Orthologue Selectivity

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Orthologue selectivity and ligand bias:translating the pharmacology of GPR35

Graeme MilliganMolecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences,

University of Glasgow, Glasgow G12 8QQ, UK

GPR35 is a poorly characterized G protein-coupled re-

ceptor (GPCR) that has been suggested as a potential

therapeutic target for the treatment of diabetes, hyper-

tension and asthma. Two endogenously produced

ligands have been suggested as activators of GPR35,

although the relevance of these remains unclear. Recent-

ly, a series of surrogate agonist ligands and the first

antagonists of GPR35 have been identified. However,

marked differences in the potency of agonists at speciesorthologues of GPR35 have been noted, and this pre-

sents substantial challenges in translating the pharma-

cology at the cloned human receptor to ex vivo and in

vivo studies of the physiological function of this receptor

in animalmodels. Currently identified agonists will prob-

ably not display high selectivity for GPR35. By contrast,

comparisons of the potency of ligands at species ortho-

logues of GPR35 have provided insight into the nature of

the ligand binding pocket and could result in the identi-

fication of more potent and selective ligands.

Introduction

GPR35 is a poorly characterized 7-transmembrane domainG protein-coupled receptor (GPCR) first identified more

than 10 years ago. It was derived from an open reading

frame corresponding to 309 amino acids located in humans

on chromosome 2, region q37.3[1]. In these initial studies,

expression was examined in a range of tissues but was

detected only in the intestine of the rat; it was also reported

to be lacking in a number of regions of human brain [1].

Subsequently, this same sequence (and a further sequence

encoding a second form of GPR35 that appears to be a

differentially spliced isoform containing an N-terminal

extension of 31 amino acids) (Figure 1) was identified from

a cDNA library produced from human gastric cancer cells

[2]. Again, expression was also detected in normal intesti-

nal mucosal cells [2], and because these cDNAs were able to

transform NIH-3T3 cells, it was suggested that GPR35

might be oncogenic and play a role in the generation of

gastric cancers [2]. The significance of the N-terminally

extended form of GPR35 remains to be defined, but mes-

senger RNA encoding this variant has been reported to be

present at higher levels than the shorter form [2].

In humans, the GPR35 gene displays significant poly-

morphic variability, with a number of non-synonymous

variants within the open reading frame resulting in altera-

tions in amino acid sequence[3,4](Figure 1). However, to

date, apart from a very cursory examination of the Ser294-

Arg variant [5], which has been associated with the propen-

sity to develop coronary artery calcification [6] (Table 1),

effects of these variations on signal transduction and phar-

macologyhave yet to be reported. A further single nucleotide

polymorphism, located in the 50 untranslated

region of the GPR35 gene, has been linked to early-onset

inflammatory bowel disease in a genome-wide association

study [7]but no further information on this is currentlyavailable.

Potential endogenous agonists of GPR35

The first endogenously produced chemical that was shown

to be able to activate GPR35 was the tryptophan metabo-

lite kynurenic acid [8]. When human GPR35 was expressed

along with a mixture of promiscuous and chimeric G

proteins[9,10](Box 1) in CHO cells, addition of kynurenic

acid elevated [Ca2+]iin a concentration-dependent fashion

[8]. Importantly, other intermediates of tryptophan metab-

olism, including the non-carboxylate kynurenine, were

inactive[8]. This demonstrated the probable importance

of the acidic moiety of kynurenic acid for binding and/orfunction. Furthermore, although each of the human, rat

and mouse orthologues of GPR35 was activated by kynure-

nic acid, it was already noted that kynurenic acid was less

potent at human GPR35 than at the rodent orthologues [8].

These observations were difficult to interpret fully, howev-

er, because the studies were performed after transient

transfection of CHO cells and without any indication of

the relative expression levels of the orthologues of GPR35

[8]. Further studies indicated that GPR35 was probably

able to couple to pertussis toxin-sensitive Gi-family G

proteins, because chimeric G protein a subunits containing

only the C-terminal five or nine amino acids from such G

proteins were able to transduce signals. Furthermore,

kynurenic acid-stimulated binding of [35S]GTPgS to mem-

branes of CHO cells expressing GPR35 was prevented by

pretreatment with pertussis toxin[8], which blocks signal

transduction via this class of G proteins. A series of further

studies has confirmed the agonist action of kynurenic acid

at GPR35[5,1113]. Moreover, the initial report of varia-

tion in potency of kynurenic acid at human versus rodent

orthologues of GPR35 has been confirmed and extended.

For example, Oka et al. [14] struggled to generate a re-

sponse to kynurenic acid at human GPR35 in Ca2+ assays,

whereas Jenkinset al.[13]reported the EC50of kynurenic

acid as >1103 M at human GPR35 but 7105 M at the

rat orthologue using a bioluminescence resonance energy

Review

Corresponding author: Milligan, G. ([email protected]).

0165-6147/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2011.02.002 Trends in Pharmacological Sciences, May 2011, Vol. 32, No. 5 317
mailto:[email protected]://dx.doi.org/10.1016/j.tips.2011.02.002http://dx.doi.org/10.1016/j.tips.2011.02.002mailto:[email protected]


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transfer (BRET)-based GPR35-b-arrestin 2 interaction as-

say (Box 2) (Figure 2). Indeed, the very low potency of

kynurenic acid at human GPR35 prompted Jenkins and

colleagues[13]to question the potential relevance of this

ligand as a functional endogenous agonist, at least in

humans. Given the higher potency of kynurenic acid at

rodent orthologues of GPR35 and reported micromolar

concentration of kynurenic acid in rat small intestine

[15], effects of this ligand via GPR35 in rodents should

be anticipated.

A second group of endogenously produced ligands that

have been reported to activate GPR35 are lysophosphatidic

acids[14], particularly 2-acyl lysophosphatidic acids [14].

Responses to such ligands were difficult to assess in [Ca2+]ielevation assays because vector transfected cells also pro-

duced a robust stimulation[14]. This probably reflects en-

dogenous expression of one or more members of thelysophospholipidreceptorgroup of GPCRs [16,17]. However,

2-oleoyl lysophosphatidic acid caused internalization of an

epitope-tagged form of human GPR35, whereas kynurenic

acid had little effect[14]. Furthermore, in cells expressing

human GPR35, 2-oleoyl lysophosphatidic acid promoted

GTP loading on to the small GTP binding protein Rho A,

and this was maintained over a substantially longer

time period than in vector-transfected cells [14]. This is

particularly interesting given recent information on the

G protein-coupling profile of GPR35 (see below). Although,

on phylogenetic trees of GPCR sequences expressed in

humans and rodents, GPR35 does not reside in the same

region as the lysophospholipid receptors, it is most closely

related to GPR23. This receptor has been reported to re-

spond to lysophosphatidic acid and has previously been

referred to as both the P2Y9 receptor and the lysophospha-

tidic acid LPA-4 receptor[18]. Furthermore, another rela-

tively closely related receptor is GPR55. GPR55 was

originally discussed in terms of being a potential atypical

cannabinoid receptor [19], but it is certainly able to respond

to lysophosphatidylinositol[20]. Lysophosphatidic acids or

other endogenously produced lipids might represent true

endogenous ligands for GPR35 and for other related recep-

tors such as GPR87 and GPR92[21].

Surrogate ligands for GPR35

Although identification of endogenously produced chemi-

cals with agonist action at GPR35 is of considerable im-

portance, the ligands described above are far from ideal toprobe the roles of GPR35. Surrogate ligands are therefore

required. Until recently, the key GPR35 agonist has been

zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one) (Table

1). Zaprinast was first identified as a GPR35 agonist by

Tanaguchi et al. [22]. Like kynurenic acid, zaprinast was

considerably more potent at rat than human GPR35, an

observation that has also subsequently been confirmed by

others[13,23](Figure 2). Importantly, however, zaprinast

is substantially better known as an inhibitor of cGMP

phosphodiesterases (PDEs), particularly PDE5 and

PDE6, for which it displays low micromolar potency. If

zaprinast is used as the probe, it could be difficult in many

[

MLSGSRAVPTPHRGSEELLKYMLHSPCVSLT

GPR35b: 31 aa insert at N-terminus

NH2

A25T 3.32

Y

R

3.36

T108M

R125S

COOHS294R

T253M

3.36

V29I V76M

TRENDS in Pharmacological Sciences

Figure 1. Important structural features of human GPR35. Two isoforms of human GPR35 differ by the presence (sequence bar) or absence of a 31 amino extracellular

N-terminal sequence. Non-synonymous polymorphic variations in sequence within the open reading frame are shown as red circles with the alternative amino acids

defined by their one letter code. The Ser294Arg (i.e. S294R) variation has been associated with the propensity to develop coronary artery disease [6]. Arginine (R) (position

3.36) and tyrosine (Y) (position 3.32) residues in transmembrane domain III that play an important role in ligand recognition and/or function are highlighted in yellow.

Review Trends in Pharmacological Sciences May 2011, Vol. 32, No. 5

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settings to disentangle the contribution of the elevation of

cGMP from that resulting from the activation of GPR35.

As a consequence, two groups screened the Prestwick

Chemical Library1 of 1120 small molecule marketed

drugs and drug-like molecules for ligands able to act as

agonists at human [12] orboth human and rat [23] GPR35.

Although both groups employed GPR35-b-arrestin 2 in-

teraction assays, the bases of these were distinct (Box 2).

Zhaoet al. [12]reported two hits from the primary screen:

the previously characterized ligand zaprinast and oxantel

pamoate. However, in follow-up studies pamoate

(4,40-methylenebis(3-hydroxy-2-naphthoic acid)), rather

than the supposed active ingredient oxantel (1-methyl-2-

(3-hydroxyphenylethenyl)-1,4,5,6-tetrahydropyrimidine),

was identified as the GPR35 active ligand, displaying an

EC50value of 80 nM [12]. By comparison, Jenkinset al. [23]

reported a wider range of hits at human GPR35 in

their primary screen. These included zaprinast but also

Table 1. Chemical structures of a range of GPR35 ligands

Structure Action Comments References

Zaprinast

[

Full a gonist Ke y surrogate li ga nd: potency at rat > human [12,13,22]

Kynurenic acid

[

Full agonist Potential endogenous agonist: potency at rat > human [8,13,34]

Pamoic acid/pamoate

[

Partial agonist Highest potency ligand at human. Low potency at rat [12,23]

Cromolyn

[

O O

O

HO

OH

O

OH

OO

OO

Full agonist Clinically used anti-asthma medication [23,33]

Dicumarol

[

O

O

OH

O

O

HO

High efficacy agonist Equipotent at human and rat [23]

Luteolin

[

OHO

OOH

OH

OH

Partial agonist at rat Limited activity at human [23]

CID2745687

[

O

N

F

F

O

N

N N

S

NHH

Antagonist Action only described at human [12]


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cromolyn (5,50-(2-hydroxypropane-1,3-diyl)bis(oxy)bis(4-

oxo-4H-chromene-2-carboxylic acid)) disodium, dicumarol

(3,30-methylenebis(4-hydroxy-2H-chromen-2-one)), niflu-

mic acid (2-{[3-(trifluoromethyl)phenyl]amino}nicotinic ac-

id) and, importantly, both oxantel pamoate and pyrvinium

(4-[(3-carboxy-2-hydroxynaphthalen-1-yl)methyl]-3-hydro-

xynaphthalene-2-carboxylic acid; 1-methyl-2-[(E)-2-thio-

phen-2-ylethenyl]-5,6-dihydro-4H-pyrimidine)) pamoate(Table 1).

The presence of the supposed inactive drug congener

pamoate in two separate primary screen hits alerted these

researchers to the possibility that this was the common

link in the responses and subsequent confirmation of pamo-

ate as an agonist at human GPR35 with an EC50 value

of 50 nM [23]. The recognition that supposedly inactive

components of a mixture might have activity at a distinct

targethasbeendiscussedfurtherinlightoftheseresults[24].

Importantly, previous studies by Jenkinset al. [13] that had

confirmed the species orthologue selectivity of zaprinast

encouraged the authors to repeat the primary screen using

rat GPR35 [23]. Althougha number of hits at human GPR35

were also identified at rat GPR35, two novel hits, the

closely related flavenoids luteolin (2-(3,4-dihydroxyphe-

nyl)-5,7-dihydroxy-4-chromenone) and quercetin (2-(3,4-

dihydroxyphenyl)- 3,5,7-trihydroxy-4H-chromen- 4-one),

were now also identified. Furthermore, neither oxantel

pamoate nor pyrvinium pamoate was identified when

screening against rat GPR35 [23]. This suggested thatpamoate might be significantly selective for human

GPR35, and when pamoate was assessed in parallel at

the two species orthologues, selectivity of at least 1000-fold

was observed. It was clear that pamoate was not an antago-

nist at the rat orthologue [23] because pamoate failed to

alter the potency of zaprinast to promote interactions be-

tween rat GPR35 and b-arrestin 2[23]. This is one of the

most notable examples to date of ligand selectivity at mam-

malian GPCR species orthologues.

Interestingly, certain other hits at GPR35 (such as

niflumic acid) also displayed substantial selectivity for

the human orthologue [23]. This was not universal; a

Box 1. Promiscuous and chimeric G proteins

Promiscuous G protein a subunits (e.g. Ga16in humans[9,10]and the

related rodent homologue Ga15, as well as a wide range of chimeric G

protein a subunits [9,10]) have been used widely in GPCR de-

orphanization studies and in ligand identification campaigns.

Although expressed endogenously in only limited sets of immune

cells, heterologous expression of Ga15and/or Ga16has been shown to

allow a wide range of GPCRs to elevate [Ca2+]i [10,39], an endpoint

favoured in many ligand screening campaigns, at least in part

because fluorescent kinetic plate readers and liquid-handling technol-ogy has allowed massive throughput[4042].

Although often described as promiscuous or universal G proteins

[9,10], Ga15and Ga16do not interact with all GPCRs, including GPR35

[6]. Thedesire to develop robustand generic assays that employ GPCR-

induced elevation of intracellular [Ca2+] as the end point has, therefore,

resulted in the widespread use of chimeric G protein a subunits (in

which the extreme C-terminal region of non-Gq-family G proteins is

used to replace the equivalent region of Gaq, to produce Ga subunits

that couple different GPCR groups to this end point). This reflects the

fact that the extreme C-terminal region plays a crucial role in

determining which GPCRs interact productively with a G protein a

subunit, whereas the downstream signalling mechanisms are regu-

lated by more internal sections of the G protein a subunit sequence.

Usually, such chimeras involve replacement of between five and nine

amino acids from the C-terminal tail. Mixtures of such chimeric

constructs are often transfected in combinations in de-orphanization

studies because, inherently, little or nothing is known in advance about

the G protein preference of the GPCR being studied. This was the

strategy employed in the first study to identify kynurenic acid as anagonist at GPR35[6]. This basic concept hasbeen adapted to produce a

family of Ga16-Gaxchimeras to attempt to extendthe utility of Ga16 [42]

and a family of Gas-Gax chimeras[43]to switch signal output to the

elevation of cAMP levels. A further variation on the same basic concept

that has been used to screen for ligands at GPR35 reflects the limited

repertoire of G protein-coupled signals in the yeast Saccharomyces

cerevisiae [44]. Following replacement of the yeast GPCR Ste2 with

human GPR35 and of the yeast G protein a subunit Gpa1 with a Gpa1-

Ga13 chimera, GPR35 agonists promoted yeast cell growth and b-

galactosidase activity via an appropriate gene reporter construct[23].

Box 2. b-Arrestin-based ligand identification

If occupied by agonist for a significant period of time, the vast majority

of GPCRs are able to interact effectively with a b-arrestin [45,46].Although believed initially only to provide a means to terminate G

protein signalling by preventing access of the GPCR to G protein,

interactions between GPCRs and b-arrestins are now believed to also

trigger alternative signal pathways[45,46]. Regardless of the relevance

of this, because such interactions occur in an agonist-dependent

manner, they have been recognized to offer a means to identify ligands

that occupy GPCRs in assays that are independent of G protein-

coupling preference [47]. In early studies, such assays invariably

employed visual detection of the cellular translocation of a b-arrestin

tagged with an autofluorescent protein, usually green fluorescent

protein. Although effective, such studies required the parallel develop-

ment of highcontent imaging hardwareand software [48,49] to provide

reasonable throughput and robust pharmacology. This approach was

used by Zhao et al. [12] to identifypamoateas a high potency agonist at

human GPR35. The need for high content screening equipment led to

the development of other assays that detect interactions betweenGPCRs and b-arrestins. The most commonly employed are based

on either enzyme complementation [50] or BRET [51]. Both b-

galactosidase complementation [13] and BRET [13,23] based GPR35-

b-arrestin 2 interaction assays have been used to either confirm the

activity of previously reported GPR35 agonists [13] or identify novel

GPR35 ligandsfrom small scale chemical libraries [23]. The BRET-basedGPR35-b-arrestin 2 assay was reported to have a high signal to

background ratio and excellent screening statistics for both human

and rat orthologues of GPR35[23]and to identify agonists with varying

efficacy [23]. A feature ofb-arrestin-based assays of particular use in

screens at orphan or poorly characterized GPCRs is that, at least

theoretically, the potency of the ligands in such assays is anticipated to

provide a good measure of ligand affinity, because there should be a

direct correlation between GPCR occupancy and potency. This can

generate a structureactivity relation profile to underpin medical

chemistry in programmes targetingthe development of agonist ligands

without the need for direct affinity measurements. These would

normally be provided via ligand binding assays but such studies might

be impractical if the ligand series is of low potency/affinity. For a

number of GPCRs a range of ligands has been shown to promote

receptor-b-arrestin interaction but has not been observed to activate G

protein-dependent signalling pathways. Such ligand bias or functionalselectivity [52,53]could have therapeutic implications but also implies

that ligands detected in such assays must be re-examined in more

conventional G protein-dependent assays to fully appreciate their

capacity for signal regulation.


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number of compounds were essentially equipotent at these

two orthologues (including the anti-asthma medication

cromolyn disodium and the vitamin K antagonist dicuma-

rol) [23]. However, like zaprinast, luteolin displayed sig-

nificant selectivity for the rat receptor[23]. A further feature

of the various ligands was that they were partial agonists

whencompared to zaprinastas the reference compound[23].

For example, increasing concentrations of pamoate reduced

human GPR35-b-arrestin 2 interactions produced by maxi-

mally effective concentrations of zaprinast[23], while the

partial agonist nature of luteolin and quercetin at rat

GPR35 wasalso demonstrated[23]. Thepotential selectivity

of these compounds for GPR35 over other related GPCRs

and potential therapeutic targets remain to be determined.

This is an important issue that needs to be explored before

roles for GPR35 are defined based onex vivo or in vivo use of

such ligands. This is also the case for a series of thiazolidi-

nedione ligands originally described as GPR35 agonists in a

patent from Arena Pharmaceuticals[25]and confirmed assuch by Jenkins and colleagues [13]. Currently the only

described antagonists ofGPR35arebasedon methyl- 5-

[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-

difluorophenyl)pyrazole-4-carboxylate; that is, CID2745687

[12] (Table 1), an nM inhibitor of the human orthologue. Use

of this ligand and identification of further antagonists from

distinct chemical series will probably be central in defining

the key functions of GPR35.

The mode of binding of ligands to GPR35

As noted above, although kynurenic acid is an agonist at

GPR35, this is true for neither kynurenine [8] nor

kynurenic acid ethyl ester [13]. This implicates a key

role for the carboxylate group in binding and/or activa-

tion of GPR35. Importantly, in studies of the L-lactate

receptor GPR81 [26], a number of receptors related to

GPR35 (and which have acidic ligands) were noted to

have a conserved arginine in transmembrane domain III.

This is at position 3.36 in the nomenclature of Ballesteros

and Weinstein[27](in which the most conserved residue

in transmembrane domain X is designated X.50, whereas

the amino acid X.49 is one residue closer to the N

terminus and X.51 is one closer to the C terminus). This

residue was predicted to provide an ionic interaction with

the carboxylate[26]. Following alteration of this residue

to alanine, neither rat nor human GPR35 responded to

kynurenic acid[13](Figure 3). Furthermore, the agonist

action of zaprinast at each orthologue was also eliminat-

ed by this mutation [13] (Figure 3). Although lacking a

formal negative charge, zaprinast does contain an acid

bioisostere (a group with similar physical or chemicalproperties that provides functional characteristics broad-

ly similar to a chemical compound). Furthermore, alter-

ation of the tyrosine residue to alanine at position 3.32,

which is predicted to be on the same face of transmem-

brane domain III but one turn of the helix further to-

wards the extracellular face of the receptor, also

eliminated responses to both kynurenic acid and zapri-

nast[13]. Although very preliminary, these studies have

begun to identify key residues of the binding pocket of

GPR35, and this will be investigated further by muta-

genesis and analysis of the effects of a wider range of

ligands at such mutants.

[

arr2

+ coelentrazine h

BRET

YFP

GPR35

Rluc

arr2GPR35

Rluc

Rluc

GPR35

YFParr2

+ agonist

YFP

-11 -10 -9 -8 -7 -6 -5 -4 -3

-50

0

50

100

150

200

250

Rat

Human

Log [zaprinast]M

NETBRET(m

BRET)

< 80A

Key:


Figure 2. Developing a BRET-based GPR35-b-arrestin 2 interaction assay. Representation of the GPR35-b-arrestin 2 interaction assay.(a)GPR35 tagged at the C-terminal tail

with enhanced yellow fluorescent protein (YFP) is cotransfected into cells along with a Renilla luciferase (RLuc) tagged form ofb-arrestin 2. Following addition of a GPR35

agonist (black triangle) GPR35-YFP interacts with b-arrestin 2-RLuc. With addition of the luciferase substrate coelentrazine-h, light emitted upon substrate oxidation by the

luciferase is transferred to YFP and subsequently re-emitted at a longer wavelength if GPR35 and b-arrestin 2 have brought YFP and RLuc within a BRET-compliant distance

(


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G protein-coupling profile of GPR35

Although some of the earliest studies of GPR35 detected

ligand activation via transfection of a mixture of chimeric

and promiscuous G proteins [8,22], they noted selective

interaction of GPR35 with chimeric G proteins containing

the receptor recognition regions of Gao and Gai [8]. By

contrast the promiscuous G protein Ga16 (Box 1) did not

appear to couple to GPR35[8]. Standard [35S]GTPgS bind-

ing studies are most suited to detect activation of Gi-family

G proteins [28,29]. Prevention of stimulation of

[35S]GTPgS binding by kynurenic acid in membranes

of CHO cells expressing human GPR35, by prior treatment

of the cells with pertussis toxin [8], was consistent with

this. Furthermore, following heterologous introduction of

the human isoforms of GPR35 into rat sympathetic neu-

rons, the ability of both kynurenic acid and zaprinast to

inhibit N-type calcium channels was blocked by prior

treatment with pertussis toxin [5]. The ability of endoge-

nously expressed GPR35 to inhibit forskolin-stimulated

cAMP levels in rat dorsal root ganglion was also ablated

by pertussis toxin pretreatment[11]. Despite these obser-

vations, Jenkinset al.[13]reported that, following expres-

sion in HEK293 cells, human GPR35 generated only very

modest increases in binding of [35S]GTPgS in response to

kynurenic acid; therefore, they explored possible interac-

tions with other G proteins. Although they were unable to

record elevation of [Ca2+]i in cells cotransfected with Gaqand either human or rat GPR35, the presence of a Gaq-

Ga13chimera generated robust [Ca2+]iresponses to zapri-

nast via both orthologues, whereas equivalent experiments

with a Gaq-Ga12chimera did not[13]. Use of an antibody

able to identify only the GTP-bound, active state of Ga13provided further support for interaction with this G protein

[13]. Subsequent development of an immunocapture assay

using an epitope-tagged form of Ga13 confirmed ligand

stimulation of [35S]GTPgS binding to this G protein [23]

(Figure 4). The ability of GPR35 expressed in HEK293 cells

to promote binding of GTP to Rho A[14]is also consistent

with a role for Ga13because activation of Ga13is generally

upstream of this effect[30]. By contrast, pertussis toxin-

mediated inhibition of interleukin 4 release from alpha-

galactosylceramide-activated human invariant natural

killer T cells via GPR35 [31], and of ERK activation in

U2OS cells expressing GPR35[12], both support a role for

Gi-family G proteins (as does the capacity of both kynure-

nic acid and zaprinast to reduce forskolin-elevated cAMP

levels in cultured mouse glial cells[32]). It appears, there-

fore, that GPR35 can couple to both Ga13 and pertussis

toxin-sensitive Gi-family G proteins. It will be instructive

to determine whether there is ligand bias (Box 2) between

these pathways or predominance of one over another in

different cells and tissues, because such effects might

generate distinct signals from GPR35 in different cell

types. Although interactions between GPR35 and

b-arrestin-2 have been employed to develop assays to iden-

tify novel GPR35ligands, and presumably occur in cellsthat

express GPR35 endogenously, their possible role in gener-

ating G protein-independent signals also remains to be

investigated.

Expression profile of GPR35

As noted above, initial studies indicated expression of

GPR35 in rat intestine [1] and stomach [2]. Subsequent

studies have confirmed significant expression levels in the

small intestine, colon and stomach, and this might be

relevant in the association between a GPR35 polymorphicvariant and early-onset inflammatory bowel disease [7].

GPR35 is also expressed in a range of other rat tissues

including lung, uterus, dorsal root ganglion and spinal cord

[22,32]. Wanget al. [8] were the first to record expression in

the spleen and white cells in both humans and mice,

whereas Yang et al. [33] have demonstrated that GPR35

is expressed in human mast cells, basophils and eosino-

phils, and that GPR35 mRNA is upregulated upon chal-

lenge with IgE antibodies. Furthermore, Barthet al. [34]

have suggested that GPR35 is highly expressed by human

peripheral monocytes, and messenger RNA encoding

GPR35 is upregulated substantially in primary human

[

-8 -7 -6 -5 -4 -3

-50

0

50

100

150200

250

300

350

rGPR35 WT

hGPR35 WT

hGPR35 R(3.36)A

rGPR35 R(3.36)A

Log [kynurenic acid] M

NETBRET(m

BRET)

-11 -10 -9 -8 -7 -6 -5 -4 -3

-50

0

50

100

150

200

250

300

350

400

rGPR35 WT

hGPR35 WT

hGPR35 R(3.36)A

rGPR35 R(3.36)A

Log [zaprinast] M

NETBRET(m

BRET)

(a) (b)OH

HN

CO2HN N

N

N

O

O

HN

Key:

Key:


Figure 3. The role of arginine 3.36 in orthologues of GPR35. YFP tagged forms of wild-type (filled symbols) and Arg3.36Ala (open symbols) human (red) or rat (blue) GPR35

were cotransfected with b-arrestin 2-RLuc into HEK293 cells. BRET measurements as in Figure 1were then performed in the presence of kynurenic acid (a) or zaprinast(b).

The chemical structure of these ligands is shown. Data are adapted with permission from [13].


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macrophages exposed to benzo(a)pyrene [35]. The func-

tional significance of this has not yet been reported.

Physiological roles of GPR35 and potential therapeutic

opportunities

The expression of GPR35 in pancreatic b-cells, coupled

with the ability of thiazolidinedione ligands that have

agonist action at GPR35 to enhance glucose-stimulated

insulin release in model cell lines and to improve oral

glucose tolerance tests [25], has suggested a potential

use for agonists of GPR35 in the treatment of diabetes

and related metabolic disorders (Table 2). Expression of

GPR35 in the intestine and colon might also be relevant

because other GPCRs expressed on b-cells that regulate

insulin secretion, such as FFA1 (also designated GPR40),

are also expressed on enteroendocrine cells and regulate

the secretion of incretins such as GLP-1 [36], hence pro-

viding dual pathways of control. This is also true of the free

fatty acid receptor GPR120 [37], and could be a general

property of GPCRs that link nutrient sensing and energy

homeostasis. Although the specific distribution of GPR35

within the gut remains unclear, this is important to un-

derstand. Further studies using GPR35 agonists unrelated

to the thiazolidinediones will also be vital to better define

the contribution of GPR35 to the control of insulin

secretion.

GPR35 has also been implicated recently in the control of

blood pressure. GPR35 was identified in an effort to explore

genes associated with heart failure [38], but this was a

poorly powered study involving only 12 patients with a

variety of underlying conditions and marked differences

in disease severity. More interestingly and convincingly,

the blood pressure of mice lacking expression of GPR35 was

reported to be elevated by 37.5 mmHg compared with wild-

type littermates [38]. GPR35 agonists might therefore be

anticipated to lower blood pressure. Substantial numbers of

patientshave poorly controlled hypertension despite the use

of combinations of current front-line therapies, so new

therapeutictargetsare needed for thispopulation.Although

evidence of involvement of GPR35 is preliminary, this re-

ceptor is clearly worthy of study in this area.

The identification of both cromolyn disodium [23,33]

and nedocromil sodium[33] as GPR35 agonists (Table 1)

is of particular clinical interest. Both of these drugs are

approved anti-asthma medications and regulators of mast

cell sensitization and histamine release. However, they are

[

180

160

140

120

100

80

Vehi

cle

Zaprin

ast

Vehi

cle

Zaprin

ast

Pamoa

te

L D K L G E P D Y I P S Q Q D I L L A R

L D K L G E P E Y M P T E Q D I L L A R

%o

fBa

ckground[35S]GTPS

***

***

pcDNA

G13(EE)

pcDN

A

G13(E

E)

G13

G13 (EE) (181-199)

(a) (b)Key:


Figure 4. Activation of Ga13by human GPR35. HEK293 cells were transfected to express human GPR35 with or without a form of G a13containing a modified sequence to

incorporate the so called Glu-Glu (EE) epitope tag. (a) Membrane preparations from these cells were resolved by SDSPAGE and immunoblotted to detect Ga13(EE). The

sequence of both wild -type Ga13and the modified EE form of Ga13is shown for amino acids 181199.(b) Membranes were then used in a [35S]GTPgS binding assay[29]

into which was added vehicle, zaprinast or pamoate. Ga13(EE) was subsequently immunoprecipitated with anti-EE and, after washing, binding of [35S]GTPgS assessed. Data

are adapted with permission from [23].

Table 2. Therapeutic potential for GPR35 ligands

Disease indication Supporting evidence References

Diabetes Thiazolidinediones with agonist action at GPR35 promote glucose-dependent

insulin secretion Such ligands also improve glucose handling

[25]

Hypertension GPR35 knockout mice have markedly elevated blood pressure [38]

Coronary artery disease Association with Ser294Arg polymorphism [6]

Asthma Anti-asthma medications cromolyn disodium and nedocromil sodium are

agonists of GPR35

[23,33]

Pain Expression of GPR35 in mouse dorsal root ganglion and spinal cord Effects

of agonist ligands in acetic acid-induced writhing models

[12,32]

Early-onset inflammatory

bowel disease

Genetic linkage to a 5 0 untranslated single nucleotide polymorphism of GPR35 [7]


323


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considered orphan drugs because their mode of action has

been unclear. The fact that they can be shown to have

agonist action at GPR35 in cells transfected to express this

receptor does not mean that their mechanism of actionin

vivo has been defined. However, as noted above, various

white blood cells, including mast cells, do express GPR35

and the growing availability of both agonist and antagonist

ligands will allow the contribution of GPR35 to the thera-

peutic actions of cromolyn disodium and nedocromil sodi-um to be more fully assessed.

Concluding remarks

Several orphan GPCRs have expression profiles that indi-

cate they are worthy of consideration as therapeutic tar-

gets. This view can be supported via various transgenic

techniques, and it would be interesting to have wide-

ranging phenotypic information on GPR35 knockout mice.

Based on the number of GPR35 active compounds identi-

fied recently in very small-scale screens[12,23,33], there is

reason to hope that more extensive screens, along with

follow-up medical chemistry programmes, will rapidly in-

crease the pharmacological uses of this receptor. Such toolswill allow investigations of the function of GPR35. The

disease areas highlighted in this review (Table 2) are all

active topics for research with unmet clinical need. This is

likely to result in rapid progress in efforts to validate

GPR35 as a therapeutic target. Although only approxi-

mately 20 publications directly address the expression,

pharmacology and function of this receptor, this number

is likely to increase as pharmacological tools that modulate

the activity of GPR35 become widely available and the

potential of GPR35 as a therapeutic target becomes better

appreciated.

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