (NKG2C-TM) that encoded the TM domain of NKG2C as well as three N- and C-terminal flanking residues for proper membrane insertion. A methionine initiation codon as well as an SBP tag were placed N-terminal to the TM domain, and complexes were isolated by two-step snIP targeting the SBP tag of NKG2C-TM and the HA-tag on DAP12 (Figure 5). DAP12 CD and the NKG2C-TM protein were only isolated when the aspartic acids in the TM domains of DAP12 were in the wt configuration (Figure 5A, lane 1, DD), but not when they were mutated to asparagine or serine (NN and SS combinations, lanes 2 and 3). The mixing control in lane 4 demonstrated that this interaction was specific, and analysis of an aliquot of all reactions without IP (Figure 5B) indicated that similar amounts of radiolabeled proteins were present in all reactions. A similar construct was generated to examine the KIR TM domain, which included a signal peptide N-terminal to the SBP tag for this type I membrane protein. A complex representing the TM domain of KIR and the DAP12 dimer could be isolated in the same fashion, and assembly was again sensitive to substitution of the DAP12 TM aspartic acids. Taken together, the experiments shown in Figures 3–5 demonstrate that the assembly of KIR or NKG2C with DAP12 is based on a three-chain interaction in the membrane that is remarkably similar even though the receptors belong to protein families with distinct membrane topology.
Figure 5. The TM Domains of NKG2C or KIR Are Sufficient for Assembly with the DAP12 Dimer.
Assembly experiments were performed with NKG2C or KIR TM constructs with an N-terminal SBP tag, and analyzed by two-step snIP targeting the SBP tag attached to the KIR or NKG2C TM domains and the HA tag on DAP12. DAP12 CD and associated TM domains were visualized by SDS-PAGE under nonreducing conditions (A). Aliquots of all reactions were also analyzed without IP to confirm the presence of similar quantities of radiolabeled proteins in all reactions (B). TM peptides were resolved on a 12% NuPAGE Bis-Tris gel. The experiment shown is representative of three experiments.
Limited Changes in the TM Domain Are Sufficient to Convert an Inhibitory KIR into a DAP12-Interacting Protein
All KIR genes show striking sequence similarities in the EC, TM, and cytoplasmic domains, indicating that they arose from a common ancestor(s) by gene duplication (Vilches and Parham, 2002; Biassoni et al., 1996; Colonna and Samaridis, 1995; Wagtmann et al., 1995), and the activating KIR2DS2 and inhibitory KIR2DL3 proteins differ in the EC domains only at a few positions. Particularly revealing is an analysis of their cytoplasmic domains: the inhibitory KIR2DL3 has a long cytoplasmic tail (L designation) with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (YAQL and YTEL, underlined in Figure 6A), whereas KIR2DS2 has a shorter cytoplasmic domain. This is due to the presence of a stop codon within the sequence segment representing the first ITIM in the inhibitory receptor (YA*L in KIR2DS2 and YAQL in KIR2DL3) (Biassoni et al., 1996). Importantly, the striking degree of sequence similarity between the two proteins extends beyond this stop codon; even the four-residue motif of the second ITIM is present in the KIR2DS2 sequence (Figure 6A). These findings indicate that these KIR genes originated from a common ancestor with a long cytoplasmic tail. Activating KIR may thus be the product of limited changes that inactivated the inhibitory cytoplasmic motifs (such as the introduction of a stop codon, as in KIR2DS2) and permitted association with the activating DAP12 dimer.
Figure 6. Limited Changes in the TM Domain Are Sufficient for Conversion of an Inhibitory KIR to a Receptor that Assembles with DAP12.
(A) Alignment of the cytoplasmic domains of the activating KIR2DS2 and the inhibitory KIR2DL3 receptors.
(B) The differences in the TM domains of the activating KIR2DS2 (left) and the inhibitory KIR2DL3 (right) are highlighted on a helical wheel representation (green, hydrophobic residues; blue, basic residue; and gray, other changes).
(C and D) Conversion of KIR2DL3 to a DAP12-interacting protein. Assembly reactions were performed with the activating KIR2DS2 receptor (lane 1, positive control), the inhibitory KIR2DL3 receptor (lane 3) and KIR2DL3 constructs in which one to three residues at positions 9, 11, and 13 (I-L-I) in the TM domain were changed to those found in the activating KIR (K-P-T), as indicated in the table beneath the gel. In addition, a construct in which a stop codon was placed into the first ITIM at the same position as in KIR2DS2 was tested in combination with the positions 9 I→K substitution (lane 8). Complexes were isolated by two-step snIP (PC→HA) that targeted the epitope tags attached to DAP12 chains. The KIR2DL3 protein and its variants with a long cytoplasmic tail (lanes 3–7) were expressed at a lower level than KIR2DS2 (lane 1) and the KIR2DL3 variant with a truncated cytoplasmic tail (lane 8), as shown by SDS-PAGE analysis of an aliquot of solubilized membranes not subjected to IP (C). These differences in expression level were taken into account in the quantification of the IP experiments (average of two experiments).
KIR2DL3 and KIR2DS2 differ at four positions within the TM domains (Figure 6B), and we examined which changes in the TM domain of the inhibitory KIR2DL3 protein would be sufficient to permit interaction with DAP12. The TM sequences of all activating human KIR are highly related and carry a lysine at position 9 of the TM domain, whereas an isoleucine is located at this site in all inhibitory KIR sequences. The inhibitory KIR2DL3 protein failed to assemble with DAP12 (Figure 6D, lane 3), in agreement with studies in cellular systems (Lanier et al., 1998a). Conversion of the isoleucine at position 9 of the TM of KIR2DL3 to a lysine (lane 4) was sufficient for assembly with DAP12 (44% relative to wt). Additional substitutions at TM positions 11 and 13 to those residues observed in activating KIR (lane 5–7) or placement of a stop codon within the cytoplasmic tail of KIR2DL3 at the same position as in the activating receptor KIR2DS2 (lane 8) did not increase the level of assembly. The KIR2DL3 protein and its variants with the long cytoplasmic tail (lanes 3–7) were expressed at a lower level than KIR2DS2 (lane 1) and the KIR2DL3 mutant with the shortened cytoplasmic tail (lane 8), and the expression levels were taken into account in the quantification shown in Figure 6D. Introduction of a basic TM residue at the appropriate site and introduction of a stop codon in the cytoplasmic domain thus represent the minimal changes for the conversion of an inhibitory KIR to a receptor that assembles with the DAP12-signaling module and lacks the inhibitory motifs in the cytoplasmic domain.
The Three-Helix Assembly Mechanism Is Relevant for Receptors Expressed by Diverse Cell Types of Hematopoetic Origin
The receptors described thus far are expressed by NK cells and subpopulations of T cells. We examined whether this mechanism also directs the assembly of receptors expressed by other cell types of hematopoetic origin. We chose the Fc receptor for IgA (FcαRI) that is expressed by cells of the myeloid lineage (neutrophils, eosinophils, interstitial dendritic cells, and the majority of monocytes/macrophages) (Monteiro and Van De Winkel, 2003; Maliszewski et al., 1990) as well as GPVI, the major collagen receptor responsible for platelet activation (Clemetson et al., 1999). The signaling dimer (Fcγ) that associates with these receptors has a TM sequence distinct from that of DAP12, and the locations of the aspartic acid pair in the Fcγ dimer and the basic residue (arginine) in these receptors differ from DAP12 and its associated receptors (Figure 1B).
Assembly experiments utilizing the same approaches as described above for KIR and NKG2C demonstrated that the TM arginine is critical for association of FcαRI as well as GPVI with the Fcγ dimer (Figure 7A and Figure S2). Both aspartic acid residues of the Fcγ dimer contributed to assembly with FcαRI, as the yield of complex was substantially reduced when only one of the aspartic acids was substituted by another polar residue (to 16.5% and 19% relative to wt for the DN and DS combinations, respectively) (Figure 7B). Nevertheless, small quantities of complex (5.2% relative to wt) were formed when both aspartic acids were mutated to alanine, as shown by two-step snIP targeting the SBP tag attached to FcαRI and the HA-tag attached to Fcγ (Figure 7C). This situation resembles the interaction of TCRα with the ζ-ζ dimer in which small quantities of complex were recovered when mutations were introduced at this interaction site (Call et al., 2002). In both cases, an arginine is located in the N-terminal part of the TM domain, closer to the surface of the membrane than the lysines that mediate the interaction of KIR and NKG2C with DAP12. The TM aspartic acid pair of Fcγ was also essential for assembly with the platelet receptor GPVI, but this protein was translated at lower levels than the other three receptors and therefore studied in less detail (Figure S1).
Figure 7. The Structural Requirements for Assembly of FcαRI with the Fcγ-Signaling Dimer Are Similar to Those Defined for Interaction of KIR and NKG2C with DAP12.
(A) Mutation of the arginine located at position 3 of the predicted TM domain of FcαRI prevents assembly with the Fcγ dimer. Assembly reactions were set up with human FcαRI as well as PC- and HA-tagged Fcγ chains and analyzed by two-step PC→HA snIP. Controls were as described in Figure 2.
(B) The contribution of both aspartic acid residues of Fcγ was examined as in Figure 3 for the KIR-DAP12 interaction. Assembly reactions were performed with PC- and HA-tagged Fcγ chains in which one or both chains had the wt TM sequence or a substitution of the aspartic acid residue; complexes were isolated by two-step PC→HA snIP. The experiment shown is representative of three experiments.
(C) The formation of small quantities of complex in the absence of both aspartic acid side chains was confirmed in an assembly experiment with SBP-tagged FcαRI and HA-tagged Fcγ chain in which complexes were directly isolated by two-step SBP→HA snIP. Small quantities of complex were recovered when both aspartic acids of the Fcγ dimer were substituted by asparagine (NN) or alanine (AA). The experiment shown is representative of two experiments.
Discussion
These data establish that the same structural mechanism is responsible for the assembly of a variety of activating immune receptors with their signaling-transducing subunits, despite their diversity in sequence and membrane topology. The key feature of this assembly mechanism is the formation of a three-helix interface in the membrane that is created by a basic TM residue of the ligand binding subunit and a pair of acidic TM residues of the relevant signaling dimer. Such three-helix interactions ensure formation of the appropriate receptor structures. Assembly based on protein interactions among TM helices rather than extracellular domains also permits substantial structural diversity of extracellular domains that bind diverse groups of ligands, ranging from soluble proteins (Fc receptors) to membrane proteins (TCR, NKG2C/CD94) and extracellular matrix proteins (GPVI). This assembly mechanism is compatible with large conformational changes of the EC domains upon ligand binding, as well as lateral interactions of EC domains with other membrane proteins.
How do the three polar TM residues interact in the assembly process? Experiments on model TM domains demonstrated that placement of a single aspartic acid in a hydrophobic segment results in formation of dimers and trimers, indicating that acidic residues can interact with each other in the membrane environment (Zhou et al., 2001; Gratkowski et al., 2001; Senes et al., 2004). An interaction between acidic residues may require prior protonation of at least one of the carboxyl groups or acquisition of a structural water molecule that is hydrogen bonded between the two carboxyl groups during partioning into the lipid bilayer. There may not be a charge imbalance in the assembled structures, because an aspartic acid pair with a shared proton would have a net charge of −1, rather than −2 (Engelman, 2003; Senes et al., 2004). The experimental data clearly demonstrate that the charge of the acidic groups is relevant to the assembly process: substitution of both aspartic acids by asparagine abrogated assembly for all receptors studied. This substitution is informative because the side chains of asparagine and aspartic acid have the same size and can serve both as hydrogen bond donor and acceptor. Assembly was observed, albeit at a reduced level, when one of the aspartic acid residues was substituted by asparagine. Such an asparagine-aspartic acid pair may be similar to an aspartic acid-aspartic acid pair (with loss of a certain degree of symmetry) in which one of the acidic groups is protonated because the CONH2 group of asparagine and a COOH group of aspartic acid could both donate a proton for interaction with a deprotonated COO− group. The mechanism delineated here applies to the majority of, but not all, activating receptors in the immune system, because basic residues are not present in the TM domains of the B cell receptor, the Fc receptor for IgE, and CD16. Polar residues are present in the TM domains of these proteins, but whether and how they contribute to assembly is not yet known.
Because a similar structural arrangement underlies the formation of a variety of different receptors, it is important to ask which mechanisms contribute to specificity. The following aspects are particularly relevant. (1) Restricted expression of ligand-recognition modules and signaling dimers in the relevant cell type(s). Relevant examples are the T cell-specific expression of the TCRα/β or TCRγ/δ chains and mega-karyocyte/platelet-specific expression of the GPVI collagen receptor. Several of the signaling adaptors are only expressed by a particular cell type (CD3γ, δ, and ε) or a limited group of hematopoetic cells (ζ, DAP10). (2) Matching of the location of the TM aspartic acid pair to the position of the basic TM residue in the relevant receptor(s). In DAP10 and DAP12, the pair of acidic residues is located close to the center of the TM domains, matching the position of the basic TM residue of NKG2D, NKG2C, and KIR. In contrast, the TM arginine of FcαRI and GPVI is located in the N-terminal segment of the membrane-spanning segment, corresponding to the location of the TM aspartic acid pair of the Fcγ dimer. (3) Packing interactions among the three TM helices resulting in steric hindrance for mismatched receptor-signaling dimer combinations. This mechanism accounts for the specificity of DAP10 for NKG2D: due to sequence differences between the DAP10 and DAP12 TM domains, the human NKG2D receptor assembles with DAP10, but not DAP12, even though the TM aspartic acid pair is located at the same position in both signaling dimers (Wu et al., 2000). (4) Contribution of EC domains to specificity of assembly. In the TCR-CD3 complex, CD3δε interacts with TCRα, whereas CD3γε binds to TCRβ. The CD3γ TM domain can be replaced with the corresponding segment from CD3δ, but replacement of the CD3γ EC domain with the CD3δ EC domain interferes with assembly (Wegener et al., 1995; Call et al., 2002).
Utilization of the same structural mechanism for the assembly of type I and type II membrane proteins represents a clear-cut example of convergent evolution. The TM domains of the type I membrane protein KIR2DS2 and the type II membrane protein NKG2C are sufficient for assembly with the DAP12 dimer. Only a single residue within the TM domain of the inhibitory KIR2DL3 receptor needed to be changed to enable assembly with DAP12 (Figure 6), and such a limited change may be sufficient due to the dominant contribution of the ionizable TM residues to the assembly process. In other cases, additional substitutions may be required to prevent steric hindrance and improve surface complementarity among the three interacting TM helices, but the sequence requirements do not appear to be strict. Modification of an ancestral gene at a limited number of positions may thus represent a common theme in the emergence of activating immune receptors. For several receptors (KIR, CD94/NKG2, and murine Ly49 families), both activating and inhibitory counterparts have been defined that apparently arose by gene duplication and diversification (Blery et al., 2000; Vilches and Parham, 2002; Natarajan et al., 2002). The changes required for association with an activating signaling subunit may thus have occurred independently in each of these families as part of the diversification process that created both inhibitory and activating forms. The difference in the topology of Ig and C type lectin receptors discussed above excludes the possibility that all activating receptors arose from a common precursor with a basic TM residue.
Study of the murine Ly49 genes has provided a fascinating example of pathogen-driven evolution of activating and inhibitory immune receptors. Mouse cytomegalovirus (MCMV) causes severe pathology in 129/J, but not C57BL/6, mice. The virus encodes a MHC-like protein (m157) that engages an inhibitory NK cell receptor in MCMV-susceptible mice (Ly49I), blocking NK cell-mediated cytolysis and cytokine production. In MCMV-resistant mice, the viral protein instead binds to a related receptor, Ly49H, which assembles with DAP12 and thus serves an activating function (Arase et al., 2002; Smith et al., 1998). The genes encoding these inhibitory and activating forms are highly homologous in their sequence, suggesting that one evolved from the other in response to selective pressure exerted by a pathogen. Sequence comparison of activating (Ly49H and D) and inhibitory (Ly49A, C, G2, and I) Ly49 receptors shows striking parallels to the KIR system discussed above. The two activating Ly49 receptors have an arginine at position 13 of the predicted TM domain, whereas a hydrophobic amino acid is present at this position in the inhibitory Ly49 receptors. The TM sequences are otherwise very similar, with three or four differences at other TM positions between activating and inhibitory Ly49 receptors. The cytoplasmic domains of the inhibitory receptors have a single ITIM; this motif is not functional in activating receptors, because the key tyrosine is changed to a phenylalanine. The viral m157 gene may have evolved to provide a selective advantage for the virus by inhibiting NK cell function, but limited changes in the Ly49 gene, including the introduction of a basic TM residue required for assembly with DAP12, may have converted it to an activating receptor that instead enhances the immune response of the host to this pathogen. Because Ly49 represents a homodimer with a basic TM residue in each TM domain, it is possible that it assembles with two DAP12 dimers, a feature that may increase the sensitivity of receptor signaling.
These results demonstrate that this assembly mechanism is responsible for the formation of a wide variety of activating receptors with diverse functions in many different cell types of hematopoetic origin.
Experimental Procedures
cDNA Constructs and In Vitro Transcription
Human NKG2C, CD94, KIR2DS2, KIR2DL3, GPVI, FcαRI, DAP12, and Fcγ sequences were amplified from peripheral blood by RT-PCR. All sequences were cloned into a modified pSP64 vector (provided by M. Kozak) with the murine H-2Kb signal sequence (for type I membrane proteins). Mutations were introduced by PCR using overlapping primers, and epitope tags were added as in-frame fusions, usually with a three amino acid flexible linker; the sequences of epitope tags and sources of epitope tag reagents were as previously described (Call et al., 2002). The KIR, NKG2C, GPVI, and FcαRI constructs encoded C-terminal SBP tags, and three methionine residues were introduced N-terminal to PC or HA tags in the Fcγ construct for radiolabeling. The first of two cysteines in the EC domain of DAP12 was mutated to serine to prevent formation of disulfide-linked multimers. In vitro transcription was performed from linearized cDNA constructs by using RiboMax T7 Large-Scale RNA Production Kit and methyl-7G cap analog (Promega; Madison, WI).
In Vitro Translation and Immunoprecipitation
Each 25 μl reaction contained 17.5 μl nuclease-treated rabbit reticulocyte lysate (Promega), 0.5 μl amino acid mixture minus methionine or methionine/cysteine (Promega), 0.5 μl SUPERase-In RNase inhibitor (Ambion, Austin, TX), 1–2 μl [35S]-labeled methionine or methionine/cysteine (Amersham; Piscataway, NJ), RNA (400 ng for FcαRI, 800 ng for GPVI, and 100–200 ng for all other chains, based on translation efficiency), and 2.0 μl ER microsomes from a murine hybridoma (IVD12) as previously described (Call et al., 2002). All in vitro translation and assembly reactions were performed at 30°C. An initial translation period of 30 min under reducing conditions was followed by a 1.5–3 hr assembly period after addition of oxidized glutathione to 4 mM. Reaction volumes were 25–75 μl as required for optimal signal with multistep snIP procedures.
Immunoprecipitation, Electrophoretic Analysis, and Densitometry
Translation and assembly reactions were stopped by dilution with 1 ml ice-cold, Tris-buffered saline (TBS)/10 mM iodoacetamide, and microsomes were pelleted (10 min/20,000 × g/4°C) and rinsed. Pellets were resuspended in 400 μl solubilization/IP buffer (TBS + 0.5% digitonin [Biosynth International, Naperville, IL], 10 mM iodoacetamide, 0.1% BSA, 5 μg/ml leupeptin, and 1 mM PMSF; with 1 mM CaCl2 when anti-protein C mAb was used), then rotated for 30 min at 4°C. Lysates were precleared for 1 hr with Tris/BSA-blocked Sepharose 4 beads, and primary captures were performed overnight at 4°C. Primary IP products were washed twice in 0.5 ml wash buffer (TBS + 0.5% digitonin, 10 mM iodoacetamide; with 1 mM CaCl2 for anti-protein C mAb binding). Nondenaturing elution with EDTA (PC tag) or biotin (SBP tag) was performed as described (Call et al., 2002), and eluted complexes were incubated with subsequent antibodies and protein G-Sepharose 4 beads (Amersham, Piscataway, NJ) for 2 hr at 4°C and washed. Final precipitates were digested for 1 hr at 37°C with 500 U endoglycosidase H (New England Biolabs, Beverly, MA), separated on 15% SDS-PAGE (all experiments, except Figures 5 and 6) or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) (reducing conditions for Figures 3C and 4, nonreducing conditions for all other experiments), transferred to PVDF membranes, and exposed to phosphor imager plates. Densitometry was performed by using the Wide Line Tool in the Image-Quant software package (Molecular Dynamics).
Supplementary Material
Acknowledgments
This work was supported by the National Institutes of Health (RO1 AI54520 to K.W.W.).
Footnotes
Supplemental Data include two figures and are available online at http://www.immunity.com/cgi/content/full/22/4/427/DC1/.
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