From the Departments of a 7TMR Systems Research, c Target Bioinformatics, e Gene Expression and Protein Biochemistry, h Cellular Genomics, and i Cardiovascular and Urinary Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom, the g Department of Metabolic Diseases, Metabolic and Viral Diseases Centre of Excellence for Drug Discovery, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, and f Gene Cloning and Expression Proteomics, GlaxoSmithKline, King of Prussia, Pennsylvania 19406-0939
Received for publication, October 18, 2002, and in revised form, January 3, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nicotinic acid has been used clinically for over
40 years in the treatment of dyslipidemia producing a desirable
normalization of a range of cardiovascular risk factors, including a
marked elevation of high density lipoprotein and a reduction in
mortality. The precise mechanism of action of nicotinic acid is
unknown, although it is believed that activation of a
Gi-G protein-coupled receptor may contribute.
Utilizing available information on the tissue distribution of nicotinic
acid receptors, we identified candidate orphan receptors. The selected
orphan receptors were screened for responses to nicotinic acid, in an
assay for activation of Gi-G proteins. Here we describe the
identification of the G protein-coupled receptor HM74 as a low affinity
receptor for nicotinic acid. We then describe the subsequent
identification of HM74A in follow-up bioinformatics searches and
demonstrate that it acts as a high affinity receptor for nicotinic acid
and other compounds with related pharmacology. The discovery of HM74A
as a molecular target for nicotinic acid may facilitate the
discovery of superior drug molecules to treat dyslipidemia.
Nicotinic acid has been used in the treatment of dyslipidemia for
many years, producing a very desirable modification of multiple cardiovascular risk factors, increasing high density
lipoprotein, and decreasing very low density lipoprotein, low density
lipoprotein, triglycerides, and lipoprotein (a), which results in a
reduction in mortality (1). Despite its long history of clinical use, the precise mechanism of action of nicotinic acid is unknown, although
it is believed that inhibition of adipocyte lipolysis via the
activation of a Gi-coupled receptor may contribute (2-4). It has been postulated that a reduction in free fatty acids liberated from adipose tissue results in a reduction of hepatic triglycerides available for very low density lipoprotein and low density lipoprotein synthesis, which in part explains the hypolipidemic effects observed during nicotinic acid therapy. Because the identification of a molecular target for nicotinic acid would facilitate our understanding of its mode of action and potentially enable the discovery of superior
drug molecules, we instigated a strategy to identify this receptor. To
identify the Gi-G protein-coupled receptor for nicotinic
acid, orphan receptors were selected based on their tissue expression
profiles for a rational screening exercise. Recently, the
pharmacological sites of action of nicotinic acid were shown to be
largely restricted to adipose tissue and spleen (5). Therefore, to
identify this nicotinic acid receptor, we selected a subset of 10 orphan G protein-coupled receptors, which by mRNA distribution
analysis (TaqMan) exhibited significant expression levels in both
adipose tissue and spleen. These receptors were then expressed in an
appropriate mammalian cell line to allow measurement of a functional
response (GTP Materials--
Nicotinic acid, nicotinuric acid, and
nicotinamide were obtained from Sigma-Aldrich, 5-methyl nicotinic acid
was from Maybridge, and pyridine-3-acetic acid was from ICN.
LipofectAMINE, Dulbecco's modified Eagle's medium, and fetal calf
serum were from Invitrogen. [35S]GTP Molecular Biology--
The HM74 expressed sequence tag was
identified from the public data base as a potential seven
transmembrane-spanning receptor, and the predicted open reading frame
was amplified using human placenta cDNA as template. Comparison of
the nucleotide sequence of HM74 with that of the published sequence
revealed 15 nucleotide differences as well as a 5-nucleotide insertion
at the 3' end of the clone that resulted in a different 3' coding
sequence. The cloning procedure was performed twice more to confirm the changes in the amino acid sequence. To confirm the correct initiation methionine, a cDNA clone containing the entire coding region and the 5'-untranslated region was isolated using human placenta cDNA library. Sequence analysis of the clone, which we termed HM74A, showed
the presence of a stop codon prior to the first initiation methionine.
A murine sequence with significant homology to human HM74 was
identified by searching public domain data bases with the peptide
sequence for human HM74 taken from GenBankTM accession
number D10923. A TBLASTN search produced significant alignment with
accession numbers AJ300198 and AJ300199, which encode the Mus
musculus PUMA-G gene for a putative seven transmembrane-spanning
receptor (termed mHM74A). Using the human and murine sequence
information, the PCR was used to amplify the corresponding rat
gene. The accession number for human HM74A is AY148884. The cDNA
sequence of rat HM74A is partially represented by EMM_patAR098624.
TaqMan mRNA Analysis--
Poly(A)+ RNA from 20 tissues of
four different individuals (two males, two females except prostate) was
prepared, reverse transcribed, and analyzed by TaqMan quantitative PCR
as described previously (6). Briefly, 1 µg of poly(A)+ RNA was
reverse transcribed using random priming, and the cDNA produced was
used to make up to 1,000 replicate plates with each well containing the
cDNA from 50 ng of poly(A)+ RNA. TaqMan quantitative PCR (Applied
Biosystems, Warrington, UK) was used to assess the level of each gene
relative to genomic DNA standards. The data are presented as the means of mRNA copies detected per ng of poly(A)+ RNA from four
individuals ± S.E. (n = 4). The gene-specific
reagents were: HM74, forward primer,
5'-ACTACTATGTGCGGCGTTCAGAC-3', and reverse primer,
5'-GGCGGTTCATGGCAAACA-3'; TaqMan probe, 5'-ACCAGCCGGCAAGGGATGTCC-3';
HM74A, forward primer, 5'-ACAACTATGTGAGGCGTTGGGA-3', and reverse
primer, 5'-TGGCGGTTCATAGCCAACA-3'; TaqMan probe,
5'-ATCAGCCGGCAAGGGATGTCC-3'; GPR81, forward primer, 5'-TCGGATGAAGAAGGCGACC-3', and reverse primer,
5'-GCTGGGCAGGTAGCATGTG-3'; and TaqMan probe,
5'-TGAACACAATTGCCACCACCATGTG-3'.
Cell Biology--
For transient transfections, HEK293T cells
(HEK293 cells stably expressing the SV40 large T-antigen) were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum and 2 mM glutamine. The cells were seeded
in 90-mm culture dishes and grown to 60-80% confluence (18-24 h)
prior to transfection with vectors containing the relevant DNA inserts
using LipofectAMINE reagent. For transfection, 9 µg of DNA was mixed
with 30 µl of LipofectAMINE in 0.6 ml of Opti-MEM (Invitrogen) and
was incubated at room temperature for 30 min prior to the addition of
1.6 ml of Opti-MEM. The cells were exposed to the LipofectAMINE/DNA
mixture for 5 h, and 6 ml of 20% (v/v) fetal calf serum in
Dulbecco's modified Eagle's medium was then added. The cells were
harvested 48 h after transfection. Pertussis toxin treatment was
carried out by supplementation into the medium at 50 ng
ml
For the generation of stable cell lines, the above method was used to
transfect CHO-K1 cells seeded in six-well dishes grown to 30%
confluence. These cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% fetal calf serum and
2 mM glutamine. 48 h post-transfection the medium was
supplemented with 400 µg/ml G418 for selection of antibiotic
resistant cells. Clonal CHO-K1 cell lines stably expressing HM74A were
confirmed by [35S]GTP P2 Membrane Preparation--
Plasma membrane-containing P2
particulate fractions were prepared from cell pastes frozen at
[3H]Nicotinic Acid Binding--
Saturation binding
assays were carried out on plasma membrane-containing P2 particulate
fractions from HEK293T cells transiently co-expressing HM74A and
G [35S]GTP Oocyte Methods--
Capped cRNA (20-50 ng/oocyte) was injected
into stage V-VI defolliculated oocytes (8), and two microelectrode
voltage clamp recordings were made 3-7 days post-RNA injection from a
holding potential of Yeast--
Human HM74A was subcloned into p426GPD adjacent to
the promoter (9), transferred to pRS306, and integrated into the
ura3 locus of MMY16 (10). Nicotinic acid-mediated stimulation of [35S]GTP
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S1 binding)
following nicotinic acid treatment. This paper describes the
identification of HM74 as a low affinity receptor for nicotinic acid
and the subsequent indication of HM74A, a high affinity receptor for
nicotinic acid. The identification of HM74A has allowed us to test
additional compounds that have been reported to possess a similar
pharmacology to nicotinic acid.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (1160 Ci/mmol)
and [5,6-3H]nicotinic acid (50-60 Ci/mmol) were
purchased from Amersham Biosciences and Biotrend, respectively.
Pertussis toxin was from Sigma-Aldrich. Acipimox, Acifran, and 5-methyl
pyrazole-3-carboxylic acid were synthesized by chemists within GlaxoSmithKline.
1 for 16 h. All of the transient transfection
studies involved co-transfection of receptor together with the
Gi/o G protein, G
o1.
S binding measurements, following
the addition of nicotinic acid.
80 °C after harvest. All of the procedures were carried out at
4 °C. The cell pellets were resuspended in 1 ml of 10 mM
Tris-HCl and 0.1 mM EDTA, pH 7.5 (buffer A), and by
homogenization for 20 s with a Ultra Turrax followed by passage (5 times) through a 25-gauge needle. The cell lysates were centrifuged at
1,000 × g for 10 min in a microcentrifuge to pellet
the nuclei and unbroken cells, and P2 particulate fractions were
recovered by microcentrifugation at 16,000 × g for 30 min. P2 particulate fractions were resuspended in buffer A and stored at
80 °C until required.
o1 using 3H-labeled nicotinic acid as
described (5). Briefly, the membranes (10 µg/point) were incubated
with increasing concentrations of [5,6-3H]nicotinic acid
(60 Ci/mmol; Biotrend) for 3 h at room temperature with agitation.
The assay was performed in 50 mM Tris-HCl pH 7.4 binding
buffer containing 1 mM MgCl2 in a total volume
of 500 µl. Nonspecific binding was assessed in the presence of 1 mM nicotinic acid. Membrane-bound ligand was recovered onto
presoaked GF/B filters using a Brandel 48-well harvester, washed
four times with 1 ml of ice-cold binding buffer, and measured by liquid
scintillation counting. [3H]Nicotinic acid (20 nM) displacement assays were performed using plasma
membrane-containing P2 particulate fractions, prepared from either a
stable CHO cell line expressing recombinant human HM74A or human
adipocytes (Zen-Bio) as described (5) and above.
S
Binding--
[35S]GTP
S binding assays were performed
at room temperature in 96-well format as described previously (7).
Briefly, the membranes (10 µg/point) were diluted to 0.083 mg/ml in
assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4) supplemented with saponin (10 mg/l) and preincubated with 10 µM GDP. Various
concentrations of nicotinic acid or related molecules were added,
followed by [35S]GTP
S (1170 Ci/mmol; Amersham
Biosciences) at 0.3 nM (total volume of 100 µl), and
binding was allowed to proceed at room temperature for 30 min.
Nonspecific binding was determined by the inclusion of 0.6 mM GTP. Wheat germ agglutinin SPA beads (Amersham Biosciences) (0.5 mg) in 25 µl of assay buffer were added, and the
whole was incubated at room temperature for 30 min with agitation. The
plates were centrifuged at 1500 × g for 5 min, and
bound [35S]GTP
S was determined by scintillation
counting on a Wallac 1450 Microbeta Trilux scintillation counter.
60 mV. The oocytes were superfused with ND96
solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,
5 mM HEPES, pH 7.5 at 25 °C) at a flow rate of 2 ml
min
1. To facilitate the recording of GIRK1/GIRK4
potassium currents, the extracellular solution was switched to a high
potassium solution (90 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 5 mM HEPES). The recording electrodes had a resistance of
0.5-1.0 M
when filled with 3 M KCl. The measurements of
potassium currents were made from two batches of oocytes harvested on
different days from different toads. Nicotinic acid was applied by
addition to the superfusate, and cumulative concentration response
curves were constructed for each individual oocyte tested.
-Galactosidase assays to
measure FUS1-lacZ reporter gene induction were performed as
described (11) except that nicotinic acid was omitted from the assay
mix, and the substrate fluorescein-
-D-galactopyranoside
(Molecular Probes; final concentration, 20 µM) was used
in place of chlorophenol
red-
-D-galactosidase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S
binding was observed only in membranes from HEK293T cells
co-transfected with the cDNA for HM74 and the G protein
G
o1 (Fig. 1A).
The nicotinic acid-induced stimulation was
concentration-dependent and was found to be abolished
following pretreatment of cells with pertussis toxin (50 ng
ml
1 for 16 h), suggesting that the effect was
Gi/o G protein-mediated (Fig. 1B). However, the
half-maximal effector concentration for nicotinic acid was estimated to
be in excess of 1 mM, over 1000-fold higher than that
previously reported in rat adipose tissue and spleen membranes (5).
Subsequent to the identification of HM74 as a low affinity receptor for
nicotinic acid, we utilized a molecular biology approach to identify a
novel paralogue of HM74, termed HM74A. Comparison of the nucleotide
sequences of HM74A and HM74 revealed 15 base changes as well as a
5-nucleotide insertion at the 3' end of the clone resulting in HM74A
possessing a shortened C-terminal tail (Fig.
2A). The two receptors are
highly homologous, displaying 96% identity at the protein level and
differing by only 15 amino acids. A third gene, GPR81, previously
identified by customized searching of the GenBankTM high
throughput genomic sequences data base (12), was also found to exhibit
substantial homology to HM74 and HM74A (57 and 58% amino acid sequence
identity, respectively). Despite their high degree of similarity, HM74
and HM74A are not simply polymorphic variants but are separate genes
being co-located with GPR81 at chromosome 12q24.31 (part of this region
of chromosome 12 is represented by accession number AC026362).
Messenger RNA expression profiling of HM74 and HM74A using TaqMan
quantitative reverse transcriptase-PCR analysis with probes designed
and confirmed to discriminate between these two homologous receptors
(data not shown) showed that both exhibited similar distribution
patterns that were largely restricted to adipose tissue and spleen
(Fig. 2B). Hence, the expression patterns of HM74A and HM74
are concomitant with that of a nicotinic acid receptor. Interestingly,
GPR81 appears to be highly restricted to adipose tissue.
View larger version (26K):
[in a new window]
Fig. 1.
HM74 is a G protein-coupled receptor that
responds to nicotinic acid. A, application of 300 µM (hatched columns) and 1 mM
(filled columns) nicotinic acid to membranes from HEK293T
cells expressing a variety of orphan G protein-coupled receptors was
found to stimulate [35S]GTP S binding in membranes from
cells expressing HM74 only (open columns, basal conditions).
B, nicotinic acid stimulated a dose-dependent
increase in [35S]GTP
S binding in cells expressing HM74
(filled circles) that was ablated by pretreatment with 50 ng
ml
1 pertussis toxin for 16 h prior to harvest
(triangles). All of the transient transfection studies
involved co-transfection of receptor together with the Gi/o
G protein, G
o1.
View larger version (29K):
[in a new window]
Fig. 2.
HM74 and HM74A are highly homologous proteins
with similar mRNA tissue distribution patterns and chromosomal
location. A, amino acid sequences of human HM74 and
human and rat HM74A aligned for comparison. Residues sharing identity
between human and rat HM74A but not with HM74 are indicated with
asterisks. B, TaqMan quantitative reverse
transcriptase-PCR analysis of mRNA levels in human tissues. The
cDNA from the reverse transcription of 1 ng of poly
(A)+ RNA from multiple tissues for four different
nondiseased individuals was assessed for its HM74, HM74A, and GPR81 and
housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), mRNA levels. The data are presented as
the means (± S.E.) mRNA levels from four individuals for each
tissue.
Expression of HM74A together with Go1 in HEK293T cells
gave robust concentration-dependent responses to nicotinic
acid with a half-maximal concentration (EC50 = 250 ± 27 nM) similar to that observed in rat adipose tissue and
spleen membranes (5) (Fig. 3A). Conversely, GPR81
responded to nicotinic acid only at relatively high concentrations (10 mM). Expression of HM74A, but not HM74 or GPR81, also
produced saturable specific binding of 3H-labeled nicotinic
acid with an affinity (Kd 95.8 ± 9.5 nM) similar to that recorded from rat adipose tissue and
spleen membranes (5) (Fig. 3B). We have not been able to
quantify the levels of expression of HM74 and GPR81 because of their
low affinity for nicotinic acid. Next, we expressed HM74A and HM74 in
Xenopus oocytes to study their coupling to the
Gi-G protein-regulated potassium channels GIRK1 and GIRK4
(13). Concentration-dependent responses to nicotinic acid
with a half-maximal concentration (EC50) of 130 ± 50 nM were observed for HM74A (four oocytes). Responses to
nicotinic acid were also obtained at concentrations of 100 µM and above following expression of HM74 (5 oocytes)
(Fig. 3C). Finally, we investigated coupling to the
pheromone response pathway of Saccharomyces cerevisiae,
measuring receptor activation with a reporter gene. Using nicotinic
acid-free growth medium, we demonstrated
concentration-dependent activation of HM74A in response to nicotinic acid (EC50 = 904 ± 28 nM; Fig. 3D). The optimal agonist responses were
observed with chimeric yeast/mammalian G
subunits having the
C-terminal 5 amino acids of Gi or the promiscuous G
,
G16 (Fig. 3D and data not shown) (11). The yeast
data confirm that HM74A is sufficient to confer the nicotinic acid
response, because these cells lack endogenous G protein-coupled
receptors capable of activating this pathway.
|
A number of nicotinic acid analogues were employed to characterize
HM74A using the [35S]GTPS binding assay (Table
I). Similar rank orders of potency were
found at HM74A compared with those previously described in native rat
tissue (5), whereas all of the analogues displayed either no or very
weak activity at HM74 (data not shown). All of the analogues were also
inactive at GPR81 (data not shown). Furthermore, we also cloned the rat
orthologue of HM74A, which was found to exhibit 82% identity at the
protein level with its human counterpart (Fig. 2A). As
expected, no significant pharmacological differences were observed
between recombinantly expressed rat and human HM74A (Table I).
|
Acipimox (Olbetam) and Acifran (AY-25,712) are two molecules that have
been reported to produce a pharmacological profile resembling that of
nicotinic acid in rat and human studies (14-17) (Fig.
4A). Using the
[35S]GTPS binding assay, we found that
Acipimox was a full agonist at HM74A (EC50 = 6 ± 1 µM) and exhibited weak activity at HM74 and no activity
at GPR81. Acifran acted as a full and relatively potent agonist at both
HM74A (EC50 = 2.1 ± 0.2 µM) and HM74
(EC50 = 20 ± 4 µM) but showed no
significant agonism at GPR81 up to 1 mM (Fig.
4B). Acifran also activated HM74A in yeast (EC50 = 3.0 ± 0.08 µM). The fact that Acifran has been
identified as a high affinity ligand for HM74 (Fig. 4B)
strongly suggests that our transfection system is sufficiently
efficient to allow agonist profiling. Furthermore, the signal to noise
ratios observed with Acifran at both HM74A and HM74 are similar, which
suggests that these receptors are expressed at similar levels. In
addition, we have expressed HM74A and HM74 in a range of different
systems (mammalian, yeast, and oocyte), and in all of these expression systems there is an ~1000-fold separation in the potency of nicotinic acid, suggesting that this is a real observation.
|
Acifran and Acipimox were included in a group of molecules with
structural or pharmacological similarities with nicotinic acid that
were tested in a [3H]nicotinic acid displacement assay
performed in membranes from either a stable CHO cell line expressing
recombinant human HM74A or human adipocytes (Table
II). The rank order of potency for the
displacement of [3H]nicotinic acid binding was nicotinic
acid > 5-methyl pyrazole-3-carboxylic acid = pyridine-3-acetic acid > Acifran > 5-methyl nicotinic
acid = Acipimox nicotinuric acid = nicotinamide. This
rank order of potency was the same in both the stable CHO cell line
expressing recombinant human HM74A and human adipocytes.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HM74 was identified as a low affinity receptor for nicotinic acid following the screening of a panel of orphan receptors selected because of their tissue expression profile. HM74 is an orphan receptor that had been previously cloned from a cDNA library derived from human monocytes (18). The half-maximal effector concentration for nicotinic acid at HM74 was estimated to be in excess of 1 mM, ~1000-fold higher than that previously reported in membranes produced from rat adipose tissue or spleen (5). We considered three possible explanations for this discrepancy in nicotinic acid potency. First, close homologues of HM74 may act as higher affinity nicotinic acid receptors. Second, because G protein-mediated nicotinic acid effects on native tissue have almost always been recorded from rat, variation between human and rodent receptors may explain this phenomenon. Finally, differences in the pharmacological integrity of the recombinantly expressed receptor and its endogenously expressed counterpart may explain potency changes.
A molecular biology approach resulted in the identification of a novel
paralogue of HM74, termed HM74A. Despite their high degree of
similarity, HM74 and HM74A are not simply polymorphic variants but are
separate genes being co-located with GPR81 at chromosome 12q24.31.
TaqMan analysis confirmed that the expression pattern of HM74A was very
similar to HM74. When expressed in a variety of test systems, HM74A was
confirmed as a high affinity receptor for nicotinic acid. The activity
and affinity of nicotinic acid was in good agreement with that
previously reported in the literature (5). Furthermore, following the
cloning of the rat orthologue of HM74A, we found no significant
pharmacological differences between nicotinic acid derivatives tested
against either human or rat HM74A. The murine variant of HM74A, PUMA-G,
was recently reported to be an interferon -inducible gene in
macrophages, suggesting a possible role in macrophage function (19).
This finding is further supported by a recent report describing a
nicotinic acid receptor in a murine macrophage cell line (20). Based on the TaqMan data generated for the distribution of human HM74A, there
appears to be little or no expression in macrophages (Fig. 2B). This may indicate that species differences in the
distribution of HM74A exist or is a reflection of the activation state
of the macrophages used in this experiment. It will be of interest to determine whether the expression of HM74A can be up-regulated in human
macrophages following incubation with interferon
.
In the [3H]nicotinic acid displacement assay, both the absolute potency and the rank order of potency of the HM74A ligands studied was the same, whether tested against the stable CHO cell line expressing recombinant human HM74A or human adipocytes. These data strongly suggest that HM74A is the Gi-G protein-coupled nicotinic acid receptor on human adipocytes. Acipimox and Acifran have also been identified as full agonists at HM74A. These compounds have also been reported to produce a pharmacological effect resembling that of nicotinic acid in rat and human studies (14-17). The other compounds identified that displace nicotinic acid from HM74A, 5-methyl pyrazole-3-carboxylic acid, pyridine-3-carboxylic acid, and 5-methyl nicotinic acid, have all previously been shown to inhibit adipocyte lipolysis (2, 23). Nicotinamide, which unlike nicotinic acid produces no alteration in lipoprotein profiles (22), acted only as a very weak agonist at HM74A. Indeed, nicotinamide was ~1000-fold less potent than nicotinic acid, a level of activity that could be due to contaminant nicotinic acid (e.g. 0.1%). It would appear that activation of HM74A would account for the inhibition of lipolysis observed with these compounds. Therefore, of the compounds that have been tested in man, it would appear that potency at HM74A is linked with their efficacy at normalizing lipoprotein profiles.
We have demonstrated that HM74A is a high affinity receptor for nicotinic acid and believe that this receptor is a likely candidate as a molecule target for the beneficial therapeutic effects observed with nicotinic acid. Nicotinic acid is an effective therapeutic agent; however, it has to be administered at high doses and has a characteristic side effect profile defined by intense, but transient, prostaglandin-mediated cutaneous vasodilation ("flushing") that affects patient compliance (21, 22).
Unlike HM74A, we were unable to identify rodent orthologues of HM74
using conventional gene cloning strategies and bioinformatics searches.
This suggests that in humans HM74 may be the result of a relatively
recent gene duplication event. Furthermore, of the compounds tested,
only Acifran exhibited activity at HM74. In fact, Acifran is the first
molecule that we have identified to date that exhibits significant
potency at HM74. Because of the high degree of homology between HM74A
and HM74 and the existence of highly selective ligands, site-directed
mutagenesis may be a useful strategy in determining which amino acid
residues play a key role in ligand binding. Indeed, 11 amino acid
residues are conserved in human and rat HM74A but not in HM74 (Fig.
2A). Such residues may play key roles in determining the
differences in ligand binding affinities between HM74A and HM74. The
identification of HM74A as a molecular target for nicotinic acid will
facilitate the discovery of potent and selective ligands for this
receptor and may expedite the discovery of improved anti-hyperlipidemic drug molecules.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of Discovery and Genetics Research and the Cardiovascular and Urinary Centre of Excellence for Drug Discovery for support. We also thank Dr. R. Ravid (Netherlands Brain Bank, Amsterdam, The Netherlands) for the arrangement/donation of brain tissue, Jean-Philippe Walhin for expert technical assistance, and E. Koppe for the provision of the human HM74A stable cell line.
![]() |
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY148884 and EMM_patAR098624.
b To whom correspondence should be addressed: 7TMR Systems Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Rd., Stevenage, Hertfordshire, SG1 2NY, UK. Tel.: 1438-764988; Fax: 1438-768091; E-mail: alan.x.wise@gsk.com.
d Present address: Pharmaceutical Discovery, Lexicon Genetics Inc., 8800 Technology, Forest Place, The Woodlands, Houston, TX 77381-1160.
j Present address: University of Cambridge, Dept. of Pharmacology, Tennis Court Rd., Cambridge CB2 1QJ, UK.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M210695200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GTPS, guanosine
5'-(
-thio)triphosphate;
GIRK, G protein-regulated
potassium channel;
CHO, Chinese hamster ovary.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tavintharan, S., and Kashyap, M. L. (2001) Curr. Atheroscler. Rep. 3, 74-82[Medline] [Order article via Infotrieve] |
2. | Aktories, K., Schultz, G., and Jakobs, K. H. (1980) Arzneimittelforschung 33, 1525-1527 |
3. | Aktories, K., Schultz, G., and Jakobs, K. H. (1983) FEBS Lett. 156, 88-92[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Green, A.,
Milligan, G.,
and Dobias, S. B.
(1992)
J. Biol. Chem.
267,
3223-3229 |
5. |
Lorenzen, A.,
Stannek, C.,
Lang, H.,
Andrianov, V.,
Kalvinsh, I.,
and Schwabe, U.
(2001)
Mol. Pharmacol.
59,
349-357 |
6. | Chapman, C. G., Meadows, H. J., Godden, R. J., Campbell, D. A., Duckworth, M., Kelsell, R. E., Murdock, P. R., Randall, A. D., Rennie, G. I., and Gloger, I. S. (2000) Mol. Brain Res. 82, 74-83[Medline] [Order article via Infotrieve] |
7. | Wieland, T., and Jakobs, K. H. (1994) Methods Enzymol. 237, 3-13[CrossRef][Medline] [Order article via Infotrieve] |
8. | Goldin, A. L. (1992) Methods Enzymol. 207, 266-279[Medline] [Order article via Infotrieve] |
9. | Mumberg, D., Muller, R., and Funk, M. (1995) Gene (Amst.) 156, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Olesnicky, N. S.,
Brown, A. J.,
Dowell, S. J.,
and Casselton, L. A.
(1999)
EMBO J.
18,
2756-2763 |
11. | Brown, A. J., Dyos, S. L., Whiteway, M. S., White, J. H. M., Watson, M.-A., Marzioch, M., Clare, J. J., Cousens, D. J., Paddon, C. J., Plumpton, C., Romanos, M. A., and Dowell, S. J. (2000) Yeast 16, 11-22[CrossRef][Medline] [Order article via Infotrieve] |
12. | Lee, D. K., Nguyen, T., Lynch, K. R., Cheng, R., Vanti, W. B., Arkhitko, O., Lewis, T., Evans, J. F., George, S. R., and O'Dowd, B. F. (2001) Gene (Amst.) 275, 83-91[CrossRef][Medline] [Order article via Infotrieve] |
13. | Uezono, Y., Bradley, J., Min, C., McCarty, N. A., Quick, M., Riordan, J. R., Chavkin, C., Zinn, K., Lester, H. A., and Davidson, N. (1993) Receptors Channels 1, 233-241[Medline] [Order article via Infotrieve] |
14. | Lovisolo, P. P., Briatico-Vangosa, G., Orsini, G., Ronchi, R., and Angelucci, R. (1981) Pharmacol. Res. Commun. 13, 163-174[Medline] [Order article via Infotrieve] |
15. | Crepaldi, G., Avogaro, P., Descovich, G. C., DiPerri, T., Postiglione, A., Sirtori, C. R., Strano, A., Ventura, S., and Musatti, L. (1988) Atherosclerosis 70, 115-121[Medline] [Order article via Infotrieve] |
16. | Cayen, M. N., Kallai-Sanfacon, M. A., Dubec, J., Greselin, E., and Dvornik, D. (1982) Atherosclerosis 45, 267-279[Medline] [Order article via Infotrieve] |
17. | Hunninghake, D. B., Edwards, D. G., Sopko, G. S., and Tosiello, R. L. (1985) Clin. Pharmacol. Ther. 38, 313-317[Medline] [Order article via Infotrieve] |
18. | Nomura, H., Nielsen, B. W., and Matsushima, K. (1993) Int. Immunol. 5, 1239-1249[Abstract] |
19. | Schaub, A., Futterer, A., and Pfeffer, K. (2001) Eur. J. Immunol. 31, 3714-3725[CrossRef][Medline] [Order article via Infotrieve] |
20. | Lorenzen, A., Stannek, C., Burmeister, A., Kalvinsh, I., and Schwabe, U. (2002) Biochem. Pharmacol. 64, 645-648[CrossRef][Medline] [Order article via Infotrieve] |
21. | Stern, R. H., Spence, J. D., Freeman, D. J., and Parbtani, A. (1991) Clin. Pharmacol. Ther. 50, 66-70[Medline] [Order article via Infotrieve] |
22. | Morrow, J. D., Awad, J. A., Oates, J. A., and Jackson-Roberts, L. (1992) J. Invest. Dermatol. 98, 812-815[Abstract] |
23. | Credner, K., Tauscher, M., Jozic, L., and Brenner, G. (1981) Arzneimittelforschung 31, 2096-2100[Medline] [Order article via Infotrieve] |