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The Counterbalance Theory for Evolution and Function of Paired Receptors

A. Neil Barclay1,* and Deborah Hatherley1 1Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom *Correspondence: [email protected] DOI 10.1016/j.immuni.2008.10.004

Paired receptors are families of membrane containing similar extracellular regions but differing in their potential for signaling with one type able to give inhibitory signals and the other activating. Inhibitory receptors could be good targets for pathogens to restrict immune responses against them. Here we suggest that activating members may have evolved to counterbalance pathogens utilizing the inhibitory pathway. Thus, if a pathogen utilizes any part of the inhibitory receptor to downregulate responses against itself, it may, because of similarities in structure, also bind the activating receptor and give an opposing signal. We evaluate recent structural data on SIRPa (signal regulatory ) and LILRB1 (leukocyte immunoglob- ulin-like receptor subfamily B member 1) showing evidence of pathogen pressure in nonligand-binding regions of these receptors together with data on pathogen binding to PIRs (paired Ig-like receptors) to provide support for this theory.

Introduction data available for several pairs, e.g., effect (Hatherley et al., 2008). We discuss The term ‘‘paired receptor’’ is commonly CD94-NKG2 (Vales-Gomez et al., 1999), recent structural data on the SIRP family used to describe families of membrane re- CD200R (Hatherley et al., 2005), SIRP and LILRB1 together with pathogen bind- ceptors that have very similar extracellular (Barclay and Brown, 2006), and PILR ing data for other paired receptors with regions but different transmembrane and (Tabata et al., 2008). respect to this model. cytoplasmic regions. Indeed, the latter are The inhibitory receptors generally inter- so different that they can give opposite act with self proteins and provide a mech- The Structure of SIRPa signals (Lanier, 2001). One type can give anism to limit cell activity as shown in NK SIRPa (also known as SHPS-1, BIT, inhibition through immunoreceptor tyro- cells (Lanier, 2005) and myeloid cells (Bar- CD172a [van den Berg et al., 2005]) is sine-based inhibition motifs (ITIM) in the clay and Brown, 2006). The roles of the the inhibitory member of the SIRP family, cytoplasmic region. The other can activate activating receptors are less clear, espe- SIRPb is the activating form associating through signaling proteins like DAP12 cially those on cells other than NK cells. with DAP12, and SIRPg is a third form that contain immunoreceptor tyrosine- Many of the paired receptor families are that does not signal (Barclay and Brown, based activating motifs (ITAM) that are as- evolving rapidly, indicative of pressure 2006). The N-terminal immunoglobulin sociated with the receptor via interactions from pathogens (Vilches and Parham, superfamily (IgSF) domain of SIRPa (d1) through their transmembrane regions 2002). Although paired receptors on NK interacts with the single IgSF domain of (Dietrich et al., 2000; Lanier, 2005). Paired cells are heavily involved in the recogni- CD47, a widely distributed membrane receptors are often expressed by NK tion of pathogen-infected cells, others protein. X-ray crystallography structures cells, others are restricted to myeloid such as CD200R and SIRPa are involved have been determined for the SIRP family cells, but some are found on other leuko- in the control of myeloid cell activity members and CD47 (Hatherley et al., 2007, cytes and also neuronal cells (Lanier, (Barclay and Brown, 2006; Foster-Cuevas 2008; Nakaishi et al., 2008). SIRPa binds 2005). Paired receptors include SIRP, et al., 2004). How might pathogens drive CD47 through loops in a manner analo- CD200R, KIR, , CD300, DCIR, PIR, this evolution? The targeting by patho- gous to binding of antigen by immunoglob- PILR, TREM, LILR, , etc., with many gens of inhibitory receptors involved in ulins and the receptor, and the failure alternative names summarized in Yamada cell regulation is clearly a sensible strat- of SIRPb to bind to CD47 is due to subtle and McVicar (2008). egy from the pathogen’s point of view. differences in these loops (Hatherley et al., If the outcomes of engagement of We suggest a mechanism for paired 2008). paired receptors are so different and the receptors by which activating receptors extracellular regions so similar, then if have evolved to interact with those patho- Polymorphisms in Human SIRPa their ligands are the same, one gets the gens that target inhibitory receptors i.e., and Binding confusing situation of two outcomes for the activating receptors act as a counter- SIRPa shows extensive polymorphism the presence of the same ligand. Often balance. Thus for paired receptors such with 10–12 amino acid differences in a cell will express both inhibitory and acti- as SIRP, if a pathogen targets the inhibi- domain 1 but only 0–2 differences in vating members. In most cases, ligands tory receptor, it is probable that the domains 2 and 3 between three mouse for the inhibitory receptors are known pathogen also binds the activating recep- strains (Sano et al., 1999) and even more and the activating receptors bind more tor because of its similar extracellular differences between the NOR (non-obese weakly or not at all with quantitative regions, and hence nullifies the inhibitory resistant) and NOD (non-obese diabetic)

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residues in SIRPa d1 are strikingly distant from the contact site for CD47 (Figure 1A). However, the three residues near the contact site are unlikely to affect binding because they are present in one or other of the two sequences that bind CD47 equally. The five residues not in either of the two standard sequences are distant from the binding site (shown in magenta in Figures 1A and 1B). Given this and the similarity of structures between the SIRPs away from the binding site (see Hatherley et al., 2008) and the failure of mutants outside of the binding site to affect CD47 binding (Hatherley et al., 2007; Liu et al., 2007), it is unlikely that the polymor- phisms in human SIRPa will markedly affect binding to CD47. CD47 itself lacks extensive polymorphisms. So what is driving the extensive SIRPa d1 polymorphism? In both human and mouse SIRPa d1 (see above), the majority of polymorphisms are nonsynonomous (Sano et al., 1999; ENSEMBL database), indicating selective pressure. The side chains of the polymorphic residue (16 out of 17) are out-pointing and hence are likely to be involved in interactions with other molecules, whereas in-pointing res- idues might be expected to have affects on the folding of the domain. This is con- sistent with evolutionary pressure through interactions in this region. One possibility is that these variants have been selected not to react with particular pathogens (i.e., avoiding downregulation of the myeloid cell). Another comparable exam- ple is the Nkrp1 lectin-like paired receptor where the receptor but not the ligand shows extensive polymorphism, and in- Figure 1. Polymorphisms and Structure of SIRPa deed it has recently been suggested this (A) Alignments of the amino acid sequences of domain 1 of the two commonly studied SIRPa sequences (accession numbers CAA71403 and NP_542970 for sequences 1 and 2, respectively) together with is to avoid virus decoys targeting this polymorphisms identified in Takenaka et al. (2007). The positions of polymorphic residues are indicated receptor (Carlyle et al., 2008). Thus, two by giving the residue for each sequence and indicating those that differ from SIRPa (1) by green or for those mechanisms are involved in avoiding a a different from either SIRP (1) or SIRP (2) by magenta. The residues that form contacts with CD47 are downregulation of activity—a high degree highlighted in red and the positions of the beta strands and one helical region are shown above the alignment. of polymorphism in the inhibitory receptor (B) The positions of the polymorphic residues are mapped onto the SIRPa domain 1-CD47 extracellular together with evolution of an activating re- domain cocrystal structure (Hatherley et al., 2008). The side chains of the polymorphic residues are ceptor. One important concept is that the indicated with spheres in green and magenta to distinguish the polymorphic residues as in (A). pathogen does not necessarily need to bind to the ligand binding site or indeed, mice (20 differences in domain 1 [Taken- domain 1 and the two alleles of SIRPb domain, to perturb the SIRPa and give aka et al., 2007]). In humans, 37 different have extensive differences throughout a signal—thus the sequence conservation individuals showed 9 different SIRPa the sequence. Most researchers studying in the remainder of the extracellular region domain 1 sequences (Figure 1; Takenaka SIRPa have used one or other of two se- is relevant (there is about 90% sequence et al., 2007), making this one of the most quences (1 and 2 in Figure 1A). We have identity between the extracellular regions polymorphic in the shown that both proteins bind CD47 with of SIRPa, SIRPb, and SIRPg)(Figure 2). after MHC and KIR antigens (Vilches and the same affinity (KD = 1 mM[Hatherley There are no binding data for pathogens Parham, 2002). In contrast, SIRPb and et al., 2008] and unpublished data). The to SIRPs but there are data for other SIRPg lack extensive polymorphism in majority (14 out of 17) of the polymorphic paired receptors (see below).

676 Immunity 29, November 14, 2008 ª2008 Elsevier Inc. Immunity Perspective

Pathogen Binding to Paired binding site and in support of the counter- Receptors balance theory. The concept that pathogens could bind It is likely that activating receptors paired receptors has been established evolved from the inhibitory receptor and with extensive functional data available they show much shorter half lives in evolu- for the Ly49 NK receptors found in mice. tion with greater variation in numbers Resistance of B6 mice to mouse cyto- (Abi-Rached and Parham, 2005; Carlyle megalovirus (mCMV) was found to be et al., 2008; Vilches and Parham, 2002; conferred by the expression of Ly49H, Wilson et al., 2006). The advantage of an activating receptor that binds to the greater numbers of activating receptors in mCMV MHC-like protein m157. This m157 protecting against more pathogens is bal- protein also binds the inhibitory Ly49I anced by greater risks for autoimmunity. receptor found in a susceptible mouse Another safeguard against unwanted strain (129) but does not bind the Ly49I re- membrane receptor signaling may be ceptor found in B6 mice (Arase et al., 2002; the utilization of synapse-like structures. Cheng et al., 2008; Smith et al., 2002). It is notable how many receptors including Arase et al. (2002) suggested that whereas many paired receptors are likely to span the inhibitory receptors have evolved for around four IgSF domains compatible immunoregulation and preventing autoim- with these interactions occurring at an munity, the activating receptor may have immunological synapse (Barclay and evolved to recognize pathogen encoded Brown, 2006; Tsai and Discher, 2008; ligands. Given that pathogens evolve Wright et al., 2000). If an inhibitory recep- faster than hosts, one might imagine that tor is active only in the immunological the pathogen could easily evade these synapse, for instance because of the activating receptors. Our suggestion dis- close proximity of other receptors and Figure 2. Sites Distant from Ligand-Binding cussed above (that pathogens might bind Regions of Inhibitory Receptors Are to ensure phosphorylation of the both types of paired receptors and the ac- Attractive Targets for Pathogens cytoplasmic region, then it is likely to be tivating receptors act as a counterbalance) The cartoon shows SIRPa (inhibitory) together with inaccessible to pathogens that would be its ligand CD47 with its binding face indicated in would fit with the data in this case and red. The arrows indicate how pathogens might excluded from the immunological syn- provide a general rationale for at least target the regions of SIRPa not involved in CD47 apse because of their size. However, some paired receptors. With regard to binding, and then react with equivalent sites in once a virus has infected a cell, virally Ly49, genetic divergence has led to strains the activating SIRPb. N-linked glycosylation sites encoded proteins can be synthesized are shown in black but the area covered by carbo- with either inhibitory or activating recep- hydrate would be much larger in 3D (Barclay et al., and reach the surface and subvert tors giving dramatic functional differences. 1997). The close proximity of the membranes in immune responses against the infected A variety of bacterial strains can bind immunological synapses when SIRPa engages cell. This appears to be the case in Kapo- CD47 would also hinder access by pathogens. both the inhibitory mouse paired Ig-like si’s sarcoma virus that has acquired receptor (PIR-B) and at least one activat- a CD200 homolog that can mimic the ing counterpart (PIR-A1) (Nakayama et al., Although the evolution of KIRs (killer cell host protein by interacting with the inhibi- 2007); a different repertoire of bacteria immunoglobulin-like receptor) is tied to tory CD200R to downregulate macro- binds PIR-B (inhibitory) orthologs in MHC class I (the ligand) divergence phage activity (Foster-Cuevas et al., humans, namely leukocyte immunoglob- (Vilches and Parham, 2002), recent data 2004). Nevertheless, for initial virus infec- ulin-like receptor subfamily B1 (LILRB1) on the LILRB1 family support the concept tions and bacterial infections, the immu- and LILRB3 (or ILT2 and ILT5) (Nakayama that evolutionary pressures may act on nological synapse may have a general, et al., 2007). For the LILRB1, it seems sites other than the ligand binding as sug- underappreciated role to prevent subver- likely that the bacterial binding site is dis- gested above for SIRPa. The N-terminal sion of receptors once they have been ac- tinct from the ligand binding as indicated two IgSF domains of LILRB1 form a bind- tivated in this specialized contact region. by the fact that monoclonal antibody can ing site for MHC class I antigens. LILRB1, block the former and not affect the latter like SIRPa, is polymorphic and 4 out of the Conclusion (Nakayama et al., 2007). Another example 5 amino differences are in the ligand- The theory that the activating partner of of pathogen binding is the involvement binding domains; three of these have no a paired receptor might act as a counter- of the inhibitory receptor PILRa in the up- affect on ligand binding affinity as tested balance to pathogens that are utilizing the take of (Satoh et al., with a variety of MHC class I ligands and inhibitory receptor is strengthened by 2008); however, no data were given on the the polymorphic residues are shown by three sets of recent data: first, structural activating counterpart. X-ray crystallography to be outside the data on SIRP and LILRB1 recognition binding site (Kuroki et al., 2005). This establish the basis of ligand binding; sec- Evolution of Paired Receptors parallels the observation discussed ond, genetic studies on SIRPs and other There are data indicating the fast evolu- above for the SIRPs where there is evi- paired receptors reveal the extent of poly- tion of paired receptors both in terms of dence for pressure of change in the inhib- morphisms outside of the ligand binding gene numbers and sequence diversity. itory receptor away from the ligand domain, indicating evolutionary pressure

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