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The Molecular Interaction of CAR and JAML Recruits the Central Cell Signal Transducer PI3K

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Science  03 Sep 2010:
Vol. 329, Issue 5996, pp. 1210-1214
DOI: 10.1126/science.1187996

Abstract

Coxsackie and adenovirus receptor (CAR) is the primary cellular receptor for group B coxsackieviruses and most adenovirus serotypes and plays a crucial role in adenoviral gene therapy. Recent discovery of the interaction between junctional adhesion molecule–like protein (JAML) and CAR uncovered important functional roles in immunity, inflammation, and tissue homeostasis. Crystal structures of JAML ectodomain (2.2 angstroms) and its complex with CAR (2.8 angstroms) reveal an unusual immunoglobulin-domain assembly for JAML and a charged interface that confers high specificity. Biochemical and mutagenesis studies illustrate how CAR-mediated clustering of JAML recruits phosphoinositide 3-kinase (P13K) to a JAML intracellular sequence motif as delineated for the αβ T cell costimulatory receptor CD28. Thus, CAR and JAML are cell signaling receptors of the immune system with implications for asthma, cancer, and chronic nonhealing wounds.

The coxsackievirus and adenovirus receptor, CAR (1, 2), plays a crucial role in both virus-related pathology and adenoviral gene therapy (35). The tissue distribution of CAR is broader than the observed virus tropism (6), and substantial efforts have been devoted to characterizing and modifying CAR-virus interactions to improve the efficacy and safety of adenoviral gene delivery (7). The high conservation of CAR among vertebrates and its low genetic diversity in humans have resulted in its frequent occurrence as a target in the evolution of virus-host interactions (6). Such conservation indicates that CAR is physiologically important. However, although roles in embryonic development, cell adhesion, tumor cell growth, inflammation, and tissue regeneration have been reported, the underlying mechanisms remain elusive (8, 9). The junctional adhesion molecule–like protein, JAML (10), present on a variety of effector cells of both adaptive and innate immunity (including neutrophils, monocytes, memory T cells, and epithelial γδ T cells) was recently identified as an endogenous ligand of epithelial and endothelial CAR (1114). CAR and JAML are both type I transmembrane glycoproteins composed of a globular ectodomain (two immunoglobulin domains), a stalk (CAR: six residues, JAML: 26 residues), a single transmembrane helix, and a cytoplasmic domain. The CAR-JAML interaction is important for leukocyte migration and for activating γδ T cell responses (1114), with implications for epithelial challenges, such as wound repair or inflammation (15). CAR binding to JAML on epithelial γδ T cells induces cytokine and growth factor production, mitogen-activated protein (MAP) kinase pathway activation, and, ultimately, cell proliferation (14), which indicates that JAML and CAR constitute an important cell-signaling complex of the immune system. To elucidate the mechanisms underlying CAR and JAML physiology, we determined the crystal structure of this receptor-ligand pair.

We expressed the mouse JAML ectodomain (residues 1 to 260) in SC2 cells, and purified and crystallized the protein (16) (tables S1 and S2). The structure was determined initially by multiwavelength anomalous dispersion phasing at 3.4 Å, by using a heavy atom derivative, and then extended to 2.2 Å by molecular replacement (MR) of the experimental model into a higher-resolution native x-ray data set. In JAML, two V-set immunoglobulin domains (D1 and D2) associate into a compact assembly (Fig. 1A), where their relative orientation is constrained by an interdomain, parallel β-sheet interaction of the D1 B strand and the D2 B′ strand, numerous H bonds, a salt bridge, hydrophobic packing, and a short interdomain linker (Fig. 1, B to D, and fig. S1). The interface between D1 and D2 buries a total molecular surface area of 1050 Å2 (D1: 540 Å2, D2: 510 Å2). The extent of the interface, combined with identical relative orientations of D1 and D2 in four different crystal forms (17), indicates that the highly compact JAML globular domain acts as a single rigid unit in contrast with typical, elongated structures of tandem immunoglobulin-domain receptors, such as CAR, CD2, or CD58 (fig. S2) (18), where flexibly linked immunoglobulin domains facilitate protein recognition.

Fig. 1

Crystal structure of the unliganded mouse JAML ectodomain reveals a novel prototype for tandem immunoglobulin-domain receptors. (A) Ribbon diagram of JAML D1 (membrane-distal, residues 10 to 121, light blue) and D2 (membrane-proximal, residues 122 to 236, salmon) immunoglobulin V-set domains comprising nine β strands arranged into two antiparallel β sheets (A′GFCC′C′′:DEB) linked by canonical disulfide bridges (Cys25 to Cys99 and Cys138 to Cys216, green). D1 tightly packs its BED β sheet against the D2 B′C and FG loops. The D1 A strand (residues 1 to 9) and the C-terminal stalk region (residues 234 to 260, dashes) which tethers the JAML immunoglobulin domains to the cell membrane were disordered. Carbohydrate moieties attached to the three N-linked glycosylation sites (yellow sticks) are labeled. (B to D) Several interdomain interactions stabilize the relative orientation of D1 and D2 (see inset boxes). (B) A hydrophobic cluster is formed by Leu22, Val74, Leu85 of D1, and Ile140, Val220, and Leu223 of D2. Arg147 forms a salt bridge with Asp81, whose side-chain orientation is restrained by H bonds to Ser83. (C) A short β strand (B′, red) in the BC loop of D2 with the B strand of D1 creates a four-stranded DEBB′ interdomain β sheet. Additional interdomain H bonds (some water-mediated) are not shown: Arg14 to Gln141, Glu19 to Gln141, Glu19 to Ser139, Glu19 to Glu122, Ser20 to Ser225, Gln87 to Glu224, and Gln87 to Ser225. (D) A short, rigid linker (residues 120 to 123) directly connects the D1 G strand to the D2 A′ strand, as D2 lacks an A strand. The linker is stabilized by two prolines, an H-bond network, and a salt bridge between Glu122 and Arg137.

Carbohydrate moieties are present at all potential N-linked glycosylation sites (Asn59, Asn69, and Asn105) of JAML (Fig. 1A). However, these sites are not entirely conserved across species, which suggests that N-glycosylation is not directly correlated with JAML function, but might aid protein stabilization and prevent nonspecific interactions on the cell membrane. The stalk, which tethers the extracellular immunoglobulin domains of JAML to the cell membrane, and the C-terminal His tag are disordered in the crystal. This stalk is rich in serine, threonine, proline, and alanine (fig. S3A) and is likely to be O-glycosylated in vivo. The glycosylation would confer some rigidity to the otherwise flexible stalk and would result in a distance of <60 Å between the globular JAML domain and the membrane (Fig. 1A).

JAML can thus be considered an unconventional immunoglobulin-domain receptor where the more compact and rigid assembly of its immunoglobulin domains provides the ligand-binding site, and its extended stalk provides sufficient flexibility to facilitate interaction with CAR on the opposing cell surface. To gain insight into this interaction, we determined the crystal structure of the ectodomain complex of JAML and CAR.

The entire mouse CAR ectodomain (residues 1 to 217) was produced in SF9 cells and crystallized in an equimolar ratio with mouse JAML. The crystal structure was determined at 2.8 Å resolution by MR with unliganded JAML and human CAR D1 [Protein Data Bank accession no. 1EAJ (19)] as MR templates. The partially disordered CAR D2 domain was manually built using the nuclear magnetic resonance (NMR) structure of human CAR D2 [2NPL (20)] as a model (table S1). CAR and JAML form a 1:1 complex in the crystallographic asymmetric unit and, as expected for interaction of cell surface receptors from opposing cells, their C-terminal stalks protrude in opposite directions (Fig. 2A). Well-defined electron density was observed for JAML and the V-set CAR D1 immunoglobulin domain (3, 19) (including the N-linked glycan at Asn87). The C2-set CAR D2 immunoglobulin domain (20) was partially disordered, which indicated flexible linkage of the two CAR immunoglobulin domains and classified CAR as a typical tandem immunoglobulin-domain receptor. At the cell surface, CAR D2 is tethered to the membrane by a short stalk, and its motion may be additionally restricted by an N-glycan (Asn182) in a membrane-proximal loop (fig. S4); thus, the flexibly linked D1 appears to facilitate interaction with JAML and might also aid in attachment of CAR to coxsackie and adenoviruses (3, 4).

Fig. 2

CAR-JAML ectodomain complex structure. (A) Ribbon diagram of the CAR-JAML complex. JAML (D1, residues 11 to 121, light blue; D2, residues 122 to 236, salmon; disordered stalk, dashes), CAR [D1, residues 3 to 120, light green; D2, residues 121 to 216, gold; note that CAR D2 loops that were disordered in the crystal structure were grafted on from the CAR D2 NMR structure (2NPL, dots)], and carbohydrates (gray sticks). CAR and JAML interact with their D1 A′GFCC′C′′ β sheets in a face-to-face, hand shake–like fashion. (B) An antiparallel β sheet is formed between the G strand (Ser107 to Lys111) of JAML and the C′ strand (Ile47 to Val51) of CAR, which moves ~6.2 Å as compared with unliganded CAR (fig. S7). (C) The interface is characterized by an array of interdigitated charged amino acids. Specifically, JAML Arg37 forms salt bridges with CAR Asp35 and Glu37 and JAML Asp36. CAR Glu37 also interacts with JAML Arg102, which forms a salt bridge to JAML Asp36. CAR Lys102 interacts with JAML Glu100 and Asp39, which in turn forms bidentate interactions with CAR Lys104. (D) The functional hotspot of the CAR-JAML interaction: JAML Asp39, Tyr52, Phe55, and Tyr57 and CAR Lys104. (E) Open-book representation of the CAR-JAML interface. Atoms that form contacts are colored green and blue on the molecular surfaces of JAML and CAR, respectively. Above, the electrostatic potential was mapped onto the molecular surface and contoured at ±35 kT/eV (blue/red). CAR residues contacting JAML are shown as green sticks above the JAML surface. JAML residues contacting CAR are shown as blue sticks above the CAR surface. The surfaces of CAR and JAML are highly complementary, which ensures high ligand specificity. The strong electrostatic component of this interaction accounts for rapid kinetics and likely a high sensitivity to the environment.

The CAR-JAML interface is restricted to the membrane-distal D1 domains of both receptors (Fig. 2A and table S4) in contrast to previous suggestions (11). Domain-deletion experiments demonstrate that JAML D1 is sufficient for CAR binding (14); however, JAML D2 exhibits indirect effects through enhancing both JAML expression levels and CAR-binding affinity to the JAML D1 domain (fig. S5). The D1 GFCC′C′′ sheets of CAR and JAML pack face-to-face, comparable to the variable regions of the light and heavy chain (VL-VH) arrangement in antibodies or variable regions Vα-Vβ in T cell receptors. Additionally, the CAR A strand interacts with the JAML CC′-loop. The CAR-JAML interface buries a surface area of 1460 Å2 (CAR: 740 Å2, JAML: 720 Å2), which is in the typical range for protein-protein complexes. Although its shape complementarity (Sc = 0.64) is on the low side for protein-protein interactions, it is consistent with the relatively weak CAR-JAML affinity (Kd ~5 μM) in solution (table S3), as in other cell-cell recognition systems (21). However, avidity and concentration effects for cell membrane–tethered receptors likely result in a greater effective molarity on the cell surface (2123).

The CAR-JAML interface is unusually hydrophilic (Fig. 2E) and characterized by an array of interdigitated charged residues (Fig. 2C and fig. S6), which are conserved among homologs for both proteins (figs. S3 and S4). Two hydrophobic clusters (Fig. 2D) and an antiparallel β-sheet interaction between the CAR C′ strand and the JAML D1 FG loop (Fig. 2B and fig. S7) provide additional stabilization. A total of 110 protein-protein interactions between CAR and JAML include 13 salt links, 6 H bonds, and 91 van der Waals’ contacts (table S4). Of the total buried surface area, 36% (CAR: 270 Å2, JAML: 260 Å2) is contributed by charged residues (Asp35, Glu37, Asp49, Lys102, and Lys104 of CAR; Asp36, Arg37, Asp39, Glu100, Arg102, and Lys111 of JAML) (Fig. 2E and fig. S6). The exceptional number of interdigitating salt bridges contributes binding energy but, more important, imparts high ligand specificity as demonstrated by mutational analysis (figs. S8 and S9). JAML Asp39, Tyr52, Phe55, and Tyr57, and CAR Lys104 compose the functional hotspot of the interaction (Fig. 2D). JAML Tyr52 orients Asp39 such that it binds CAR Lys104 in an extended conformation in which its aliphatic portion packs against JAML Phe55 and Tyr57. The Tyr57 hydroxyl also forms crucial H bonds to the CAR backbone. The charged interface and functional hotspot of the JAML-CAR complex show significant similarities to the CD2-CD58 interaction (18) (fig. S10), although low sequence identity (14% for the D1 domains of JAML and CD2, 20% for the D1 domains of CAR and CD58) and different interface topology do not suggest any evolutionary relation between those receptor-ligand pairs.

The JAML-binding site on CAR overlaps with the receptor-binding site for adenoviruses (3, 4) (Fig. 3). Adenoviruses interact with up to 16 of the 18 CAR residues that mediate JAML binding (Fig. 3B and fig. S11) and thus inhibit JAML-CAR interaction (14) and induction of γδ T cell signaling (fig. S15). In contrast, only six residues of the Coxsackie B virus–binding site overlap with the functional CAR interface, but notably these include hotspot residue Lys104. The high degree of conservation and the sensitivity of the key residues of the CAR-JAML interface to mutation support the idea that some viruses ensure long-term entry into host cells by interaction with highly conserved receptor-binding sites, as any mutations would compromise receptor function (9, 19).

Fig. 3

Comparison of functional and viral epitopes of CAR. (A) Schematic representations of CAR complex structures: from left to right: mCAR-mJAML [PDB no. 3MJ7, this work], hCAR-hAd12 [1KAC (3)], hCAR-hCVB3 [1JEW (4)], and hCAR homodimer [1EAJ (19)]. (B) Molecular surface of the CAR D1 domain in the various CAR complex structures. CAR atoms that are part of the epitope for the respective ligands are colored blue (CAR-JAML), red (CAR-Ad12), brown (CAR-CVB3), and light green (CAR homodimer). The CAR-JAML binding site mostly coincides with both the adenoviral and the homodimerization interface of CAR, whereas the coxsackievirus B–binding site is located at the periphery of the functional CAR interfaces.

The CAR-JAML ectodomain interaction induces cell signaling events at the JAML intracellular domain (ICD) (Fig. 4A and fig. S3A) (14), but the mechanism of signal transduction across the cell membrane has not been determined previously. Although no significant structural rearrangements occur in the JAML ectodomain on CAR ligation, JAML and CAR can apparently exist as both monomers and dimers (12, 19). In the crystal, a dimer of the CAR-JAML complex, with an interface size (~1400 Å2) and shape complementarity (Sc = 0.73) within the range of typical protein-protein interactions (Sc = 0.70 to 0.76), is assembled on the crystallographic two-fold axis (figs. S12 and S13). Although the biological relevance of this particular assembly remains to be established (24), JAML dimerization and/or clustering as a mechanism for signal transduction is strongly supported by the finding that binding of dimeric, but not monomeric, CAR or antibody ligands recruits phosphoinositide 3-kinase (PI3K) to the JAML ICD (Fig. 4, D and E).

Fig. 4

Ligand-induced PI3K recruitment to the JAML ICD. PI3K-specific antibody Western blots of JAML immunoprecipitated with anti-JAML HL4E10 immunoglobulin G (IgG) from epithelial γδ T cell lysates after stimulation with ligands (A to E) or from JAML mutant-transfected CHO cell lysates (F). (A) Time course of stimulation with CAR-Fc. After 1 min, PI3K association to JAML is significantly elevated over the basal level. (B) PI3K recruitment is rapidly switched off once CAR is released from the JAML ectodomain. γδ T cells were incubated with buffer (DPBS) or adenovirus F5 protein (F5) or were stimulated with CAR (CAR-Fc) followed by an excess of F5 (which competes with JAML for CAR-binding). F5 alone does not alter basal PI3K levels (DPBS), whereas a 1-min addition of F5 (CAR-Fc+1min F5) significantly decreases PI3K recruitment in CAR-stimulated γδ T cells (CAR-Fc+1min). (C) Functional consequences of mutations in the CAR-JAML interface on downstream signaling by JAML. wt CAR-Fc and an N49A mutant induce significant PI3K recruitment, consistent with their comparable JAML-binding characteristics (fig. S9) and location of CAR Asp49 at the periphery of the JAML-binding site (Fig. 2E), where mutations would be expected to cause smaller effects on binding. Mutation of CAR Asp35 induces less, but does not entirely abolish, PI3K binding to JAML, consistent with its interaction with JAML Arg37, for which mutations are well tolerated. In contrast, CAR mutants E37A, K102A, and K104A, all of which interact with JAML hotspot residues (Fig. 2C), do not induce significant PI3K recruitment. (D and E) Dimeric, but not monomeric, ligands induce PI3K recruitment to JAML. PI3K levels are increased by JAML binding of bivalent HL4E10 IgG, bivalent HL4E10 Fab′2 fragment, and dimeric CAR (CAR-Fc fusion protein), but not by monovalent HL4E10 Fab or monomeric CAR (CAR-His). Fc- or His-tagged control proteins (CD62L-Fc and CD1d-His) do not induce PI3K recruitment. (F) Dissection of the role of conserved JAML intracellular signaling motifs in PI3K binding. PI3K constitutively binds to wild-type and Y314F JAML, whereas binding is abolished to any Y336F mutants in the YMxM PI3K-binding motif. PI3K association is greatly reduced in JAML polyproline motif mutants P340A/P343A/P346A and Y314F/P340A/P343A/P346A, even though they still exhibit an intact YMxM motif. Data are representative of at least three independent experiments.

How does the interaction between CAR and JAML activate γδ T cell responses during epithelial insults (14)? The JAML ICD is highly conserved (which suggests interaction with intracellular proteins) and contains putative serine, threonine, and tyrosine phosphorylation sites (fig. S3A). Notably, it includes a YMxMxPxxP signaling motif (25) similar to that in the major costimulatory αβ T cell coreceptor CD28 (fig. S3B). The tyrosine-phosphorylated motif in CD28 binds the universal signal transducer PI3K 1A (2628) leading to activation of MAP kinase pathways and interleukin-2 (IL-2) production (28, 29).

As resting and activated epithelial γδ T cells lack the major PI3K-binding αβ costimulatory receptors, CD28 and ICOS, and ligand binding to JAML leads to MAP kinase activation and IL-2 production (14), we tested whether JAML mediates costimulation through interaction with PI3K. Indeed, on epithelial γδ T cells, PI3K associates with JAML within 1min of CAR ligation (Fig. 4A). Once PI3K has been recruited, disruption of the CAR-JAML interaction leads to rapid decline of PI3K levels (Fig. 4B), tightly linking extracellular protein-recognition events to intracellular signaling. PI3K binding to JAML also strongly correlates with affinity of the CAR-JAML interaction, as demonstrated by decreased kinase recruitment by CAR mutants that have reduced binding to JAML (Fig. 4C and fig. S9). To further dissect the JAML-PI3K interaction, mutants of the JAML intracellular signaling motifs were generated. In JAML-transfected Chinese hamster ovary (CHO) cells, PI3K binds constitutively to wild-type JAML and a Y314F mutant, but not to a Y336F mutant, of the YMxM motif (Fig. 4F), identifying this region as the PI3K-binding site. In P340A/P343A/P346A JAML mutants, negligible PI3K association occurs, which suggests that, as for CD28 (2628), the polyproline motif serves to recruit a primary kinase to phosphorylate YMxM and that PI3K association to JAML critically depends on the phosphorylation state of the PI3K-binding motif. Despite low sequence identity (11% for JAML and CD28, 16% for CAR and B7-1) and the different domain organization of JAML and CD28, these results demonstrate a striking functional similarity between costimulation and cell signaling through CAR-JAML on epithelial γδ T cells and B7-CD28 on αβ T cells.

Thus, this delineation of the molecular details of the CAR-JAML-PI3K regulatory circuit has now uncovered a physiological role of the major virus receptor CAR and its partner JAML in cell signaling. These insights may serve as a basis for modulating cellular responses during tissue homeostasis and immunity, and have implications for treatment of chronic nonhealing wounds, asthma, and cancer.

Supporting Online Material

www.sciencemag.org/cgi/content/full/329/5996/1210/DC1

Materials and Methods

Figs. S1 to S13

Tables S1 to S5

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. JAML crystallized in space group P43 in two forms with 1mol/ASU and with 2mol/ASU, in F222 with 1mol/ASU, and in P3112 as the JAML-CAR complex.
  3. Although the high protein concentration in the crystal may facilitate CAR-JAML heterotetramer formation analogous to the expected elevation of the effective concentration arising from 2D diffusion of receptors on the cell membrane, the weak affinity constant for oligomerization may explain the difficulty in isolating a CAR-JAML heterotetramer in biochemical experiments. Analytical ultracentrifugation (AUC), coimmunoprecipitation of differentially tagged JAML and CAR, and Bimolecular Fluorescence Complementation (BiFC) assays were used to investigate the formation of CAR-JAML heterotetramers in solution and on the cell surface. Although there was some indication of a CAR-JAML heterotetramer from AUC (table S3) and for JAML dimers from cross-linking experiments and BiFC, providing definitive biochemical evidence for a very low affinity CAR-JAML heterotetramer was intrinsically hampered by experimental constraints (e.g., the limited protein concentrations that could be used in the experiments or that soluble CAR proteins are a poor probe to investigate the heterotypic interactions that naturally occur between the cell-bound JAML and CAR counterreceptors).
  4. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr; and X, any amino acid.
  5. We thank A. Schiefner for help with the structure solution; S. Ferguson, H. Lindermuth, and J. Vanhasny for technical support; L. Teyton for providing cell lines; J. G. Luz, M. A. Adams-Cioaba, J. Stevens, D. A. Shore, A. L. Corper, R. L. Stanfield, and E. Ollmann Saphire for helpful discussions; and X. Dai and K. Saikatendu for assistance on synchrotron trips. We acknowledge the Advanced Light Source, Stanford Synchrotron Radiation Lightsource, and the Advanced Photon Source for use of their synchrotron facilities. This work was supported by NIH grants AI42266, CA58896 to I.A.W., and AI52257 and AI064811 to W.L.H., an Erwin-Schrödinger Fellowship of the Austrian Science Fund to P.V., and the Skaggs Institute. Atomic coordinates and structure factors for JAML and the CAR-JAML complex have been deposited in the Protein Data Bank with accession numbers 3MJ6 and 3MJ7 respectively. This is manuscript 18641-MB from The Scripps Research Institute.
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