In this work, I refer to multichain activating receptors () that share a common organizing principle—their EC recognition module(s) and intracellular signaling module(s) are found on separate subunits that are noncovalently associated through their TM domains, as to multichain immune recognition receptors (MIRRs). It should be noted, however, that members of this family are not necessarily immune-related (an example is the major collagen receptor on platelets, GPVI).
The MIRR-mediated activation signal can be divided into four parts: (1) the EC recognition of a multivalent ligand/antigen resulting in the aggregation, or clustering, of the MIRRs, (2) MIRR triggering and TM signal transduction, (3) phosphorylation of the ITAM or YxxM tyrosine residues by protein tyrosine kinases (PTKs) and activation of specific intracellular pathways and (4) the activation of genes in the nucleus. The EC recognition of a ligand/antigen, the MIRR-triggered biochemical cascades and the mechanisms of gene activation are understood in significant detail. However, the mechanism by which the MIRR transduces the recognition information via receptor TM and juxtamembrane (JM) regions into intracellular biochemical events (part 2) has been a long-standing mystery. In other words, the key question remained unanswered: what is the molecular mechanism by which clustering of the EC recognition domains of MIRRs leads to receptor triggering and tyrosine phosphorylation of the intracellular ITAMs or YxxMs, thus initiating specific pathways and resulting in immune cell functional outcomes? It was also not known how this putative mechanism could explain the intriguing ability of immune cells to discern and differentially respond to slightly different ligands.
MIRR-mediated signal transduction plays an important role in health and disease, making these receptors attractive targets for rational intervention in a variety of immune disorders.1,13,16,19,122–125
Thus, future therapeutic strategies depend on our detailed understanding of the molecular mechanisms underlying MIRR triggering and subsequent TM signal transduction. In addition, understanding these mechanisms would give us a new handle in dissecting the basic structural and functional aspects of the immune response.
Despite numerous models of MIRR-mediated TM signal transduction suggested for particular MIRRs (e.g., TCR, BCR, Fc receptors, NK receptors, etc.), no current model fully explains how ligand-induced TM signal transduction commences at the molecular level. As a consequence, these models are mostly descriptive, do not explain mechanistically a vast majority of the specific processes behind “outside-in” MIRR signaling, and do not reveal clinically important points of therapeutic intervention. In addition, since the term “MIRR” was first introduced in 1992,3
and MIRR-triggered signaling pathways were hypothesized to be similar,3,70,126–128
no general mechanistic model of MIRR-mediated immune cell activation has been suggested to date with the exception of the SCHOOL model.4,49,97,129,130
This impeded our advanced understanding of the immune response, and more importantly, prevented the potential transfer of therapeutic strategies between seemingly disparate immune disorders.
Basic concept and major stages.
A novel unusual biophysical phenomenon, the homointeractions of intrinsically disordered CYTO domains of ITAM-containing MIRR signaling subunits, has recently been discovered.78
Demonstrating that intrinsically disordered proteins do not necessarily undergo a transition between disordered and ordered states upon interaction,44,79,81
this finding opposes the generally accepted view on the behavior of IDPs. Perhaps even more intriguing is the fact that no chemical shift changes and significant changes in peak intensities are observed in the 1
N heteronuclear single quantum coherence (HSQC) spectra of 15
Later, the natural propensity of the random-coil TCRζ CYTO domain to homodimerize has been independently confirmed by other investigators.131
Interestingly, the homooligomerization of CYTO domains of MIRR signaling subunits is best described by a two-step monomer-dimer-tetramer fast dynamic equilibrium with dissociation constants in the micromolar affinity range.78,81
As mentioned above, the overall binding affinity between proteins depends on the function of the protein complex and proteins that associate and dissociate in response to changes in their environment, such as the majority of signal transduction mediators, tend to bind more weakly. In this context, micromolar binding affinities in combination with a rapid association and dissociation kinetics78
make the homotypic CYTO interactions between MIRR signaling subunits a valid candidate for involvement in MIRR-mediated signal transduction.
Uncovering a crucial physiological role of these unique homointeractions, the SCHOOL model suggests that formation of competent MIRR signaling subunit oligomers is necessary and sufficient to trigger the receptors and induce TM signal transduction and the downstream signaling sequence ( and
Intracellularly, the need for MIRR dimerization is consistent with the suggested structural hypothesis of cross-phosphorylation70,132
that assumes that (1) the kinase(s) responsible for catalyzing ITAM Tyr residue phosphorylations exist associated with the receptors, and (2) for steric reasons, these kinases cannot phosphorylate tyrosine residues on chains of the same receptor complex. Upon dimerization/oligomerization, these kinases phosphorylate the tyrosines of a distinct receptor complex (cross-phosphorylation, or transphosphorylation), thus triggering the receptor.70
Figure 6 Within the model, formation of competent signaling oligomers in cytoplasmic milieu is necessary and sufficient to generate the activation signal, thus triggering downstream signaling pathways. Ligand-induced MIRR clustering and reorientation (in pre-existing (more ...)
Figure 7 Within the model, formation of competent signaling oligomers in cytoplasmic milieu is necessary and sufficient to generate the activation signal, thus triggering downstream signaling pathways. Receptor clustering and reorientation (and/or receptor reorientation (more ...)
Within the model, MIRR engagement by multivalent antigen () or anti-MIRR antibodies (e.g., anti-CD3ε and anti-TCRβ for TCR or anti-Igβ antibodies for BCR; ) leads to receptor clustering coupled with a multi-step structural reorganization driven by the homooligomerization of MIRR signaling subunits ( and ). Ligand-induced MIRR clustering leads to receptor reorientation and formation of a dimeric/oligomeric intermediate in which signaling chains from different receptor units start to trans-homointeract and form signaling oligomers ( and , stage 1). Upon formation of signaling oligomers, PTKs phosphorylate the tyrosine residues in the ITAMs located on the CYTO tails of MIRR signaling subunits, leading to the generation of intracellular activation signal(s), dissociation of signaling oligomers and internalization of the engaged MIRR ligand-binding subunits ( and , stage 2). Signaling oligomers then interact with the signaling subunits of nonengaged receptors resulting in formation of higher-order signaling oligomers, thus propagating and amplifying the activation signal and resulting in internalization of the non-engaged MIRR recognition subunits ( and , stages 3 and 4).
Similar to SRs, some MIRRs such as TCR and major platelet collagen receptor GPVI, can exist as pre-assembled oligomers on the cell surface.71,133,134
In these oligomers, multivalent ligand binding or antibody stimulation results in re-orientation of receptors to adopt an interunit geometry permissive for further receptor activation ( and
The model also assumes that the diversity of the immune cell response is partly provided by the combinatorial nature of MIRR-mediated signaling. Signal diversification may be achieved through different patterns of MIRR signaling subunit oligomerization4,49,97
in combination with distinct activation signals provided by different MIRR signaling modules135–146
and/or different ITAMs located on the same signaling module (e.g., TCRζ chain).147
Thus, according to the model, the diversity of cell functional outcomes in response to different ligands is higher with the more different signaling subunits the MIRR complex has.
According to the SCHOOL model, MIRR triggering and TM signaling induced by binding to multivalent ligand/antigen or anti-MIRR antibodies can be divided into four major stages ( and
- Dynamic lateral clustering and rotation with subsequent formation of the intermediate complex. Ligand/antigen/antibody brings two or more MIRRs together in sufficient proximity and correct relative orientation toward each other to promote the interreceptor homointeractions between signaling subunits. Once initiated, these homointeractions weaken the intrareceptor TM interactions between recognition and signaling subunits. A signaling-competent oligomeric intermediate complex is formed, bringing together the CYTO domains of the signaling subunits, protein kinases and various adaptor/effector proteins, to create a competent, activated receptor complex. In the signaling subunit oligomers formed, the ITAM Tyr residues become phosphorylated, thus starting the signaling cascade.
- Dissociation and internalization. Signaling oligomers dissociate from the engaged ligand-recognition subunits, which are then internalized.
- Interactions with nonengaged receptors, lateral signal propagation and amplification. Signaling oligomers interact with the signaling subunits of nonengaged receptors resulting in formation of higher-order signaling oligomers, thus propagating and amplifying the activation signal.
- Dissociation and internalization. Signaling oligomers dissociate from the nonengaged ligand-recognition subunits, which later are internalized.
Major driving forces.
As described above, within the SCHOOL model, there are three major driving forces of MIRR signaling: antigen/ligand-MIRR EC interactions, intrareceptor TM interactions and interreceptor CYTO interactions (see also and
), and an outcome of the interplay between these forces defines MIRR triggering and activation. Antigen/ligand-MIRR interactions are generally of low affinity (micromolar range) and have rapid association and dissociation kinetics (reviewed in ref. 92
). This low-affinity binding, in combination with fast kinetics, allows immune cells to recognize and discriminate a variety of antigens/ligands with high specificity, selectivity and sensitivity in order to respond with a variety of biological responses. Considering that EC and TM regions of MIRRs are well-ordered receptor segments while MIRR signaling CYTO domains are intrinsically disordered,2,78,79,81
an important and intriguing question is raised: how do MIRRs transduce highly ordered antigen recognition/discrimination EC information across the cell membrane into intracellular biochemical events, triggering specific pathways and resulting in a specific functional outcome?
Despite intensive studies of MIRR-mediated TM signal transduction, the only model that can answer this question and even more important, mechanistically explain how this signaling starts, is the SCHOOL model.4,49,97,130
As described above, all three major protein-protein interactions that define MIRR signaling are characterized by micromolar affinity and relatively rapid kinetics.78,89–95
Thus, this conjugated and well-balanced system of interprotein interactions can explain the molecular mechanisms of the ability of MIRRs to transduce the recognition/discrimination information across the cell membrane and translate it into different activation signals, thus triggering different intracellular pathways and resulting in different cell responses. Within the model, the MIRR-generated intracellular activation signals are combinatorial in nature and involve multiple components such as different ITAM Tyr phosphorylation patterns135–147
as well as formation of functionally different competent signaling oligomers formed by the CYTO homooligomerization of different MIRR signaling subunits.4,49,97,98,130
This system also explains mechanistically high specificity, selectivity and sensitivity of immune cells in recognition and discrimination of different antigens/ligands and how this recognition/discrimination results in different functional outcomes. This is particularly important for the TCR148
that has four different signaling subunits, namely ζ, CD3ε, CD3δ and CD3γ, known to play different roles in T cell biology.130,149
In addition, in contrast to other MIRR signaling subunits, ζ has three ITAMs that can provide differential Tyr phosphorylation patterns in response to different ligands, initiating different intracellular signaling pathways. Thus, within the model, TCR-mediated signaling and cell activation has the highest combinatorial potential as compared to other MIRRs, explaining a high variability of distinct TCR-triggered intracellular signaling pathways and therefore distinct T cell functional responses depending on the nature of the stimulus.4,49,97,98,130
Interactions between TM helices of recognition and signaling MIRR subunits maintain receptor integrity in unstimulated cells ()4,15–17,19,24,25,27,32–43,49,97,98,130,150–155
and determine the relative positions of these subunits in the receptor complex (angles, distances, etc.,), thus dictating the overall geometry and topology of MIRRs. Within the SCHOOL model, this overall structural architecture of MIRRs, in combination with the requirement to initiate interreceptor CYTO homointeractions between receptor signaling subunits (, and
), impose several restraints for multivalent antigen/ligand-induced MIRR triggering ():4,49,97,98
- sufficient interreceptor proximity in MIRR dimers/oligomers,
- correct (permissive) relative orientation of the receptors in MIRR dimers/oligomers,
- long enough duration of the MIRR-ligand interaction that generally correlates with the strength (affinity/avidity) of the ligand, and
- sufficient lifetime of an individual receptor in MIRR dimers/oligomers.
The importance of these factors for productive MIRR triggering is strongly supported by a growing body of evidence10,38,48,54,56,62,67,73,94,95,142,156–184
and is discussed in detail below.
The restraints imposed by the SCHOOL model play an especially important role during the first stage of MIRR triggering ( and ), at which point these spatial, structural and temporal requirements (correct relative orientation, suf- ficient proximity, long enough duration of the MIRR-ligand interaction and lifetime of MIRR dimers/oligomers) should be fulfilled to favor initiation of trans-homointeractions between MIRR signaling subunits and formation of competent signaling subunit oligomers. If these requirements are not fulfilled at this “final decision-making” point, the formed MIRR dimers/oligomers may dissociate from the ligand and remain signaling incompetent and/or break apart to its initial monomeric receptor complexes. Also, at this stage, slightly different ligands may bring two or more MIRRs in different relative orientations that favor homointeractions between different signaling subunits and result in formation of different signaling oligomers or combinations, thus initiating distinct signaling pathways. This mechanism can explain the ability of MIRRs to differentially activate a variety of signaling pathways depending on the nature of the stimulus.
Within the proposed model, the signaling oligomers formed dissociate from ligand-binding chains, which later are internalized ( and
, stage 2). This dissociation mechanism provides a structural and mechanistic basis for our improved understanding of many immunological phenomena, such as adaptive T cell tolerance or anergy,185–191
differential biological role of CD3 chains,192
ligand- or antibody-induced exposure of a cryptic polyproline sequence in the CYTO domain of CD3ε,165,193–195
human cytomegalovirus (CMV) escape from NK attack200
and others. The dissociation mechanism also allows the initially formed signaling oligomers to sequentially homointeract with the signaling subunits of nonengaged receptors ( and
, stages 3 and 4) resulting in formation of higher-order signaling oligomers, thus propagating and amplifying the signal. This leads to dissociation and subsequent internalization of the nonengaged ligand-binding subunits. Thus, as with bacterial chemoreceptors,201–203
the SCHOOL model-based mechanism of MIRR-mediated cell activation suggests spreading (propagation) activation signal from engaged to nonengaged receptors within receptor clusters.
Finally, it should be noted that similar spatial, structural and temporal restraints are imposed within the proposed model for MIRR triggering by not only antigen () but also the anti-MIRR (), antibodies such as anti-TCRα, anti-TCRβ, anti-CD3ε, anti-Igβ and others. This can explain differential immune cell functional outcomes mediated by MIRRs depending on the specificity of the antibodies.159,160,163–165,204–208
The plausible and easily testable SCHOOL model is fundamentally different from those numerous models that have been previously suggested for particular MIRRs and has several important advantages.4,49,97,98,130
- This is the first general mechanistic model for all MIRRs known to date, including TCR, BCR, Fc receptors, NK receptors, ILTs, LIRs, SIRPs, DCAR, BDCA-2, MDL-1, NITR, TREMs, GPVI and others, and for those that will be discovered in the future. Thus, assuming that the general principles underlying MIRR triggering and TM signaling mechanisms are similar for all MIRRs, the SCHOOL model can be easily applied to any particular receptor of the MIRR family.
- This is the first model that is based on specific protein-protein interactions ()—biochemical processes that can be influenced and controlled,209–213 and specific inhibition and/or modulation of these interactions provides a promising novel approach for rational drug design, as revealed by recent progress in the design of inhibitory antibodies, peptides and small molecules.129,213–220
- Introducing the CYTO homointeractions between MIRR signaling subunits as one of the key elements of MIRR triggering and signaling, the SCHOOL model imposes functionally important restraints () and suggests molecular mechanisms for the vast majority of unexplained immunological observations accumulated to date.4,49,97,98,130
- Unraveling the molecular mechanisms underlying MIRR triggering and subsequent TM signaling, the model suggests unique and powerful tools to study the immune response and a means to control and/or modulate it.4,49,97,98,130,221,222
- Based on specific protein-protein interactions, the SCHOOL model reveals new therapeutic targets for the treatment of a variety of disorders mediated by immune cells.4,44,49,97,98,129,221–223
- Finally, an important application of the SCHOOL model is that similar therapeutic strategies targeting key protein-protein interactions involved in MIRR triggering and TM signal transduction may be used to treat diverse immune-mediated diseases.44,98,129,222 This assumes that clinical knowledge, experience and therapeutic strategies can be transferred between seemingly disparate immune disorders or used to develop novel pharmacological approaches and that a general pharmaceutical approach may be used to treat diverse immune disorders.