From the Departments of a 7TMR Systems Research, n Target Bioinformatics Europe, d Gene Expression and Protein Biochemistry, k Cellular Genomics, and h Cardiovascular and Urinary Centre of Excellence for Drug Discovery, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom, the Departments of c Genomic Histology, e Metabolic and Viral Disease Center of Excellence for Drug Discovery, and i Cellular Genomics, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, the j Department of Cellular Genomics, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, and the l Department of Safety Assessment, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire AL6 9AR, United Kingdom
Received for publication, November 14, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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GPR41 and GPR43 are related members of a
homologous family of orphan G protein-coupled receptors that are
tandemly encoded at a single chromosomal locus in both humans and mice.
We identified the acetate anion as an agonist of human GPR43
during routine ligand bank screening in yeast. This activity was
confirmed after transient transfection of GPR43 into
mammalian cells using Ca2+ mobilization and
[35S]guanosine 5'-O-(3-thiotriphosphate)
binding assays and by coexpression with GIRK G protein-regulated
potassium channels in Xenopus laevis oocytes.
Other short chain carboxylic acid anions such as formate, propionate,
butyrate, and pentanoate also had agonist activity. GPR41 is related to
GPR43 (52% similarity; 43% identity) and was activated by similar
ligands but with differing specificity for carbon chain length, with
pentanoate being the most potent agonist. A third family member,
GPR42, is most likely a recent gene duplication of
GPR41 and may be a pseudogene. GPR41 was expressed
primarily in adipose tissue, whereas the highest levels of GPR43 were
found in immune cells. The identity of the cognate physiological
ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain
pathophysiological conditions.
Within family A of the G protein-coupled receptor
(GPCR)1 gene superfamily
(also classified as family 1), there is a phylogenetically related
group of ~90 receptors that respond to an unusually wide variety of
ligand types, considering the relatively close similarity of their
primary sequences (1). The group includes receptors that respond to
purinergic or pyrimidinergic nucleotides (P2Y1, P2Y2, P2Y4, P2Y6,
P2Y11, P2Y12, and P2Y13), modified
nucleotides (UDP-glucose), lipids (platelet-activating factor
receptor), leukotrienes (BLT1 and BLT2
and CysLT1 and CysLT2), proteases
(protease-activated receptor-1-4), chemoattractants (FPR1), and
chemokines. To date, these receptors have no clear homologs in
invertebrates, unlike the monoamine or neuropeptide receptors,
suggesting a relatively recent evolutionary origin (2, 3). At least 50 GPCRs whose cognate ligands are unknown (orphans) (4) are categorized
within this group on the basis of sequence homology. Often, these
orphans fall into subsets, being more related to each other than to
receptors with known ligands; and this, combined with the ligand
diversity noted above, makes it difficult to predict the chemical
nature of their ligands. One subset comprises GPR40-43, which were
identified as tandemly encoded genes present on cosmids isolated from
human chromosomal locus 19q13.1 (5). GPR42 differs from GPR41 at only
six amino acid positions; otherwise, the four members of this subfamily
share ~30% minimum identity. BLAST searches have identified the next
most closely related receptors as the protease-activated receptors.
However, the long N-terminal extracellular domains that serve as
protease substrates and that are characteristic of protease-activated
receptors are absent in the GPR40-43 group, suggesting that they are
not activated by a similar mechanism.
By adopting a ligand fishing strategy (4) following heterologous
expression of orphan GPCRs in yeast, we found that short chain
carboxylic acid anions can activate GPR41 and GPR43 in a dose-dependent and -specific manner. These molecules
represent a novel chemical class of GPCR ligand. The known
pharmacological and pathological effects of these ligands and the
tissue distribution of GPR43 suggest potential roles in immune cell
function and hematopoiesis. The relevance of GPR41 is less
clear. Furthermore, our data support the hypothesis (5) that
GPR42 arose as a tandem duplication of GPR41 in
the human lineage and has acquired mutations since duplication that
abolish its ability to respond to carboxylate ions. Ligands for GPR40
are described in the accompanying article (6).
Materials--
Carboxylates were obtained from Sigma as sodium
salts or free acids and were prepared as 100 mM or 1 M stock solutions of the sodium salt and adjusted to pH
7.0. Peptides for antibody generation were obtained from Severn Biotech Ltd.
Yeast Strain Construction and Reporter Gene Assay--
Yeast
cells were derived from the dual-reporter gene strain MMY11
(MATa fus1::FUS1-HIS3
LEU2::FUS1-lacZ far1 Oocyte Methods--
Xenopus laevis oocyte
isolation and microinjection were performed as described previously
(12). Human GPR43 cRNA was produced using T7 polymerase
(Promega) from linearized plasmid pJG3.6-hGPR43 and co-injected with
GIRK1 and GIRK4 cRNAs into single oocytes in stage V or VI. Potassium current amplitude was measured using dual
microelectrode voltage clamp electrophysiometry (12).
Cell Biology--
Mammalian cells were maintained in Dulbecco's
modified Eagle's medium (pH 7.4) containing 10% fetal calf serum and
2 mM glutamine, except where otherwise indicated.
HEK293T cells were grown to 60-80% confluency in 90-mm dishes,
transfected with cDNA (total DNA = 9 µg) using LipofectAMINE
reagent (30 µl; Invitrogen), and collected 48 h after
transfection. Immunoblotting was performed by standard methods (13).
[35S]GTP Antibody Generation--
The antigen peptide
CEQKGGEEQRADRPAERKTSEHSQGC (SB130) corresponds to an internal sequence
shared by human GPR41 (hGPR41) and hGPR42 (amino acids 311-335) in the
predicted C-terminal tail (5). SB130 was conjugated to the carrier
protein tuberculin PPD via the cysteine residues and used to immunize
female New Zealand White rabbits. The immune response was monitored by
enzyme-linked immunosorbent assay using plates coated with the free
peptide, and immunoglobulins from high titer sera were purified using
immobilized peptide (Sulfolink, Perbio). Purified antibodies
were dissolved in phosphate-buffered saline containing 0.2% bovine
serum albumin.
Gene Expression--
Primers and probes for TaqMan analysis (PE
Applied Biosystems) were designed using Primer Express software (PE
Applied Biosystems) and compared with public databases by BLAST
searches to confirm specificity (Table
I). RNA concentrations were determined
using RiboGreen (Molecular Probes, Inc.), and PCRs were performed in an
ABI PRISM 7700 sequence detection system (PE Applied Biosystems). For
the human tissue samples shown in Fig. 5A,
poly(A)+ RNA (1 µg) from 20 tissues of four different
individuals (two males and two females except prostate) was prepared,
reverse-transcribed using Superscript II and random 9-mer priming
(Invitrogen), and diluted and plated using Biomek robotics (Beckman
Coulter, High Wycombe, UK) to produce 1000 replica plates with
each well containing the cDNA from 1 ng of RNA. TaqMan quantitative
PCR analysis of each sample was conducted as described previously (16).
For the immune cell samples shown in Fig. 5B, total RNA was
extracted from samples derived from up to three individuals using the
RNeasy maxiprep procedure (QIAGEN Inc.). Samples were treated with
DNase I (Ambion Inc.) and normalized to levels of 18 S ribosomal RNA. Reactions were run in 50 µl containing 5.5 mM
MgCl2, 1× TaqMan buffer A (PE Applied Biosystems), 300 µM dNTP mixture, 20 units of RNase inhibitor, 12.5 units
of murine leukemia virus reverse transcriptase, 900 nM each
primer, 200 nM probe, 1.25 units of Amplitaq Gold (PE
Applied Biosystems), and 50 ng of total RNA. Cycling conditions were as
follows: 30 min at 48 °C, 10 min at 95 °C, and 40 cycles of
15 s at 94 °C, followed by 1 min at 60 °C.
Immunohistochemistry--
Formalin-fixed, paraffin-embedded
human tissue sections were deparaffinized and hydrated through graded
alcohols. Immunohistochemical staining was performed using a Ventana
Nexus automated stainer with Ventana reagents (Ventana Medical Systems,
Inc., Tucson, AZ). Antigen retrieval was carried out using protease-1
for 32 min at 37 °C. Rabbit anti-hGPR41 immunoglobulins were applied at 0.2 mg/ml for 32 min at 37 °C, and horseradish
peroxidase-conjugated goat anti-rabbit secondary IgG (Ventana Medical
Systems, Inc.) was used according to the manufacturer's instructions.
An Enhanced diaminobenzidine kit was used for detection, and
sections were counterstained with hematoxylin.
The yeast Saccharomyces cerevisiae has been established
as a useful host for heterologous expression and functional
analysis of GPCRs (17). The system is engineered to allow mammalian
GPCRs to couple to the endogenous yeast signal transduction pathway that responds to mating pheromone in wild-type cells. The yeast pheromone receptor Ste2p is deleted from the strains commonly used to
provide a null background lacking endogenous host receptors. Another
advantage, particularly in the analysis of orphan GPCRs for which no
ligand is known, is that ligand-independent G protein activation
("constitutive activity") can frequently be detected, thus
confirming functional expression of the receptor (7, 18). We used a set
of plasmid constructs encoding orphan GPCRs under the transcriptional
control of strong yeast promoters. These were introduced into yeast
containing dual reporter genes and a chimeric G
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sst2
ste2
gpa1::ADE2 his3 ura3 trp1) (7). To introduce
G
subunits into this strain, expression cassettes comprising the
GPA1 promoter, G
coding sequence, and the ADH1
terminator (8) were subcloned into pRS304 and integrated into the
trp1 locus of MMY11. Integrants were selected to have low
basal levels of reporter gene activity. This produced an isogenic panel
of yeast strains containing a range of functional G
subunits as
follows: MMY12, wild-type Gpa1p; MMY14, Gpa1p/G
q; MMY15,
Gpa1p/G
s; MMY16 Gpa1p/G
16; MMY19,
Gpa1p/G
12; MMY20, Gpa1p/G
13; MMY21,
Gpa1p/G
14; MMY22, Gpa1p/G
o; MMY23, Gpa1p/G
i1; and MMY24, Gpa1p/G
i3. MMY16
and MMY23 have been described previously (7, 9). Mammalian GPCRs were
expressed using the p426GPD vector (10).
-Galactosidase activity was
measured in an in vivo bioassay during growth in liquid
culture using the chromogenic substrate
chlorophenolred-
-D-galactopyranoside (7). A library of
80 known or candidate GPCR agonists, similar to those described
previously (11), was screened at final concentrations ranging from 0.2 µM to 2.5 mM.
S binding assays were performed on plasma
membrane-containing P2 particulate fractions at room temperature in
96-well format as described previously (14). Bound
[35S]GTP
S was determined by scintillation counting.
Maintenance, transfection, and fluorometric imaging plate reader
(FLIPR) (Molecular Devices) assay of internal Ca2+
concentration in HEK293 cells were performed as described previously (15). Pertussis toxin (PTX) treatment was carried out by
supplementation of the medium at 50 ng/ml for 18 h. Swiss 3T3-L1
and 3T3-L442A fibroblasts (American Type Culture Collection) were grown
in medium supplemented with penicillin or streptomycin (100 units/ml or 100 µg/ml, respectively). Cells were differentiated 2 days after confluence by supplementation of the culture medium with
3-isobutyl-1-methylxanthine (500 mM), dexamethasone (0.25 µM), insulin (0.8 µM), and Myoclone for 2 days, followed by insulin (0.8 µM) and Myoclone
only for an additional 2 days. RNA was extracted (RNeasy, QIAGEN Inc.) from differentiated cells within eight passages from original stocks.
Oligonucleotides used in TaqMan experiments
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit in which the
five C-terminal amino acids of mammalian G
i3 were fused
to the remainder of the yeast G
protein Gpa1p (8). In the absence of
any added ligand, the orphan hGPR43 caused significant activation of
the FUS1-lacZ reporter gene, which is under the control of
the pheromone response pathway in these cells, indicating that hGPR43
was functionally expressed (Fig. 1,
A and B).
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Fig. 1.
Activation of the yeast pheromone response
pathway by hGPR43. A, a yeast strain containing the
Gpa1p/G i3 chimera and the pheromone pathway-responsive
FUS1-lacZ reporter gene was transformed with a hGPR43
expression construct.
-Galactosidase activity was assessed in a
chromogenic substrate bioassay buffered to pH 7.0. Introduction of
hGPR43 into these cells caused significant reporter gene activation in
the absence of added acetate (No acetate). Exposure to
sodium acetate further activated hGPR43 in a
concentration-dependent manner (
), whereas no activation
was detected in control cells expressing the human somatostatin SST2
receptor (
). B, the G protein coupling specificity of
hGPR43 was determined in a panel of yeast strains expressing the yeast
wild-type G
subunit Gpa1p or a range of different Gpa1p/G
chimeras. Chimeras contained the five C-terminal amino acids of the
indicated mammalian G
subunits fused to the remainder of Gpa1p.
FUS1-lacZ reporter gene induction due to the constitutive
activity of hGPR43 was determined in acetate-free assay medium
(white bars), and agonist-dependent activity was
determined in the presence of 150 µM acetate (black
bars). Results from single representative experiments are
presented. Data shown are the means ± S.D. of four independent
transformants.
Yeast cells expressing hGPR43 were then used to screen a collection of
known GPCR ligands plus compounds with demonstrated biological
activity, but no known mechanism of action. Several compounds, all
formulated as salts containing acetate counterions (19), activated
hGPR43. Further experiments using sodium acetate or ammonium acetate
buffered to pH 7.0 indicated that the acetate anion alone was
sufficient to cause concentration-dependent reporter gene
activation over and above that attributable to the constitutive activity of hGPR43. The median effective concentration
(EC50) of this response was 137 ± 24 µM. Control cells expressing the human somatostatin SST2
receptor (8) did not respond to acetate (Fig. 1A). To
investigate the G protein specificity of this response, hGPR43 was
introduced into a series of similar yeast strains containing different
yeast/mammalian G chimeras (8). Elevated reporter levels in the
absence of acetate (constitutive activity) and induction in the
presence of acetate indicated the activation of the particular chimera.
hGPR43 could activate Gpa1p/G
chimeras containing the C termini of
mammalian G
12, G
13, G
14,
G
i1, and G
i3 (Fig. 1B).
Because the G protein specificity for chimeras in yeast generally conforms to that observed in mammalian cells (8), this suggests that
GPR43 may activate the Gi, Gq, and
G12 families of G proteins.
We next determined whether acetate could activate hGPR43 expressed in
mammalian cells. HEK293 cells were transiently transfected with
hGPR43 and loaded with the calcium-sensitive fluorescent dye
Fluo-4. Intracellular calcium ion concentrations
([Ca2+]i) were measured using the FLIPR. Acetate
provoked transient increases in [Ca2+]i
(EC50 = 52 ± 10 µM). Control cells,
transiently transfected with hGPR40 or the µ-opioid
receptor, showed no change in [Ca2+]i in response
to acetate (Fig. 2A). To
determine which class of G protein was involved in this response, we
used the specific inhibitor of Gi/o family proteins, PTX.
Treatment with PTX had no significant effect on either the magnitude or
EC50 of the [Ca2+]i response to
acetate in HEK293 cells expressing GPR43 compared with vehicle-treated
cells. In contrast, PTX treatment completely abolished the
[Ca2+]i response of control cells stably
expressing the APJ receptor (20) to apelin-13 (Fig.
2B). In a separate experiment, we introduced
hGPR43 into HEK293T cells by cotransfection in combination with the Gi/o family protein Go1. Acetate
provoked dose-dependent increases in
[35S]GTP
S binding in membranes prepared
from these cells (Fig. 3). Acetate
responses mediated by hGPR43 could also be detected in X. laevis oocytes upon coexpression with GIRK G protein-regulated potassium channels. In vitro synthesized RNA encoding hGPR43
and the inwardly rectifying potassium channel subunits GIRK1 and GIRK4 (Kir3.1/Kir3.4) were co-microinjected into oocytes, and
transmembrane conductance was measured in the presence of a buffer
containing a high concentration (90 mM) of potassium ions.
Application of acetate resulted in inward shifts in holding current
(EC50 = 202 ± 68 µM) (Fig.
2C). No such shifts occurred in control oocytes injected
with GIRK1/GIRK4 alone (n = 5) (data not shown).
Together, these results confirm the original observation in yeast that
hGPR43 is activated by acetate.
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Next, we determined whether other carboxylate anions could also
activate hGPR43. The C3 carboxylate propionate stimulated [35S]GTPS binding in HEK293T cell membranes containing
hGPR43 and G
o1 with a potency similar to that of
acetate. Longer chain acid anions were also active. Relative potencies
were as follows: acetate (C2) = propionate
(C3) = butyrate (C4) > pentanoate
(C5) > hexanoate (C6) = formate (C1) (Fig. 3A and Table
II). A similar rank order of potency for
these ligands was found using the FLIPR assay (Table II). Another
member of the GPR40-43 receptor subfamily, GPR41, is closely related
to GPR43 (43% amino acid identity in human) and is encoded adjacent to
GPR43 at human chromosomal locus 19p13.1 (5).
hGPR41 was cotransfected into HEK293T cells with
G
o1, and ligand-stimulated [35S]GTP
S
binding was assessed in membranes prepared from these cells. As
expected from the close sequence similarity, hGPR41 was activated by
the same carboxylate ligands, although with a different rank order of
potency: propionate = pentanoate = butyrate > acetate > formate (Fig. 3B). Thus, hGPR41 and hGPR43
are both activated by carboxylate anions, but have differing
specificities for carbon chain length, with pentanoate (C5)
activating hGPR41 more potently than acetate, but with acetate
activating hGPR43 more potently than pentanoate. Formate and several of
the longer chain carboxylates were lower efficacy agonists of both
receptors relative to propionate. A range of saturated and unsaturated
fatty acids containing nine or more carbon atoms were also tested, but no activation was observed for either GPR41 or GPR43 (data not shown).
A third member of this family, GPR40, is more distantly related to
GPR41 and GPR43 (33% amino acid identity between GPR40 and GPR43 in
human). GPR40 is activated by carboxylate anions of longer chain length
(i.e. fatty acids), but does not appear to respond to
carboxylate anions of six carbons or less (6).
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hGPR42 is also located at 19p13.1 and differs from hGPR41 by only six amino acids (5). We performed similarity searches of public sequence databases to find mammalian orthologs of this pair. Two partially sequenced bacterial artificial chromosomes (GenBankTM/EBI accession numbers AC079472 and AC087143) from the mouse locus that is syntenic with human 19p13.1 contained GPR40-43 orthologs. In mouse, only one ortholog of the hGPR41/hGPR42 pair was detected (72% amino acid similarity to hGPR41). From the mouse sequence, we designed oligonucleotide primers and amplified a rat hGPR41/hGPR42 ortholog (accession number AX224758). We also identified a Bos taurus ortholog from overlapping expressed sequence tags (accession numbers AW632495, AW660795, BE755048, and BI541693). The amino acid sequences of the orthologs were intermediate between hGPR41 and hGPR42, but more similar to hGPR41. Four of the six amino acid positions that differ between hGPR41 and hGPR42 are also conserved among the orthologs. The orthologs match hGPR41 at two or three of these positions, but match hGPR42 at only one (Table III). This suggests that hGPR42 occurred as the result of a gene duplication of hGPR41 that occurred after the human lineage diverged from the rodent and bovine lineages. Furthermore, we found that the rat ortholog is activated by carboxylate anions with similar potencies as hGPR41 (Table II). Because hGPR42 is also reported to occur infrequently in human populations as a polymorphic insert, the mouse, rat, and bovine genes have been named Gpr41.2
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To determine the functional relevance of hGPR42, we prepared membranes
from HEK293T cells transiently cotransfected with hGPR42 and
Go1. No stimulation of [35S]GTP
S
binding could be detected in response to carboxylate ligands (Fig.
4B). Immunoblotting using
affinity-purified anti-SB130 serum, which was raised against a
conserved region of the predicted C-terminal tail and which
cross-reacts with both hGPR41 and hGPR42, confirmed that hGPR42 was
present in the transfected cell membranes at levels equal to or greater
than hGPR41 levels in control membranes (data not shown). This suggests
that hGPR42 is not activated by carboxylate ligands. We introduced
mutations into rat GPR41 (rGPR41) to investigate which of the residues
differing between hGPR41 and hGPR42 allow carboxylate responses (Table
III). These mutations had various effects on the response to propionate
(Fig. 4A), but the most profound phenotype was observed with
rGPR41(R170W), which failed to respond. The equivalent position 174 in
extracellular loop 2 is positively charged Arg174 in
hGPR41, which could potentially form a salt bridge with the carboxylate
ligand, whereas Trp174 in hGPR42 could not. As expected,
this residue is conserved between hGPR41 and mammalian orthologs. We
also demonstrated that the single amino acid change W174R in hGPR42 was
sufficient to restore responses to propionate (Fig. 4B). The
magnitude of the response of hGPR42(W174R) was significantly less than
that of wild-type hGPR41, suggesting that the amino acid differences at
the other positions also influence receptor function. In conclusion,
hGPR42 appears to have arisen by tandem duplication of
hGPR41 and to have lost the ability to activate
Gi family proteins in response to carboxylate ligands
primarily due to an amino acid change at position 174. This does not
exclude the possibility that hGPR42 may respond to other ligands.
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TaqMan quantitative reverse transcriptase (RT)-PCR (PE Applied
Biosystems) was used to determine mRNA distribution of
hGPR41. To distinguish hGPR41 from
hGPR42, we used a primer/probe set containing nucleotide
differences between the pair. In control experiments, these primers
gave large signals from genomic DNA or hGPR41 plasmid, but
not from hGPR42 plasmid, confirming their specificity for
hGPR41 (data not shown). hGPR41 mRNA was
detected in a set of samples from normal human tissues, with the
highest level in adipose tissue and lower expression across all tissues tested (Fig. 5A). A second
primer set that annealed to sequences common to both hGPR41
and hGPR42 gave the same distribution pattern (data not
shown). To confirm expression of GPR41 in adipose tissue, we
examined levels of mouse Gpr41 in 3T3-L1 and 3T3-F442A
fibroblasts upon growth factor-induced differentiation into cells with
an adipocyte-like morphology and containing cytoplasmic lipid droplets. As expected, CAAT/enhancer-binding protein- and adipsin,
which are characteristic of adipocytes (21, 22), were both induced during in vitro adipogenesis, whereas housekeeping genes
were either unchanged or mildly repressed (Fig.
6). Mouse Gpr41 was induced
10-20-fold, although the absolute levels of mouse Gpr41 mRNA detected even after differentiation were low (3.1 ± 0.5 copies/50 ng of total RNA).
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We also performed immunohistochemistry on sections from selected
tissues using antisera that cross-react with both hGPR41 and GPR42.
Staining of endothelial cells was detected consistently across the
sections examined. This was most striking in white adipose tissue, as
shown in Fig. 7A, and could be
blocked by preincubation with the peptide antigen (Fig. 7B).
Positive endothelial cell staining appeared to be mainly in arteries
and arterioles rather than in veins and lymphatic vessels. Mesenchymal
cells (either fibroblasts or preadipocytes) in the collagen layers of
white adipose were also positive. Breast adipocytes were positive,
although staining was faint (data not shown). In tonsil, the antibodies stained lymphocytes and plasma cells, primarily in the mucosa and
submucosa; and this staining could be competed with the peptide antigen. In the trachial-bronchial lymph node, there were positive fibroblasts or fibrocytes in the capsule layer, and plasma cells and a
subset of lymphocytes were also stained. This is consistent with the
RT-PCR data, which also showed hGPR41 in pancreas, spleen, and peripheral blood mononuclear cells (Fig. 5A).
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TaqMan RT-PCR for hGPR43 detected mRNA at relatively
high levels in lymphatic tissues such as spleen and adenoid as well as myometrium and breast (data not shown). A lower level expression of
hGPR43 was detected throughout a panel of non-diseased
tissues. Consistent with the presence in spleen and adenoid, purified
neutrophils and monocytes contained relatively high levels of
hGPR43 mRNA, ~50-fold greater than detected in whole
spleen (Fig. 5B). hGPR43 was also relatively
strongly expressed in peripheral blood mononuclear cells and
B-lymphocytes. No signals were detected in glioma, T-cells, or Raji
cells. The pattern of widespread, low level expression of
hGPR43 may be due to its presence in immune cells,
e.g. in infiltrating neutrophils.
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DISCUSSION |
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We have described the identification of ligands of GPR41 and GPR43. These orphan GPCRs are two members of a family of four (along with GPR40 and GPR42) that are related on the basis of primary sequence and colocalization at the same chromosomal locus (5). We originally identified acetate, present in our screening set as a counterion of several basic peptides, as an agonist of hGPR43. We then demonstrated that other short chain carboxylic acids, including propionate, also had agonist activity and that GPR41 could be activated by similar ligands. Together with evidence that GPR40 is activated by longer chain carboxylates, i.e. fatty acids (6), this identifies GPR40, GPR41, and GPR43 as a family of receptors activated by carboxylic acid anions. These findings were unexpected because this group of receptors is most closely related to the family of protease-activated receptors that are activated by an internal motif within their primary sequences following protease cleavage. There is, however, significant homology between GPR40 family receptors and the leukotriene receptor family; and because leukotrienes also contain a carboxylic acid moiety, this may reflect an evolutionary relationship and/or similar mode of ligand binding.
GPR42, which differs from hGPR41 at only six amino acid positions, is most likely the result of a recent gene duplication event. This is supported by the finding of single orthologs of hGPR41/hGPR42 in other mammalian species and by the greater sequence similarity of these orthologs to hGPR41. Furthermore, the rat ortholog responded to carboxylates with similar potencies as hGPR41, whereas GPR42 failed to respond to carboxylates. The lack of response appears to be largely due to the amino acid at position 174, which is arginine in hGPR41 and mammalian orthologs, but tryptophan in GPR42. Switching this residue by mutagenesis abolished the function of rGPR41 or partially restored function to GPR42. Thus, GPR42 appears to have acquired loss-of-function mutations since duplication. Conceivably, GPR42 may be a functional GPCR that responds to other as yet uncharacterized ligands, although this is unlikely considering that GPR42 differs from hGPR41 at so few residues. TaqMan RT-PCR using GPR42-specific primers detected no signal for GPR42 mRNA in samples from normal human tissues (data not shown), although definitive determination of whether GPR42 is transcribed may require sample genotyping because the GPR42 gene is reportedly present in only a subset of individuals as a polymorphic insert (5).
GPR41 and GPR43 activate the Gi/o family proteins, as
indicated by agonist-stimulated [35S]GTPS binding in
membranes from HEK293T cells transfected with either receptor.
Similarly, activation of GIRK channels in Xenopus oocytes by
hGPR43 is a characteristic Gi-mediated response (23). However, the [Ca2+]i response to acetate in
HEK293 cells expressing hGPR43 was resistant to blockade by PTX,
suggesting that hGPR43 may activate Gq in addition to
Gi family proteins. The G
specificity in a panel of G
chimeras in yeast was largely consistent with that observed in
mammalian cells. rGPR41 was constitutively active in yeast when
coexpressed with Gpa1p/G
i1 and Gpa1p/G
i3
chimeras (data not shown) and responded to carboxylates when
coexpressed in yeast with the Gpa1p/G
o chimera (Table
II). Other yeast G
chimeras did not support rGPR41 coupling or
constitutive activity.
GPR43 is expressed in immune cells, whereas GPR41 appears to be expressed in blood vessel endothelial cells, particularly in adipose tissue, with significant expression also in immune cells and endothelial cells of other tissues. GPR41 levels in adipocytes appear to be low because only weak staining was observed in adipocytes of human adipose tissue sections and because 3T3 cells contained very low levels of mouse Gpr41 mRNA after in vitro adipogenesis. Consistent with this, we have been unable to demonstrate reproducible inhibition of isoprenaline-stimulated lipolysis by carboxylate ligands in rat primary adipocytes (data not shown). This is in contrast to known adipose Gi-coupled receptors such as EP3 (24, 25) and suggests that GPR41 does not directly regulate lipolysis.
At least two of the GPR41 and GPR43 agonists identified here occur in humans in tissue fluid under certain physiological or pathophysiological conditions. Acetate is produced in the liver as a metabolite of ethanol, but this is rapidly absorbed from the circulation by tissues and converted to acetyl-CoA, so concentrations of acetate from this source may not reach levels high enough to activate GPR41 or GPR43. Accumulation of propionate in the blood is a characteristic feature of the disease propionic acidemia (PA) (26). This rare inherited disorder is caused by deficient activity of propionyl-CoA carboxylase (EC 6.4.1.3). This results in an inability to convert propionyl-CoA, formed through catabolism of essential amino acids and fatty acids containing odd numbers of carbon atoms, to methylmalonyl-CoA. PA causes neonatal or infantile symptoms of ketoacidosis resulting from lowering of blood pH. Another clinical feature is the impairment of immune function and frequent infection, often due to opportunistic pathogens (26). Low serum IgG and IgM, leukopenia, lymphopenia, granulopenia, and deficiency of peripheral B-cells have variously been associated with PA (27). Immunodeficiency is common to several other diseases that also result in propionate accumulation, including inherited defects in methylmalonyl-CoA mutase (26). In contrast, ketoacidoses that result from accumulation of organic acids other than propionate are not reported to have profound effects on immune function.
Propionate itself has been implicated as the immunosuppressive factor
in PA serum (28). Propionate inhibits the proliferation of
granulocyte/macrophage progenitor cells and colony formation of
T-lymphocytes in culture (29, 30). It also suppresses proliferation and
maturation of hematopoietic progenitor cells (31) and inhibits lymphocyte activation (28). These properties are apparently not due to
cytotoxicity and are specific to propionate over other organic acids
such as methylmalonate (29). Hence, there is a striking overlap between
the tissue distribution of GPR43, a receptor activated by propionate,
and the cell types in which propionate has reported pharmacological
effects in vivo. The concentrations of propionate that
elicit immunosuppressive effects in vitro are high (1-10
mM), but similarly high levels occur in the sera of PA
patients (26), and these levels are consistent with the concentrations required to activate GPR43 in recombinant assays. An involvement in PA
can also be postulated for GPR41 because this receptor is also
expressed in immune cells. Although other mechanisms for the
immunosuppressive effects of propionate have been proposed (28), the
possible role of GPR40 family receptors in this disease process should
now be considered. To our knowledge, no abnormality of other tissues
expressing GPR41, such as adipose or vascular tissue, has been
described in PA, although the severity of acidosis may mask this. For
both GPR41 and GPR43, full description of their functional roles will
await the development of specific high affinity agonist and antagonist
ligands and the evaluation of null alleles.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. Ravid (Netherlands Brain Bank, Utrecht, The Netherlands) for donation of brain tissue; Chris Plumpton (Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline) for design of immunogenic peptides; Zoe Heaton (Department of Computational and Structural Studies, GlaxoSmithKline) for liquid chromatography-mass spectroscopy analysis; and Andy Whittington (Department of Cheminformatics, GlaxoSmithKline), Richard Green (Department of Computational and Structural Studies), and Katy Gearing (Department of Gene Expression and Protein Biochemistry) for contribution to the orphan ligand fishing project.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is dedicated to the memory of Dr. Richard H. Green.
b To whom correspondence should be addressed: 7TMR Systems Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Rd., Stevenage, Hertfordshire SG1 2NY, UK. Tel.: 1438-764-020; Fax: 1438-768-091; E-mail: andrew.j.brown@gsk.com.
f Present address: Laboratory of Physiology, University of Oxford, Parks Rd., Oxford OX1 3PT, UK.
g Present address: AstraZeneca Research & Development, Charnwood, Bakewell Rd., Loughborough, Leicestershire LE11 5RH, UK.
m Present address: Dept. of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QJ, UK.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211609200
2 The rat orphan GPCR termed GPR41 and described previously by Kimura et al. (32) is homologous to hGPR30 and is distinct from the rat ortholog of hGPR41 described here.
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
GTPS, guanosine
5'-O-(3-thiotriphosphate);
FLIPR, fluorometric imaging plate
reader;
PTX, pertussis toxin;
hGPR41, human GPR41;
rGPR41, rat GPR41;
RT, reverse transcriptase;
PA, propionic acidemia.
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