g protein-coupled receptors in rheumatology

NATURE REVIEWS | RHEUMATOLOGY ADVANCE ONLINE PUBLICATION | 1

Department of Internal Medicine and Rheumatology, Justus-Liebig University of Giessen, Rheumatology and Clinical Immunology, Kerckhoff Klinik, GmbH, Benekestrasse 2–8, Bad Nauheim D-61231, Germany (E.N., K.K., U.M.-L.).

Correspondence to: U.M.-L. [email protected]

G protein-coupled receptors in rheumatologyElena Neumann, Kiran Khawaja and Ulf Müller-Ladner

Abstract | G protein-coupled receptors (GPCRs) are transmembrane receptor proteins that allow the transfer of signals across the cell membrane. In addition to their physiological role, GPCRs are involved in many pathophysiological processes including pathways relevant in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis. Two-thirds of all currently available drugs target GPCRs directly or indirectly. However, the detailed mechanism of GPCR signalling is still unclear. Selective modification of GPCR-dependent signalling cascades to inhibit disease progression in rheumatic diseases is now being investigated. One approach is to use antibodies against ligands activating GPCRs. However, several GPCRs are known to be activated by only one ligand. In this case, targeting the receptor itself is a promising approach. So far, more information is available on GPCR action in RA as compared with OA, and even less information is available for other rheumatic diseases. Additional research on the role of GPCRs involved in the pathophysiology of rheumatic diseases is required to develop specific therapeutic approaches.

Neumann, E. et al. Nat. Rev. Rheumatol. advance online publication 6 May 2014; doi:10.1038/nrrheum.2014.62

IntroductionA cell maintains homeostasis by controlling the move-ment of substances across the cell membrane. Signal transduction, whereby an extracellular molecule acti-vates a cell-surface receptor to create a physiologi-cal cell response, is one of the mechanisms by which homeo stasis is regulated. G protein-coupled receptors (GPCRs), also known as seven-transmembrane (7TM) receptors as the polypeptide chain passes through the cell membrane seven times,1 are transmembrane pro-teins that transfer extracellular signals through the cell membrane and into the intracellular environment (Figure 1). These receptors can be activated by differ-ent factors, including hormones, neurotransmitters, chemokines, ions, odorants, and some GPCRs are even activated by photons of light.1 After sensing a particular signal, signalling through GPCRs takes place via bind-ing to GTP proteins located on the cytosolic side of the cell membrane. A series of signalling cascades are initi-ated, which subsequently lead to specific cellular and physiologic al responses.2

GPCRs are very diverse and form the largest family of transmembrane proteins—more than 800 genes in the human genome encode GPCRs—with an impor-tant role in many physiological processes.2 Owing to the diverse but highly specific roles of the respective GPCRs in cellular processes, the interest of many academic and industrial researchers has focused on the characteriza-tion of the mechanisms of action of GPCRs. Currently, two-thirds of all drugs target GPCRs either directly or indirectly.1 These drugs are used to treat, for example, cancer, metabolic diseases, and a variety of inflammatory diseases, including rheumatoid arthritis (RA).3

GPCR structureGPCRs are structurally divided into three parts. The N-terminus, along with three extracellular loops (EL1–EL3), forms the extracellular region. The central trans-membrane loops consist of seven α-helices (TM1–TM7). The intracellular region consists of three intracellular domains (ICL1–ICL3), an intracellular amphipathic helix and the C-terminus (Figure 2).

Bovine rhodopsin was the first GPCR crystallized structure to be published, in 2000.4 The first human GPCR structure to be described, in 2007, was the β2- adrenergic receptor.5 Notably, the structure of the β2-adrenergic receptor was found to be very similar to bovine rhodopsin. The second extracellular loop (EL2) of the β2-adrenergic receptor forms a lid at the top of the ligand-binding site5 (Figure 2) and the 7TM helices of GPCRs are arranged to form a cavity-like structure (which is occupied by the ligand) in the cell membrane.6,7 Some ligands are able to bind to the extracellular loops instead of the usual ligand-binding site of the GPCR—ligand binding to the extracellular loops transfers the signal to the transmembrane domain, which leads to a conformational change in the orientation of the trans-membrane helices. The signal is then transferred to the intracellular domain, which leads to the activation of specific signalling cascades.2,6

GPCR familiesGPCRs are classified into four major families according to their pharmacological properties. Family A, also called the ‘rhodopsin family’, is the largest family of GPCRs and, therefore, the primary target for drug development. Family B is also called the ‘secretin and adhesion family’. It contains 15 members of the secretin-like receptors and 33 members of adhesion-like receptors. The secretin-like receptors consist of large extracellular domains, which

Competing interestsThe authors declare no competing interests.

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are involved in ligand binding. The structure of most of the adhesion-like receptors has still not been defined but some consist predominantly of the transmembrane domain. Family C, the third class of the GPCR family, is also called the ‘metabotropic glutamate family’. This family contains the GABAB receptors, the calcium- sensing receptors, and also some taste receptors. Family C members have a large extracellular domain, which is very specific for ligand binding and activation. The receptors in this family are mainly homodimers and heterodimers. The fourth family of GPCRs is also called ‘frizzled family’. GPCRs of the frizzled family show structural similarities to family A and family B GPCRs.1–3

Key points

■ G protein-coupled receptors (GPCRs) are transmembrane receptor proteins involved in many pathophysiological processes in rheumatic diseases

■ GPCRs include a wide variety of receptors relevant to arthritis pathophysiology, including adenosine, chemokines, kinin B2, complement factor, and protease-activated receptors

■ Methotrexate, the most commonly used drug to treat rheumatoid arthritis, works via the adenosine A2A GPCR signalling pathway

■ Therapeutic approaches that target GPCRs could help in reducing the disease progression of a number of rheumatic diseases

The first structure solved by X-ray crystallo graphy, as described previously, was published for bovine rhodop-sin. High-resolution structures have since been described for additional GPCRs including squid rhodopsin, β-adrenoreceptors, muscarinic acetylcholine receptors, human histamine H1 receptor, human dopamine D3 receptor, human adenosine receptor A2A (A2AR), human CXC chemokine receptor 4 (CXCR4), opioid recep-tors, rat neurotensin receptor type 1 (NTR1), human proteinase-activated receptor 1 (PAR-1), and human sphingosine-1-phosphate receptor 1 (S1P1).2 The crystal structures of these GPCRs (except for NTR1) have only been described in their inactive conformation. The active conformation of GPCRs is thermodynamically unstable and, therefore, crystal structures of activated GPCRs are much more difficult to obtain than the crystal structure of GPCRs in their inactive conformation. Stabilization of GPCRs in their active conformation generally requires either high-affinity agonists (agonists whose action is independent of physiological conditions, such as pH or ionic strength, with the exception of r hodopsin—the active state of the rhodopsin structure was obtained from crystals formed at pH 4.5–6.08), or stabilization via muta-tions or fusion proteins. Currently, only bovine rho dopsin, human β2-adrenergic receptor, human A2AR, and rat neuro tensin receptor have been structurally characterized in their active (or intermediate-active) conformation.2

GPCR signalling—a standard modelGuanine nucleotide-binding complexThe signalling cascade of GPCRs induced by ligand binding is regulated by a guanine nucleotide-binding complex (consisting of Gα, Gβ, and Gγ subunits), as well as additional regulatory proteins. In an inactive form, the Gα subunit is bound to GDP and is firmly attached to the Gβ and Gγ subunits.9 Usually, when a ligand binds to its GPCR, it causes a conformational change of the receptor, which leads to the conversion of the inactive-Gα to active-Gα (along with the exchange of GDP to GTP) (Figure 3).1,10 The signalling cascade is usually initiated when the active-Gα dissociates from the Gβγ dimer and binds with adenylate cyclase, which then leads to the acti-vation of cAMP and activation of the protein kinase A (PKA) pathway. G-proteins are also involved in the acti-vation of the phospholipase C (PLC) signalling pathway, which catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). The signal is terminated by the hydrolysis reaction of GTP, which restores the inactive form of active-Gα, which binds with the Gβγ dimer to reform the G-protein binding complex (Figure 3).1,9

The Gα subunit of heterotrimeric G proteins induces a signalling cascade in a number of cell signalling pathways. The Gα subunit is further subdivided into four families: Gαs, Gαi/Gαo, Gαq/Gα11, and Gα12/Gα13. In addition, the Gβγ dimer is made up of one of five different G protein β-subunits and one of twelve γ-subunits.9,11 Activation of GPCRs can trigger many G protein s ubtypes—the reason that one GPCR can activate various signalling cascades via Gα as well as Gβγ subunits. Among the different families of Gα subunits, the Gαi/Gαo family is known

A2AR S1P1

CXCR4 PAR-1

Figure 1 | Schematic diagram showing the structure of illustrative GPCRs known to have a role in rheumatic disease. Abbreviations: A2AR, adenosine receptor A2A; CXCR4, CXC chemokine receptor 4; PAR-1, protease-activated receptor 1; S1P1, sphingosine 1-phosphate receptor 1.

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to be expressed widely.12 As Gαi and Gαo are expressed in high amounts, a GPCR-dependent activation of Gαi/Gαo causes a very high amount of free intracellular Gβγ dimers, which leads to Gβγ-mediated signalling.9,13 Therefore, activation of Gαi and Gαo is considered to be a major part of the mechanism that leads to the additional activation of Gβγ-mediated signalling.9

G protein-coupled receptor kinasesGPCR kinases (GRKs) are a family of proteins, well-known to be involved in the regulation of GPCR signal-ling. They are grouped into six subtypes, GRK1–GRK6. GRKs mediate the phosphorylation of GPCRs, which facilitates the binding of these receptors to cytosolic pro-teins called arrestins and, ultimately, causes dissociation of GPCRs from the G protein complex. In this way, GRKs regulate GPCR desensitization and resensitization.14 Changes in GRK expression levels have been shown to affect the signalling of GPCRs.14

The expression of GRKs is reduced in peripheral blood mononuclear cells of patients with RA, although the mRNA levels remain unchanged.15 GRK levels were also reduced in a rat model of adjuvant arthritis.16 Pro-inflammatory cytokines, such as IL-6, can down regulate GRKs, as observed in immune cells in vitro. The produc-tion of proinflammatory cytokines is increased in RA17 and might be responsible for the observed GRK degrada-tion.18 Therefore, the regulation of GRK expression rep-resents an interesting parameter for GPCR regulation in the treatment of RA.16

GRKs are involved in the phosphorylation of GPCRs.18 In addition to GPCR phosphorylation, GRKs are able to induce the phosphorylation of cytosolic proteins includ-ing p38 mitogen-activated protein kinase (p38 MAPK).18 p38 MAPK activation is central to the pathophysiology of various inflammatory diseases, including RA. GRK2 levels, as outlined above, are reduced in an adjuvant arthritis model,16 as well as in human RA and multi-ple sclerosis.18 These findings, therefore, underline the relation ship between GRK2 and p38 MAPK activation. Additionally, GRK2 has a role in regulating the activa-tion of p38 MAPK, thus further contributing to various inflammatory responses.18

GPCRs and rheumatologyGPCRs have a very important role in rheumatic dis-eases, including RA, osteoarthritis (OA), psoriatic arthritis (PsA) and psoriasis. The results from many studies show that targeting GPCRs can affect the patho-physiology of these diseases (Table 1). However, the detailed mechanism behind the regulatory response is still not clear for many GPCRs. The process of ligand-binding to these GPCRs is normally strictly regulated. Ligands usually activate a specific signalling mechanism that is mediated by a particular receptor.9 This specific-ity has opened a new area of drug discovery and clari-fication of the detailed mechanisms of receptor-ligand behaviour, along with quantification of the drug effect in such a complicated system, is required.9 Here, we sum-marize some examples of GPCRs with a known effect in r heumatic diseases.

Role of adenosine receptorsAdenosine receptors belong to the rhodopsin family of GPCRs. There are four distinct types of adeno sine re ceptor—A1, A2A, A2B, and A3. The A2A ad enosine recep-tor has a crucial role in the pathophysiology of RA, as confirmed with A2AR-deficient mice. The increase in

Cytoplasm

ICL1 ICL2 ICL3

COOH

EL1

EL2

EL3

NH3

Transmembrane α-helicesGβγ binding regionLigand binding region

Gα binding

Amino acid chain

Extracellular

TM1 TM2 TM3 TM4 TM5 TM6 TM7

Figure 2 | Schematic diagram of a GPCR with seven TM domains (TM1–TM7), extracellular loops (EL1–EL3) and intracellular loops (ICL1–ICL3). The different binding regions are shown in different colours as indicated. The EL2 domain forms a lid-like structure on top of the TM3 domain. Abbreviations: EL, extracellular loop; GPCR, G protein-coupled receptor; ICL, intracellular loop; TM, transmembrane.

Ligand

GPCR

GαGDP GTP

Gα

PKA PIP2

Cytoplasm

Extracellular

γβ

γβ

ATP PPi

AC

cAMP

PLC

Figure 3 | Schematic diagram of GPCR signalling mediated by the activation of the G protein α subunit. In the inactive state, Gα is bound to the Gβ and Gγ subunits, GDP and also to the GPCR. Upon ligand binding, the Gα subunit is activated and dissociates from the Gβγ dimer and from the GPCR. GDP exchanges to GTP during this process. The activated Gα then initiates a downstream signalling cascade by the activation of AC. AC catalyses ATP to form cAMP, which then activates PKA. In parallel, PLC can be activated via active Gα, which in turn activates PIP2. Abbreviations: AC, adenylate cyclase; GPCR, G protein-coupled receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PLC, phospholipase C.

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inflammation and tissue damage (induced by concanava-lin A) is more severe in A2AR -deficient mice in compari-son with the minor damage observed in control mice.19 This research illustrates the anti-inflammatory effects of A2A receptors, and also explains that inflammatory stimuli can cause the accumulation of extracellular adenosine, which is followed by an anti-inflammatory effect by acti-vating cAMP via A2AR signalling. Thus, targeting A2AR signalling could achieve an anti- inflammatory effect.

One example currently in therapeutic use is metho-trexate, an agent commonly used for the treatment of RA that targets the A2AR signalling pathway.20 Methotrexate is the most commonly prescribed drug for the treat-ment of rheumatic diseases, particularly for RA but also OA and PsA.21 Methotrexate causes the inhibition of folate-dependent enzymes, such as dihydrofolate reductase, thymidilate synthase, or 5-aminoimidazole-4- carboxamide ribonucleotide (AICAR) trans formylase.22,23 Methotrexate also inhibits purine and pyri midine synthe-sis, which attenuates DNA and RNA synthe sis. This inhi-bition of purine and pyrimidine synthesis causes many harmful effects including stomatitis and liver toxicity.23 Moreover, methotrexate increases the extracellular levels of AICAR. AICAR inhibits AMP deaminase. There fore, high levels of AICAR lead to the accumulation of adeno-sine and AMP in the extracellular space. This extra-cellular adenosine binds to the GPCR A2AR and causes an increase of the anti-inflammatory cytokine IL-10 and inhibition of the proinflammatory transcription factor nuclear factor κB (NFκB).24

The subtype A3 of adenosine receptor is also involved in PsA pathogenesis. The A3 agonist CF101 (IB-MECA) 23 is currently under evaluation in clinical trials for RA,

PsA and dry-eye syndrome.25 Previously, treatment with CF101 was evaluated in phase II clinical trials for mod-erate to severe plaque psoriasis. CF101 was found to be safe, well tolerated, and showed a good efficacy in patients with psoriasis.26

Role of chemokine receptorsThe family A receptors include chemokine receptors that are present on various cell types.27 Approximately 50 chemokines—the ligands of chemokine receptors —and 19 chemokine receptors are known to exist.28 In RA, numerous chemokines and their receptors are involved in synovitis, including in the extravasation of leukocytes through the endothelium into the synovial tissue28 as well as for the migration of pro-destructive cells to the site of cartilage and bone destruction.29 Therefore, the inhibition of chemokines represents an interesting approach to therapeutically preventing, or reducing, disease progression in RA.

Selective targetingChemokines often act via GPCRs by binding to heparan sulphate (HS) present in proteoglycans of the cell mem-brane or extracellular matrix.30 This binding takes place between the cationic regions of the heparin-binding domains with anionic sulphate regions in HS.31 Research has shown that a specific mutation (exchange of basic amino acid residues [represented by B] to alanine within an identified 44BXBXXB49 GAG-binding motif) in the heparin-binding regions of the CC chemokines CCL5 (also known as RANTES) and CCL7 (also known as MCP3) does not have an effect on ligand binding with GPCR.32 However, the ratio of one mutant to one wild

Table 1 | Overview of GPCRs targeted in rheumatic diseases

GPCR family GPCR Disease Drug Clinical data

Adenosine receptor A2AR RA Methotrexate Standard therapy

A3R PsA CF101 Phase II completed

Chemokine receptor CXCR3 RA MDX-1100 (with methotrexate)AMG487

Phase II completed34

NA

CXCR4 RA, OA AMD3100 NA

CCR2 RA MLN1202PF-04136309

Phase II in RA failed41

Phase II in OA completed

CCR3 RA, OA No specific agents developed NA

CCR4 PsA, RA No specific agents developed NA

CCR5 RA Maraviroc (with methotrexate) Well tolerated for 12 weeks in patients with RA but failed to improve disease severity42

Bradykinin receptor B2 receptor OA Icatibant

MEN16132

Analgesic effects, but no anti-inflammatory effects51

NA

Anaphylatoxin receptor

C5aR RA Neutrazumab Phase I complete but full results not available3

S1P receptor S1P1 RA, PsA FTY720 (fingolimod)Anti-S1P mAb

NASuccessfully passed phase I clinical trial57

Protease-activated receptor

PAR-2 OA PAR2-IP NA

Abbreviations: A2AR, adenosine receptor A2A; A3R, adenosine receptor A3; C5aR, C5a anaphylatoxin receptor; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; GPCR, G protein-coupled receptor; IP, inhibitory protein; mAb, monoclonal antibody; NA, not available; OA, osteoarthritis; PAR-2, protease-activated receptor 2; PsA, psoriatic arthritis; RA, rheumatoid arthritis; S1P, sphingosine 1 phosphate; S1P1, S1P receptor 1.

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type chemokine can inhibit GPCR activation, which leads to transendothelial leukocyte migration in vitro and chemokine-mediated inflammation in vivo,30,32,33 although the mechanisms of the effects mediated by HS binding of chemokines is not clear. Targeting this interaction has been proposed as a tool to inhibit the chemokine-induced proinflammatory effects.30

Sometimes it is better to target the receptor itself, rather than targeting the ligand by using an antibody, as many GPCRs bind the same ligand. However, in some cases, tar-geting the ligand is more specific. For example, CXCL10 (IP-10) is a more selective target than its receptor CXCR3, as CXCR3 exists in different isoforms. Patients with RA receiving methotrexate treatment showed a successfully steady decrease in the disease when treated additionally with MDX-1100, a therapeutic neutralizing anti-CXCL10 antibody, in a phase II trial.3,34

Targeting the CXCR4 pathwayChemokines and their receptors are important inflam-matory mediators in many autoimmune diseases, includ ing RA, multiple sclerosis, and psoriasis.35 When a chemokine binds to its receptor, the receptor is acti-vated and an intracellular signalling cascade is induced which ultimately leads to an increase in cytosolic levels of free calcium.36 Changes in the phosphorylation status of the receptor, and hence receptor internalization, can also occur. Chemokine and receptor interactions, such as that between stromal cell derived factor (SDF)-1 and CXCR4, also have an important role in RA and OA. The levels of SDF-1 are increased in RA and OA, and SDF-1 also increases the production of IL-6 in human synovial fibroblasts via the CXCR4 receptor.37 There fore, inhibi-tion of the CXCR4 receptor could represent a potential therapeutic application for RA. A bicyclam (a class of compounds that affect HIV’s ability to bind to healthy cells, composed of two identical cyclam [1,4,8,11-tetra-azacyclotetradecane] rings connected by a phenylenebis-methylene linker) named AMD3100 acts as a CXCR4 antagonist. It can be used to inhibit the SDF-1 and CXCR4 interaction and, via this mechanism, decreases the disease progression. AMD3100 blocks the intra cellu-lar calcium signalling triggered by SDF-1–CXCR4.35 Further more, AMD3100 reduced collagen-induced arthritis in mice by blocking the leuco cyte migration towards the inflamed areas, mediated by SDF-1–CXCR4 interaction.38 There fore, AMD3100 is an attractive drug candidate for the treatment of RA.

Similar to RA, binding of SDF-1 to the GPCR CXCR4 has a very important role in the pathophysiology of OA. SDF-1 and CXCR4 interactions induce the expression, as well as release, of MMP-3, MMP-9 and MMP-13 by chondrocytes in the articular cartilage of patients with OA. Treatment with AMD3100 reduced mRNA expression and release of MMP-3, MMP-9 and MMP-13 in OA cartilage by inhibiting the interaction between SDF-1 and CXCR4. However, the levels of MMP-3, MMP-9 and MMP-13 did not reach the normal level.39 This effect of AMD3100 has been tested in vitro in human OA cartilage, as well as in vivo in male Hartley guinea pigs.40 The results from

these studies show that blocking the SDF-1–CXCR4 inter-action with AMD3100 has the potential to be an effective therapeutic approach for the treatment of OA. However, no data from clinical trials are currently available.

Targeting CCR2So far, identifying selective inhibitors or inducers of dif-ferent GPCR families or specific GPCRs has been diffi-cult. One approach is the use of antibodies against these receptors and, at present, the development of anti bodies against GPCRs is a growing field in pharmaceutical and bio technology companies, and many agents are currently being tested in clinical trials. For example, a mono clonal antibody named MLN1202 targets the CC chemo kine receptor 2 (CCR2), which is a GPCR. The drug MLN1202 passed the phase I trial, but failed a phase II clinical trial for RA.3,41

Targeting CCR5The levels of CC chemokine receptor 5 (CCR5), another GPCR, are increased in the synovial fluid of patients with RA. Blocking CCR5 was hypothesized to help in the reduction of the altered behaviour of synovial fibro-blasts, as well as chondrocyte activation, and ultimately lead to protection against joint destruction in patients with RA. Clinical trials were performed using an antago-nist of CCR5, known as maraviroc, for patients with RA who additionally received methotrexate. Maraviroc was well tolerated for 12 weeks. However, it failed to improve disease severity in patients with RA during this time period.42

Psoriasis and the D6 receptorThe D6 receptor, which is a GPCR, has now been shown to act as a scavenger receptor for inflammatory CC chemo kines in human psoriatic skin.43 The D6 receptor has a very important role in protection against inflamma-tion, and its absence contributes to disease pro gression. High expression of D6 was observed in unaffected skin areas at least 8 cm from psoriatic plaques.43 This high expression of D6 was induced in response to the increased expression of inflammatory CC chemokines in order to suppress the effect of CC chemokines. How-ever, the expression of D6 is strongly reduced in psoriatic plaque areas,43 showing the protective role of D6 receptor in progression of disease in psoriasis.

RA and CXCR3In order to treat RA, therapeutic agents are needed that can reduce disease progression without any serious adverse effects, which are often observed during long-term treatment of RA.44,45 Levels of CXCL10 are increased in patients with RA, and invading synovial fibroblasts from DA rats in an arthritis model produced high levels of CXCL10.46 The increased concentration of CXCL10 causes the synovial fibroblasts to invade in an autocrine and paracrine manner. CXCL10 functions via interaction with CXCR3, a GPCR. The invasive property of synovial fibroblasts can be reduced by using anti-CXCR3 block-ing antibodies or the CXCR3 inhibitor AMG487, which

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also affected lamellipodia formation (which is relevant for cell migration) by RA synovial fibroblasts. These results indicate that targeting CXCL10 and CXCR3 interactions reduces the synovial fibroblast invasion and hence joint damage in patients with RA.46

Targeting eotaxinThe levels of chemokine eotaxin (CCL11) are increased in patients with OA and RA compared with healthy indi-viduals.47 High levels of eotaxin not only increase the gene expression of MMP-3, but also enhance MMP-3 protein secretion in chondrocytes.47 Eotaxin acts by binding to its specific receptors CCR3 and CCR5, members of the rhodop sin family of GPCRs. Eotaxin induces the release of MMP-3 via p38 MAPK and extracellular signal- regulated kinase (ERK) pathways. Blocking these molecules showed an inhibitory effect on MMP-3 expres-sion. How ever, inhibition of cAMP by cAMP antagonists enhanced eotaxin-induced MMP-3 expression in the human chondro cyte cell line SW1353,47 indicating the regu latory effect of G protein-coupled eotaxin receptor on MMP-3 expression. Eotaxin could, therefore, be a useful tool for the treatment of OA and RA.

Targeting CCR4The macrophage-derived CC chemokine CCL22 (also known as MDC) was detected at high levels in the syno-vial fluid of patients with RA and PsA, as compared with patients with OA.48 CCL22 acts by binding to its recep-tor, CCR4, which is a chemokine receptor belonging to the GPCR family. CCR4 is expressed by memory T cells and is important for the migration of these cells towards the skin or joints. The authors suggested that high levels of CCL22 in PsA joints cause the migration of CCR4-expressing memory cells towards affected joints and, thus, disease progression. Therefore, CCL22 and CCR4 inter-actions represent an interesting therapeutic target with regard to disease protection.

Role of kinin B2 receptorsBradykinin has been known for many years to be involved in the pathophysiology of knee OA, as it was detected in the synovial fluids from patients with OA.49 Bradykinin is a peptide that acts by binding specifically to its receptors. These receptors are members of the rhodopsin family of GPCRs.49 Two bradykinin receptors are known, the B1 receptor and the B2 receptor.50 When, for example, B2 receptors are activated by bradykinin, the activa-tion of the signalling cascade is induced, leading to an algogenic effect involving activation of nociceptors that trigger the capsule and the synovium, and also inflam-matory effects that cause pain and synovitis.49 Blocking bradykinin and B2 receptor binding has been suggested as a therapeutic approach for the treatment of OA. When a B2-receptor blocker named icatibant was intra-articularly administered to patients with OA, analgesic effects, but no anti-inflammatory effects, were observed.51 Another B2 receptor antagonist, MEN16132 (also known as fasitibant),52 was able to block anti-inflammatory reac-tions, specifically the release of IL-6 and IL-8 induced in

response to bradykinin, in human synovial fibroblasts.49 However, MEN16132 has not yet been tested in patients with OA.

Role of the complement receptor C5aRThe C5a anaphylatoxin receptor (C5aR) is a receptor of the complement system component C5a, a chemotactic agent that can cause smooth-muscle contraction, oedema, and induces the release of inflammatory factors includ ing TNF, IL-1, IL-6, IL-8, and prostaglandins.53 C5aR belongs to the extensively-studied rhodopsin family of GPCRs, and is involved in the pathogenesis of many rheumatic diseases, including RA and OA. In order to treat chronic inflammation, as well as tissue damage in OA, a promis-ing approach is the use of therapeutic agents that inhibit the binding of C5a with its receptor C5aR.

Different peptides and monoclonal antibodies that target C5aR have been developed. One of the mono clonal antibody-based drugs, neutrazumab, has completed a phase I clinical trial for RA, although no full report is currently available regarding the outcome of the trial.3

One compound which was specifically developed for the treatment of OA is EP 1575606 A1 (patent pending). This compound acts as a C5aR anatagonist and has the capacity to regulate the functions of GPCRs, specifically C5aR.54 So far the compound EP 1575606 A1 has been proven to be useful against OA induced in dogs.54 No reports are currently available regarding the effectiveness of EP 1575606 A1 in humans.

Role of sphingosine 1-phosphate receptorsSphingosine 1-phosphate receptors belong to the family A of GPCRs. They are the targets of lipid signalling mol-ecule sphingosine 1-phosphate (S1P).55 S1P receptors are divided into five subtypes: S1P1, S1P2, S1P3, S1P4 and S1P5. These receptors are extensively expressed by lym-phocytes, macrophages, endothelial cells and fibroblasts.3 So far, finding selective inhibitors of S1P GPCRs has been difficult. The fungal metabolite myrocin derivative called FTY720 (also known as fingolimod), an analogue of sphingosine and known to work via S1P1, has been shown to exert immunosuppressive activity. FTY720 and related compounds are currently being examined in clini-cal trials for treatment of multiple sclerosis.3 FTY720 was additionally found to inhibit the development of arthritis in the SKG mouse model of RA. In this model, FTY720 treatment decreased IL-6 and TNF expression by syno-vial fibroblasts, as well as by other inflammatory cells.56 Inhibitors of sphingosine kinase (an enzyme that causes the phosphorylation of sphingosine to S1P) are also under investigation for the treatment of various inflammatory diseases. In this context, S1P lyase (an enzyme responsible for S1P catabolism) is a promising target, but new results from a clinical trial in RA showed only low effectiveness.57 The use of anti-S1P monoclonal antibodies is a differ-ent approach that has successfully passed phase I clinical trials.57 S1P antagonists such as FTY720, SEW2871, and VPC23019 are also being evaluated along with S1P anti-bodies, or by inhibitors of S1P lyase, to disrupt the S1P activity in lymphoid organs. This idea is proven to have

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therapeutic potential for the treatment of autoimmune diseases, such as RA.58

Role of protease activated receptorProtease activated receptors (PARs) belong to the GPCR family which are activated by serine proteases.59,60 They have been divided into four types (PAR-1–PAR-4). PAR-2 is expressed by chondrocytes in cartilage from patients with OA. When PAR-2 is activated, the level of matrix metalloproteases increases.59 Furthermore, trypsin acts on PAR-2 by exposing a hexameric–tethered peptide, which then binds with the conserved regions at EL2 of the receptor to start the signalling.60 Additional path-ways involving GPCRs are also known in OA. PAR-2 is well-known to have an important role in the progression of OA inflammation.59 OA synovial cells express PAR-2 without additional stimulation. Activation of PAR-2 is linked to NFκB activation and signalling. Treatment of OA primary synovial fibroblasts with PAR-2-inhibiting peptides (PAR2-IP) showed an inhibition of the tr ypsin-induced NFκB activation, and also a reduction of COX-2 expression. These data highlight the potential of PAR2-IP as a therapeutic tool for the treatment of OA.60

ConclusionsGPCRs are transmembrane receptors containing a GTP binding protein that allows the transfer of signals through

the cell membrane. After interaction with the respective ligand, GPCRs induce a series of downstream signalling pathways, which ultimately lead to different physiological responses. Research now shows that GPCRs are involved in many pathophysiological processes of RA, OA and PsA. Modification of the signalling cascades induced by GPCRs could help in reducing disease progression. However, most of the involved pathways are still under investigation and not yet clearly understood. More work is now required to understand the detailed phenomenon of disease progression via GPCRs, and the plausibility of targeting GPCRs in the human setting.

Review criteria

In the preparation of this manuscript, relevant articles in the PubMed database were identified using search terms including “G-protein coupled receptors”, “G-protein coupled receptors structure”, “GPCRs + rheumatoid arthritis”, “GPCRs + inhibitors + rheumatoid arthritis”, “GPCRs + osteoarthritis”, “GPCRs + inhibitors + osteoarthritis”, “GPCRs rheumatic diseases”, “GPCRs + psoriatic arthritis”, “GPCRs classification”, “GPCRs crystal structures” and “GPCR + methotrexate + rheumatoid arthritis”. The search focused on articles published since 2005, especially in the case of rheumatic diseases. For clinical trials, updates were retrieved from the ClinicalTrials.gov registry and database.

1. Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).

2. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).

3. Hutchings, C. J., Koglin, M. & Marshall, F. H. Therapeutic antibodies directed at G protein-coupled receptors. MAbs 2, 594–606 (2010).

4. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

5. Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).

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16. Lombardi, M. S. et al. Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system. J. Immunol. 166, 1635–1640 (2001).

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Systematic Reviews 1998, Issue 2. Art. No.: CD000957. http://dx.doi.org/10.1002/ 14651858.CD000957.

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25. Jacobson, K. A., Balasubramanian, R., Deflorian, F. & Gao, Z. G. G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions. Purinergic Signal. 8, 419–436 (2012).

26. David, M. et al. Treatment of plaque-type psoriasis with oral CF101: data from an exploratory randomized phase 2 clinical trial. J. Eur. Acad. Dermatol. Venereol. 26, 361–367 (2012).

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29. Neumann, E., Lefevre, S., Zimmermann, B., Gay, S. & Müller-Ladner, U. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends Mol. Med. 16, 458–468 (2010).

REVIEWS


http://dx.doi.org/10.1002/14651858.CD000957

http://dx.doi.org/10.1002/14651858.CD000957


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31. Johnson, Z. et al. Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system. J. Immunol. 173, 5776–5785 (2004).

32. Ali, S. et al. A non-glycosaminoglycan-binding variant of CC chemokine ligand 7 (monocyte chemoattractant protein-3) antagonizes chemokine-mediated inflammation. J. Immunol. 175, 1257–1266 (2005).

33. Ali, S., Fritchley, S. J., Chaffey, B. T. & Kirby, J. A. Contribution of the putative heparan sulfate-binding motif BBXB of RANTES to transendothelial migration. Glycobiology 12, 535–543 (2002).

34. Yellin, M. et al. A phase II, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of MDX-1100, a fully human anti-CXCL10 monoclonal antibody, in combination with methotrexate in patients with rheumatoid arthritis. Arthritis Rheum. 64, 1730–1739 (2012).

35. Hatse, S., Princen, K., Bridger, G., De Clercq, E. & Schols, D. Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett. 527, 255–262 (2002).

36. Garcia-Vicuna, R. et al. CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients. Arthritis Rheum. 50, 3866–3877 (2004).

37. Chen, H. T. et al. Stromal cell-derived factor-1/CXCR4 promotes IL-6 production in human synovial fibroblasts. J. Cell. Biochem. 112, 1219–1227 (2011).

38. Matthys, P. et al. AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-γ receptor-deficient mice. J. Immunol. 167, 4686–4692 (2001).

39. Li, Y. et al. Influence on matrix metalloproteinases 3, 9, and 13 levels after blocking stromal cell derived factor 1/chemokine receptor 4 signaling pathway with AMD3100 [Chinese]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 26, 652–656 (2012).

40. Wei, F. et al. Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway. Arthritis Res. Ther. 14, R177 (2012).

41. Vergunst, C. E. et al. Modulation of CCR2 in rheumatoid arthritis: a double-blind, randomized, placebo-controlled clinical trial. Arthritis Rheum. 58, 1931–1939 (2008).

42. Fleishaker, D. L. et al. Maraviroc, a chemokine receptor-5 antagonist, fails to demonstrate efficacy in the treatment of patients with rheumatoid arthritis in a randomized, double-blind placebo-controlled trial. Arthritis Res. Ther. 14, R11 (2012).

43. Singh, M. D. et al. Elevated expression of the chemokine-scavenging receptor D6 is associated with impaired lesion development in psoriasis. Am. J. Pathol. 181, 1158–1164 (2012).

44. Lindberg, M. Use of NSAIDs in rheumatoid arthritis should be limited [Danish]. Ugeskr. Laeger 175, 1039–1041 (2013).

45. Kinsey, S. G., Naidu, P. S., Cravatt, B. F., Dudley, D. T. & Lichtman, A. H. Fatty acid amide hydrolase blockade attenuates the development of collagen-induced arthritis and related thermal hyperalgesia in mice. Pharmacol. Biochem. Behav. 99, 718–725 (2011).

46. Laragione, T., Brenner, M., Sherry, B. & Gulko, P. S. CXCL10 and its receptor CXCR3 regulate synovial fibroblast invasion in rheumatoid arthritis. Arthritis Rheum. 63, 3274–3283 (2011).

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52. Meini, S. et al. Fasitibant prevents the bradykinin and interleukin 1β synergism on prostaglandin E2 release and cyclooxygenase 2 expression in human fibroblast-like synoviocytes. Naunyn Schmiedebergs Arch. Pharmacol. 385, 777–786 (2012).

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58. Graler, M. H. Targeting sphingosine 1-phosphate (S1P) levels and S1P receptor functions for therapeutic immune interventions. Cell. Physiol. Biochem. 26, 79–86 (2010).

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Author contributionsE.N., K.K. and U.M.-L. contributed equally to researching data for article, substantial contribution to discussion of content, writing and review/editing of the manuscript before submission.

REVIEWS


http://www.ncbi.nlm.nih.gov/pubmed/18576354

http://www.google.com/patents/EP1575606A1?cl=en

http://www.google.com/patents/EP1575606A1?cl=en

g protein-coupled receptors in rheumatology

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