Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors - PubMed (original) (raw)

Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors

Jianwen Feng et al. Immunity. 2005 Apr.

Abstract

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.

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Figures

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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).

Figure 7

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.

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