RAGE: a single receptor fits multiple ligands

Opinion

RAGE: a single receptor fits multiple ligands Gu¨nter Fritz University of Freiburg, Department of Neuropathology, Neurozentrum, Breisacher Str. 64 79106 Freiburg, Germany

The receptor for advanced glycation end products (RAGE) is a central signaling molecule in the innate immune system and is involved in the onset and sustainment of the inflammatory response. RAGE belongs to a class of pattern recognition receptors that recognize common features rather than a specific ligand. Recent structural information on the extracellular portion (ectodomain) of RAGE shed new light on this unusual ability. X-ray crystallographic, NMR and biochemical data suggest that ligand binding is driven largely by electrostatic interactions between the positively charged surface of the ectodomain and negatively charged ligands. In this article, I propose a putative mechanism of RAGE ligand recognition of receptor activation. The RAGE axis in inflammation The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin (Ig) superfamily of cell surface receptors [1,2]. RAGE has been recognized as a key molecule in the development of severe chronic pathologies, including diabetic complications [3], chronic inflammation [4], atherosclerosis [5], neurodegeneration and cancer [6]. In healthy animals, RAGE expression is very low in most cell types and tissues. By contrast, elevated expression is observed in different disease states. Such changes in RAGE expression elicit concurrent increases in the expression of RAGE ligands, which themselves are also diseaseassociated. These include the so-called advanced glycation end products (AGEs), members of the S100 protein family [7], high mobility group protein box-1 (HMGB1) [6], amyloid b [8–10] and fibrillar protein aggregates [10,11]. Ligand engagement of RAGE activates multiple signaling pathways, dependent on the ligand, environment and cell type and cover Ras-extracellular signal-regulated kinase 1/ 2 (ERK1/2) [12], Cdc42/Rac [13], stress-activated protein kinase/c-Jun-NH2-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinase pathways [6] resulting in the activation of transcription factors like nuclear factor (NF)-kB [14], cAMP response element-binding (CREB) protein [12] or a member of the signal transducers and activators of transcription family (STAT3) [15]. During an inflammatory response, RAGE–ligand engagement leads to an increased expression of RAGE itself. This positive feedback loop results in sustained NF-kB activation, thereby converting a transient proinflammatory response into a chronic pathophysiological state [16]. Future therapeutic approaches therefore might aim to Corresponding author: Fritz, G. ([email protected]).

disrupt this loop by blocking the receptor–ligand interaction. Such an intervention will require detailed knowledge of the molecular mechanism of RAGE–ligand interactions. Recent structural studies of the extracellular domain of RAGE using X-ray crystallography [17,18] and NMR spectroscopy [19–21] have unraveled a putative mechanism underlying RAGE–ligand recognition. Structural analyses reveal that RAGE has an unusually large positive surface charge (Figure 1) that might act as an electrostatic trap for ligands displaying a net negative charge. Moreover, the molecular organization of the receptor might play a central role in the initiation of a signaling cascade. Studies using a fluorescence-labeled receptor revealed that RAGE does not float as a single molecule in the plasma membrane but instead agglomerates into receptor assemblies [22] in accordance with structural and biochemical studies [17,23]. Although cell surface receptor activation by ligand engagement is a field of intense research, current models cannot explain how RAGE is activated by its ligands. In this article, I propose an activation model that integrates the key characteristics of RAGE, positive surface charge and preassembly, and describes how ligand engagement might initiate intracellular signaling cascades. Analysis of RAGE–ligand interactions RAGE–ligand interactions have been analyzed by a broad range of techniques. Ligand binding can be followed in vivo by positron emission tomography [24] as well as in vitro. Studies performed in vitro comprise monitoring the cellular response induced by RAGE ligands as well as protein– protein interaction assays. RAGE–ligand binding elicits immediate cellular responses. Moreover, the strength of the signal is dependent on the affinity between the interaction partners and, to a larger extent, on the duration of the active signaling complex. It is therefore necessary to resolve the association and dissociation phase of the binding process. In particular, the dissociation phase is of significant interest because it directly reflects the lifetime of a signaling-competent complex. For instance, ligands that dissociate very slowly in vitro probably induce prolonged and enhanced receptor activation. Surface plasmon resonance (SPR) is a valuable and versatile method for characterizing such interactions. In this technique, one interaction partner is immobilized and the binding of the second partner is followed in real-time, allowing assessment of the thermodynamics of binding (stoichiometry, affinity) and the kinetics of association and dissociation. Several groups have applied SPR to characterize different RAGE–ligand interactions; these experiments have

0968-0004/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2011.08.008 Trends in Biochemical Sciences, December 2011, Vol. 36, No. 12

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Figure 1. (a) Schematic depiction of RAGE and its alternative splicing products. The receptor of RAGE is composed of three extracellular immunoglobunlin (Ig)-like domains, a single transmembrane helix and a short cytoplasmic domain. The Ig domains are termed V (shown in green), C1 (light green) and C2 domain (orange). Two major alternative splicing products are known: dominant negative RAGE (DN-RAGE) is devoid of the cytoplasmic domain; endogenous secretory RAGE (esRAGE) lacking the transmembrane and the cytoplasmic region is soluble protein. (b) Surface representation model of the RAGE ectodomain. Available structural information for the V, C1 and C2 domains was combined to build a model of the entire extracellular domain of RAGE. The color code on the left-hand side is the same as in (a). The right-hand side depiction of the RAGE ectodomain illustrates the charge distribution on the surface. Areas with positive surface charge are depicted in blue, negatively charged areas are shown in red. The V domain of RAGE is strongly positively charged. The positive surface charge extends to the C1 domain and covers almost one entire side of V and C1 (right). The C2 domain is predominantly negatively charged.

revealed that multimeric ligands exhibit especially prolonged binding to the receptor [7,17,25,26]. RAGE ligands RAGE ligands, unlike those for other receptors, comprise a diverse group of molecules. It is not clear why there are so many different RAGE ligands, or what common features and molecular properties of the ligands drive RAGE activation. To provide an overview of the different ligands and their nature, a concise description of the different groups of ligands follows. Advanced glycation endproducts (AGEs) AGEs were the first RAGE ligands to be described [1]. This heterogeneous group of molecules is derived from condensation and oxidation processes between proteins or peptides and sugars [27]. The first step in AGE formation is the nonspecific reaction of sugars via their aldehyde functional group with an amine or guanidine group from lysine or arginine residues (Figure 2a). This nonspecific, non-enzymatic and frequent process is termed glycation and affects many proteins; e.g. in a healthy individual, 4–6% of total hemoglobin undergoes glycation. Increased levels of protein glycation are observed in ageing individuals and in diabetes patients [28]. The primary Schiff base products are further converted by rearrangements, oxidation and cross-linking into AGEs (Figure 2a). Moreover, this 626

transformation increases the overall negative surface charge of a protein by carboxymethylation of the amino and guanidine groups of lysine and arginine residues (Figure 2b). AGEs bind RAGE with high affinity, triggering the initiation of a pro-inflammatory signaling cascade. RAGE activation by high levels of AGEs has been identified as a direct cause of severe diabetic complications, including accelerated atherosclerosis, cardiovascular disorders [29], nephropathy [30] and chronic inflammation [31]. S100 proteins The human family of S100 proteins comprises more than 25 members that display different expression patterns, function, metal ion binding properties and oligomeric states [32]. S100 proteins are small acidic proteins (Figure 2c) that are composed of two different calciumbinding EF-hands connected by a flexible loop. Most S100 proteins form homodimers, but several are able to form heterodimers and oligomers ranging from tetramers to octamers [26,33]. Although the S100 proteins constitute an evolutionarily young group expressed exclusively in vertebrates, the protein family is characterized by a wide diversification that is reflected in different functions, expression patterns and temporal and spatial distribution in the cell. On the basis of sequence homology, several subgroups can be differentiated within the S100 family; these subgroups are involved in different cellular processes.

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Figure 2. RAGE ligands are predominantly negatively charged molecules. All RAGE ligands are characterized by a strong negative net charge that can extend over a single domain as in HMGB1 or over the entire molecule, as in native S100 proteins or in AGE-modified proteins. Where structural information is available, the surface charge properties of the ligands were calculated and are represented by color code. White areas indicate surface areas without charge, blue areas positive charge and red areas regions with negative charge. (a) The scheme shows the chemical reactions between a sugar and an amino group of a lysine residue, which leads to the formation of carboxymethylated lysine. Note that after carboxymethylation the originally positively charged lysine carries a negative charge. (b) Surface representations of human serum albumin (HSA), which is frequently modified by glucose in the blood stream resulting in carboxymethylation of surface lysine residues (AGEs). HSA modified by AGEs in the test tube has been used in numerous studies as an RAGE ligand. Here, the effect of carboxymethylation on the surface charge of serum albumin is illustrated in the figure. The left-hand side depicts the surface of unmodified HSA (PDB 1AO6). Positively charged (blue) areas alternate with negatively charged (red) areas. Modification of three arginine residues and seven lysine residues, which have been shown to be carboxymethylated in vivo [62], render the surface uniformly negatively charged. (c) Surface representation of human S100B dimer (extracted from PDB 2H61) [26] showing that S100B is negatively charged. The strong negative charge mediates high-affinity binding to RAGE. (d) Left-hand side, amyloid b1-40 (PDB 1AML); right-hand side, structure of amyloid b fibril protomer. The single peptide displays a net negative charge, whereas the fibril displays a pattern of negative charges. (e) Schematic structure of HMGB1, which is composed of two N-terminal DNA-binding domains and a C-terminal domain of unknown function. The two N-terminal domains contain several arginine and lysine residues, which are required for DNA binding. The C-terminal domain is composed only of acidic residues.

Most S100 proteins are localized in the cytoplasm, where they act as calcium sensors. However, some family members are found almost exclusively in the extracellular space where they exhibit cytokine-like properties. Moreover, some S100 proteins display both intracellular calcium signaling functions and, once secreted, cytokine-like functions. Calcium binding induces a conformational change in S100 proteins that alters the distribution of surface charge and accessibility of hydrophobic residues. In the extracellular space, where calcium concentrations are high, the S100 proteins are permanently loaded with calcium, which is a prerequisite for binding to RAGE [7]. In particular, extracellular S100 proteins form oligomeric assemblies that are thought to be crucial for their cytokine function. The following S100 proteins are known to interact with RAGE in vivo or in vitro; S100B, S100P, S100A1, S100A2, S100A4, S100A5, S100A6, S100A7, S100A8/A9, S100A12 and S100A13. These proteins display different affinities as illustrated by dissociation constants ranging from low

nanomolar to micromolar [7]. S100 proteins can induce different cellular responses. For example, S100B promotes concentration-dependent neuronal survival and axon growth, S100A7, S100A8/A9 and S100A12 act as proinflammatory molecules and S100A2 and S100A4 are associated with differentiation and cell growth. Another subgroup comprising S100A5, S100A6 and S100P can promote tumor growth. Amyloid b and amyloid fibrils A hallmark in the initiation and progression of Alzheimer disease (AD) is the extracellular deposition of insoluble extracellular aggregates of the amyloid b peptide that originates from proteolytic cleavage of the amyloid precursor protein. The two major amyloid b peptides contain 40 or 42 amino acid residues. Both peptides harbor mainly acidic and hydrophobic residues and are prone to aggregation (Figure 2d), as amyloid fibrils, which finally build up to amyloid plaques in the brain of AD patients. 627

Opinion A mouse model of AD showed that RAGE binds amyloid b and shuttles it from the blood stream across the blood– brain barrier into the central nervous system [9]. In vitro analysis of the RAGE–amyloid b interaction [34] revealed high-affinity binding of soluble amyloid b peptide. More recent results indicate that RAGE also binds amyloid fibrils [35]. High mobility group box-1 protein (HMGB1) The high mobility group box-1 protein was originally described as a nuclear DNA-binding protein. More recent research has shown that HMGB1 has an extracellular regulatory function and acts as a proinflammatory activator [36]. HMGB1 is composed of three major domains; two N-terminal domains involved in DNA binding and a Cterminal region that consists of acidic amino acid residues (Figure 2e) and that directs RAGE binding [37]. DNAbound HMGB1 binds to RAGE and Toll-like receptor 9 (TLR9), triggering the formation of an HMGB1–RAGE– TLR9 complex that activates autoreactive B cells [38]. A recent publication showed that DNA binds RAGE with high affinity, forming a stable complex in the absence of HMGB1 [18]. Yet, is still not known whether the DNA– RAGE interaction has a physiological function. b2-Integrin Mac-1 Macrophage 1 antigen (Mac-1) is a heterodimeric cell surface receptor that consists of CD11b (integrin aM) and CD18 (integrin b2) and is involved in leukocyte adhesion and migration. RAGE was recognized as a binding partner for Mac-1, mediating leukocyte recruitment that is augmented by S100B [39], S100A9 [40] and HMGB1 [41]. However, so far no in vitro binding data are available for Mac-1–RAGE interactions. The different sizes, shapes and functions of RAGE ligands put forward two central questions in RAGE signaling. What is the molecular basis for the ability of RAGE to bind all these different ligands? How do these different ligands assemble with RAGE to form signaling-competent complexes that are able to initiate intracellular signaling cascades? In the next section, common features of the ligands and unique structural features of RAGE, which might be crucial for signaling, are discussed. Common features of ligands RAGE has been described as a pattern recognition receptor [39,42] that is able to recognize a common pattern within its different ligands. At first glance, RAGE ligands do not have much similarity. Nevertheless, a closer look reveals some prominent characteristics common to all of them. First, all RAGE ligands display a net negative charge at neutral pH: S100 proteins are very acidic [43]; AGE-modified proteins accumulate negative surface charge during their transformation by glycation and oxidation (Figure 2a,b); amyloid b peptide has a net negative charge at physiological pH and amyloid fibrils expose a regular pattern of negative charges on their surface (Figure 2d) [35]; in addition, HMGB1 contains a highly acidic domain (Figure 2e). The second key feature is the tendency of most ligands to oligomerize: AGE modifications of proteins lead to multiple covalent cross-links 628


Box 1. Immunoglobulin domain structures and classification The immunoglobulin (Ig)-like domains are named according to their occurrence in antibodies (i.e. in immunoglobulins). The Ig domain is a versatile structural unit that is found in proteins of different function, including cell surface receptors, cell adhesion molecules and muscle proteins [60]. These proteins are highly modular, consisting of several Ig domains as well as other protein domains with different functions. The Ig domains are classified into four different types: variable (V-set), constant-1 (C1-set), constant-2 (C2set), and intermediate (I-set) [60]. These domains form a common core Greek-key b-sandwich structure [61] and differ in the number of strands in the b-sheets as well as in their sequence patterns.

resulting in higher molecular mass molecules; several S100 proteins (e.g. S100B, S100A2, S100A3, S100A4, S100A8/A9 and S100A12) are reported to form larger assemblies ranging from tetramers to octamers [44–48]; and amyloid b forms fibrils. The next section describes the structural characteristics of the RAGE ectodomain, highlighting that the surface properties of the RAGE ligand-binding domain are complementary to the negatively charged ligands. On the basis of these findings, I propose a mechanism for RAGE activation that suggests an important role for the oligomeric nature of its ligands. Structure of RAGE RAGE is composed of a single transmembrane-spanning helix that connects the ectodomain with a short cytoplasmic domain and an extracellular moiety required for ligand recognition and binding (Figure 1a). The extracellular moiety comprises three immunoglubulin (Ig)-like domains (Box 1). Structural analysis of the single and tandem Ig domains by X-ray crystallography and NMR have been compiled to yield a complete picture of the RAGE ectodomain. Domain organization of the RAGE ectodomain The N-terminal Ig domain (residues 23–119) has been assigned to the V-set of Ig-like molecules and is referred to as the V domain of RAGE. The two additional Ig domains belong to the C1-set and are termed the C1 (residues 120– 233) and C2 (residues 234–325) domains. The N-terminal V domain is located most distal from the plasma membrane, whereas the C2 domain resides close to the membrane. The V and C1 domain are joined together, forming a slightly bent elongated structure [17,18]. The orientation of the V and C1 Ig domains is fixed with respect to each other so that they form a structural unit rather than represent individual domains [19]. The VC1 tandem domain and the C2 domain are connected by a stretch of several amino acid residues with no apparent secondary structure, which is thought to act as a flexible linker between the VC1 and C2 domain. This proposal is strongly supported by NMR studies showing that VC1 moves as a single unit with respect to the C2 domain [19]. Surface properties of RAGE Ig domains The subdivision into the VC1 tandem domain and C2 domain is most striking with respect to the charge properties of the domains. An inspection of the amino acid sequence of the RAGE Ig domains shows that the V domain

Opinion


has an unusually high content of arginine and lysine residues, which carry a positive charge at neutral pH. In fact, the RAGE V domain contains more arginine and lysine residues than 95% of the V-set of Ig domains listed in the Pfam database (http://pfam.sanger.ac.uk/). An analysis of the available RAGE structures (V domain (PDB codes 2E5E and 2L7U), VC1 tandem domain (3CJJ and 3O3U) and C2 domain (2ENS)) revealed that arginine and lysine residues form a large positively charged patch on the surface of V and C1 (Figure 1b). The majority of basic residues are located on one side of the VC1 tandem domain, underlining the fact that the VC1 forms a defined structural and functional unit. In stark contrast to the VC1 domain, the flexibly linked C2 domain has mainly acidic residues on its surface and is negatively charged (Figure 1b). Taking together the structural subdivision into a VC1 and a C2 domain and the opposing charge distribution on the two domains, it becomes clear that the extracellular moiety of RAGE is composed of two subdomains. This subdivision of the RAGE ectodomain into VC1 and C2 domains is fully reflected in the ligandbinding properties of the different domains.

Ligand-binding domain of RAGE Most ligands have been reported to bind to either the V domain or the VC1 tandem domain [10,25,26,19,49,50]; to date, there is only one study reporting binding of a ligand to the C2 domain [49]. The majority of binding data and a recent structural study [21] point to the V domain being the primary binding site of most ligands, but the C1 domain plays a prominent role either in the recognition of the ligand [7,49] or in stabilizing the V domain [19]. The ligands show a strong preference for the positively charged VC1 tandem domain over the negatively charged C2 domain. Taking into account the charge properties of the ligands described above, it is evident that a negatively charged ligand will be repulsed from the C2 domain, but the ligand will be attracted by the complementary positive surface charge of the VC1 tandem domain. The structure of the V domain in complex with an AGE-modified peptide showed that charge–charge interactions are key for the formation of the receptor–ligand complex [21]. Moreover, it has been suggested that the positively charged ligand binding domain of RAGE recognizes a specific arrangement of negative charges as a key pattern of its ligands.

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Figure 3. Preassembly and activation of RAGE. (a) Proposed model of RAGE activation in the plasma membrane. Multimeric ligands stabilize the receptor assemblies, shifting the equilibrium to larger oligomers. Intracellular adapter molecules, e.g. diaphanous-1 (mDia1), activate diverse signal cascades that initiate and stimulate the production of proinflammatory molecules or cell motility, depending on the ligand and cell type. Moreover, in a positive feedback loop, expression of RAGE itself is stimulated, resulting in sustained RAGE signaling. (b) Preassembly of RAGE might be regulated by splice products of RAGE, esRAGE and DN-RAGE. Assembly of DN-RAGE and esRAGE with full-length RAGE will result in hetero-complexes, which can bind extracellular ligands but are not signaling-competent.

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Opinion Charge complementarity and charge–charge interaction can readily explain the diversity of ligands that are recognized by RAGE. However, further consideration of the molecular properties of RAGE is needed to develop a model for receptor activation. Receptor preassembly and ligand oligomerization RAGE forms constitutive multimers within the plasma membrane. In vitro data indicate that these multimers comprise at least four molecules but can form much larger assemblies [22,23]. In line with these data, the arrangement of VC1 molecules in protein crystals reveals extended side-by-side contacts, suggesting that assemblies of RAGE in the plasma membrane adopt a parallel orientation (Figure 3). Such a preassembly of receptors in the absence of extracellular ligands has been described for several cell surface receptors, including TNFa-receptor [51], interleukin receptor [52], erythropoietin receptor [53], epidermal growth factor receptor [54] and, recently, B-cell receptor [55]. The observed preassembly has substantial implications for the mechanism of RAGE activation by its diverse ligands. As outlined above, RAGE binds ligands of different structure, size and molecular organization. This characteristic of RAGE is in stark contrast to most cell surface receptors, which specifically bind one or a few structurally related ligands. Studies of those receptors led to a model in which the association of receptor molecules is driven by the extracellular ligand that recruits several receptor molecules. Hence, the formation of a receptor–ligand complex drives the co-localization of the cytoplasmic domains that is required for initiation of signal transduction. In these models, the geometry of the ligand is of major importance. Indeed, the geometry will control activity, because the intracellular domains of the receptor must be brought into close proximity in a specific orientation. This model has been invoked for receptor tyrosine kinases [56] in which close proximity of intracellular domains harboring intrinsic kinase activity is essential for cross-phosphorylation of the domains and initiation of the signaling cascade. The opposite scenario is suggested for RAGE, which preassembles in the plasma membrane, and in vitro data suggest that ligand binding shifts the equilibrium distribution towards higher order oligomerization states (Figure 3a) [23,26]. Thus, receptor assemblies would be stabilized predominantly by the binding of multimeric ligands such as S100 proteins, amyloid b oligomers, amyloid fibrils or AGEs. Moreover, this model explains how RAGE activation creates a positive feedback loop to sustain elevated RAGE expression; increased receptor levels at the cell surface will promote further assembly, thus explaining the hyper-activation of the RAGE pathway in the development of chronic inflammatory and neurodegenerative disorders. RAGE preassembly might be regulated by two alternative splice products; DN-RAGE (dominant negative RAGE), which lacks the intracellular domain, and esRAGE (endogenous secretory RAGE), a soluble protein comprising only the extracellular region (Figure 1a, Figure 3b) [57]. It has been proposed that soluble alternative splice products of RAGE can act as decoy receptors, decreasing the concentration of available ligands. An alternative mode 630


Box 2. Outstanding questions What are the normal physiological functions of RAGE? Have we identified all the RAGE ligands? Are there hot spots for RAGE preassembly in the plasma membrane (e.g. lipid rafts)? Are there additional signaling pathways activated by RAGE? Which other pathways might interfere with RAGE signaling? What conformational changes occur upon ligand–receptor complex formation and how do these structural alterations transform into the activation of adapter molecules? Are there other receptors with overlapping ligand binding and signaling profiles?

of action is suggested by the preassembly model. In this scenario, esRAGE and DN-RAGE might prevent the formation of signaling-competent complexes by the formation of hetero-complexes with full-length RAGE (Figure 3b). Such hetero-complexes would be able to bind the extracellular ligands, but they would lack the ability to recruit intracellular adapter molecules (Figure 3b). The proposed mechanism might provide the basis for a new therapeutic approach by means of interfering with receptor assembly in the treatment of diabetic complications, chronic inflammatory and fatal neurodegenerative disorders. Concluding remarks The recent structural and functional studies on RAGE provide detailed insights into the processes of ligand recognition and guide the way towards a mechanism of receptor activation. On this route, several key questions remain open and contributions from such fields as physiology, cell biology and biochemistry will be necessary to find the answers (Box 2). The current model, however, posits that RAGE recognizes patterns of negative charge on the surfaces of a diverse group of ligands, thereby classifying RAGE as a pattern recognition receptor. These findings represent an important step forward in the direction of pharmacological intervention in RAGE-associated diseases. Clearly, as a next step we must uncover exactly how RAGE binds its different ligands. Here, structural biology is the tool of choice. Structures of ligand–receptor complexes should provide hints regarding the pattern that is necessary to activate RAGE and, at the same time, will guide the development of antagonists that share this pattern. For now, comprehensive knowledge on the structure and function of extracellular region of RAGE is available. However, we still lack detailed information regarding the intracellular domain that is essential for signaling. Two intracellular adapter proteins, diaphanous-1 [58] and ERK [59], have been recognized so far, but whether there are more adapters or other proteins modulating these interactions remains to be answered. Indeed, future studies might provide a complete picture of the receptor in its active conformation in complex with extracellular ligands and a full set of intracellular adapter molecules bound. Acknowledgements The author is supported by a Heisenberg fellowship of the Deutsche Forschungsgemeinschaft (FR 1488/3-1). This work was further supported by a grant from the Deutsche Forschungsgemeinschaft (FR 1488/5-1).

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