(Received for publication, June 12, 1995; and in revised form, August 17, 1995)
From the
The cDNA encoding a novel P receptor was isolated
from rat aortic smooth muscle cell library and functionally
characterized. The cloned P
receptor exhibits structural
features characteristic of the G protein-coupled receptor family and
shows 44 and 38% amino acid identity with previously cloned rat
P
and chicken P
receptors, respectively. The
cloned P
receptor is functionally coupled to phospholipase
C but not to adenylate cyclase in C6 rat glioma cells transfected with
the cloned P
expression vector. The rank order of agonist
potency as judged by intracellular Ca
mobilization
responses is UTP > ADP = 2-methylthioATP > ADP
S >
ATP = ATP
S, which is not compatible with any of the
previously characterized P
receptor subtypes. The
nonselective P
antagonists, suramin and reactive blue-2,
inhibit nucleotide-induced phospholipase C activation in cells
expressing the cloned P
receptor. The cloned P
receptor mRNA is abundantly expressed in various rat tissues
including lung, stomach, intestine, spleen, mesentery, heart, and, most
prominently, aorta. The results indicate that the novel metabotropic
P
receptor has pharmacological characteristics distinct
from any of P
receptor subtypes thus far identified and
suggest the existence of a novel regulatory system by extracellular
nucleotides of potential significance.
P nucleotide receptors mediate a wide variety of
physiological responses to extracellular nucleotides, including
vascular smooth muscle contraction and relaxation, neurotransmission,
and endocrine and exocrine secretion(1, 2) . In the
vascular system, nucleotides activate P
receptors on
vascular smooth muscle cells to cause contraction(3) . On the
other hand, when nucleotides activate P
receptors on
vascular endothelial cells, it stimulates release of the vasorelaxants,
prostacyclin and NO, to cause
vasorelaxation(4, 5, 6) . Nucleotide-induced
vasoconstriction and relaxation were initially suggested to be mediated
via two distinct subtypes of P
receptors, P
and P
purinoceptors, respectively(7) .
However, recent studies on the agonist specificity and potency rank
order have provided evidence for the existence of more than a single
class of P
receptors in both vascular smooth muscle and
endothelium. For example, the pyrimidine UTP as well as the P
selective agonist
,
-methylene ATP evokes
vasoconstriction in various vascular beds, leading to the suggestion
that the third P
receptor that can interact with UTP,
P
, mediates vasoconstriction, because UTP does not serve
as a ligand for P
or P
receptors (8, 9, 10, 11, 12) .
Furthermore, activation of P
receptors with UTP and
,
-methylene ATP in smooth muscle cells was demonstrated to
lead to activation of different downstream effector molecules (i.e. phospholipase C (2) and a cation channel intrinsic to
P
receptors(13) , respectively). Several studies
also demonstrated that depending on vascular beds and animal species,
P
receptors mediate nucleotide-induced
endothelium-dependent
relaxation(11, 14, 15) . However, because of
unavailability of a radioactive P
receptor ligand and an
agonist and antagonist selective for each of the P
receptor
subtypes, definitive identification of P
receptor subtypes
that are expressed in vascular smooth muscle and endothelium has been
hampered.
Very recently, the cDNAs encoding for mouse(16) ,
human(17) , and rat (18) P receptors and
chicken brain P
receptor (19) have been isolated.
It is, however, unknown whether these P
receptor subtypes
are expressed in the vascular tissues or in fact mediate vascular
responses to nucleotides. It was reported that the cloned chicken brain
P
receptor, when expressed in Xenopus oocytes,
displays a significantly different agonist potency rank order from that
for endothelium-dependent relaxation(19) . Hence, it is still
possible that another P
receptor subtype may be responsible
for nucleotide-induced vascular responses. In the present report we
demonstrate the isolation of a cDNA encoding a novel P
receptor subtype from rat aortic smooth muscle cell cDNA library,
which is coupled to the Ca
phospholipase C messenger
system and shows a distinct agonist specificity from any other known
P
receptor. This receptor is predominantly expressed in rat
aorta and several other tissues.
The intracellular free
Ca concentration
([Ca
]
) was measured in
trypsinized cells stably expressing the cloned receptor by using a
fluorescent Ca
indicator fura-2 (Dojin, Kumamoto,
Japan). For obtaining cells that stably express the cloned receptor,
C6-15 cells were co-transfected with the receptor expression
plasmid and neomycin-resistant gene expression vector pKM3 (Dr. H.
Nojima, Osaka University) by the calcium phosphate precipitation method
and after 48 h were selected with G418 (Life Technologies, Inc.) (600
µg/ml). The fluorescence of fura-2-loaded cells was measured with a
CAF-110 spectrofluorimeter (Japan Spectroscopy, Inc., Tokyo, Japan)
with excitation at 340 and 380 nm and emission at 500 nm as described
previously(28) . The [Ca
]
was calculated from the measurements of the ratio of fluorescence
intensities as described by Grynkiewicz et al.(29) .
Figure 3:
Northern blot analysis of mRNA isolated
from rat tissues and cells with the cloned P receptor cDNA
as a probe. Poly(A)
RNA (5 µg each except aorta
where 10 µg was loaded) from rat tissues and total RNA (20 µg)
from cultured RASM cells were separated by formaldehyde/1.0% agarose
gel electrophoresis. Hybridization was performed as described under
``Materials and Methods,'' followed by
autoradiography.
Shown in Fig. 1are nucleotide and deduced amino acid sequences of this clone. An in-frame initiating codon (nucleotides 440-442 in Fig. 1) is in the context of the Kozak translation initiation consensus sequence (30) and is preceded by an in-frame stop codon. The predicted molecular mass of 36.7 kDa of this protein is substantially low among G protein-coupled receptors and close to that of A1 adenosine receptor (31) and several odorant receptors (32) so far reported to have the smallest molecular weight. Hydropathy analysis (33) of the clone reveals seven stretches of hydrophobic amino acids, predicted to represent membrane-spanning domains characteristic of the G protein-coupled receptors. The amino-terminal region preceding the putative first transmembrane domain contains a single potential asparagine-linked glycosylation site, and the third intracellular loop and cytoplasmic tail have two recognition sites (Ser-235 and Thr-320) for phosphorylation by protein kinase C(34, 35) . This protein also possesses a number of residues conserved in most of the G protein-coupled receptors such as Asp in the second transmembrane domain, Leu in the second and the seventh transmembrane domains, Arg-Tyr immediately behind the third transmembrane domain, and Pro in the fifth, the sixth, and the seventh transmembrane domains(36, 37) . A Cys in the carboxyl-terminal region conserved in many of the G protein-coupled receptors, which may be a membrane-anchoring palmitoylation site, and a Asp in the third transmembrane domain conserved in the G protein-coupled receptors for charged amines are absent in this protein(37) . All these characteristics suggest that this protein represents a G protein-coupled receptor.
Figure 1:
The nucleotide sequence and the
predicted amino acid sequence of the cloned P receptor. The double underlining indicates an in-frame stop codon preceding
the initiating codon. Potential N-linked glycosylation site
(
) in the amino-terminal region and phosphorylation sites
(
) for protein kinase C in the cytoplasmic loop and the
carboxyl-terminal region are indicated. Both symbols are below the amino acids. The putative transmembrane domains I-VII
assigned on the basis of the results of Kyte and Doolittle
hydropathicity plot are indicated by single underlining.
Poly(A)
signal AATAAA is indicated by single
underlining.
Shown in Fig. 2is alignment of the
amino acid sequences of this protein and two previously cloned
P receptors, rat P
and chicken P
.
25% of the residues are conserved among these three proteins. The
observed similarities are mostly confined to the region between the
first and the seventh transmembrane domains, whereas the amino acid
sequences and the length of the amino and carboxyl termini are variant.
The next most similar proteins found from a data base search are rat
G10D orphan receptor (29% identity in a 294-amino acid overlap), rat
thrombin receptor (28% identity in a 294-amino acid overlap), and rat
type 1b angiotensin II receptor (27% identity in a 300-amino acid
overlap).
Figure 2:
Alignment of the amino acid sequence of
the cloned P receptor with those of rat P
receptor and chicken P
receptor. The approximate positions
of the transmembrane domains are indicated by boxes, and
conserved residues are shaded. Gaps (-) are introduced
so as to maximize the alignment. Rat P
and chicken
P
were recommended to be renamed as P
and
P
, respectively, by Bernard et al.(42) .
Because previous
pharmacological studies demonstrated in vascular tissues the expression
of P receptors that respond to
UTP(8, 9, 10, 11, 12) , we
compared the expression of P
receptor and the cloned new
P
receptor in the large capacitance vessel aorta, the
mesentery, which includes lots of resistance vessels of smaller
calibers, and cultured RASM cells. The 2.8-kb P
transcript
is abundantly expressed in the aorta like the case of the cloned
P
receptor (Fig. 4). The P
transcript
is also readily detected in the mesentery and RASM cells. The signal
intensities of both P
and cloned new P
receptors transcripts in each tissue and cell appear to be
roughly similar (compare Fig. 3and Fig. 4). However,
strict quantitative comparison of expression levels of P
and cloned P
receptor transcripts in Fig. 3and Fig. 4may not be possible, although the
lengths and specific radioactivities of employed cDNA probes and
exposure times of membranes to films in the Northern analyses were
similar.
Figure 4:
Northern blot analysis of mRNA isolated
from rat aorta and mesentery and rat aortic smooth muscle cells with
P receptor cDNA as a probe. Poly(A)
RNA
(10 µg) from rat tissues and total RNA (20 µg) from RASM cells
were separated by formaldehyde/1.0% agarose gel electrophoresis.
Hybridization was performed as described under ``Materials and
Methods,'' followed by
autoradiography.
Figure 5:
Effects of nucleotides on
[Ca]
in cells stably
expressing the cloned P
receptor. A, effects of
CoCl
and removal of extracellular Ca
on
ADP-induced increases in
[Ca
]
. Test substances
were applied at arrows and were present throughout each
recording. Tracings are representative of at least four experiments. B, dose-response curve of nucleotide-induced peak
[Ca
]
increments.
, UTP;
, 2-methylthioATP;
, ADP;
, ADP
S;
, ATP
S;
, ATP. Values are expressed as a percentage
of the maximal ADP response and are the means of three
determinations.
To characterize the agonist specificity of the cloned P receptor, C6-15 cells expressing the cloned P
receptor were stimulated with various concentrations of
nucleotides, and the [Ca
]
response was monitored. As shown in Fig. 5B, UTP
is the most potent with the EC
value of approximately 1
µM, which is similar to the reported EC
values for UTP in mouse and human P
receptors.
Unlike the P
receptor, however, ATP (EC
= 500 µM) is more than two orders less potent
than UTP, and ADP and 2-methylthioATP (EC
= 50
µM for both nucleotides) are more potent for the cloned
P
receptor than ATP. AMP and adenosine are both totally
ineffective (data not shown). The P
-selective agonist
,
-methylene ATP is also ineffective. Thus, the agonist
specificity of the cloned new P
receptor does not exactly
fit with any of previously cloned P
receptors.
To
explore whether nucleotide-induced Ca mobilization is
linked to phospholipase C activation in C6-15 cells expressing
the cloned P
receptor, the production of inositol
phosphates was measured in [
H]inositol-prelabeled
cells. As shown in Fig. 6, in cells transfected with the cloned
P
receptor-expression vector, the addition of 100
µM ADP stimulates the production of total inositol
phosphates about 4.3-fold. In contrast, ADP is without effect on cells
transfected with vector alone. The results indicate that the cloned
P
receptor is coupled to phospholipase C. To examine
whether the receptor is coupled to phospholipase C via a pertussis
toxin-sensitive G protein, we studied the effect of pertussis toxin
pretreatment on ADP-induced inositol phosphate production. Pretreatment
of pertussis toxin (10 ng/ml) for 24 h does not affect ADP (100
µM)-induced inositol phosphate production at all (Table 1). Thus, the cloned P
receptor appears to be
coupled to phospholipase C via a pertussis toxin-insensitive G protein.
Figure 6:
ADP-induced inositol phosphate production
in cells transfected with vector alone or cloned P
receptor-expression plasmid. Cells prelabeled with
myo-[
-H]inositol were stimulated with 100
µM ADP for 60 min in the presence of 10 mM LiCl.
Total inositol phosphates were separated and counted as described under
``Materials and Methods.'' Values are the means ± S.D.
of three determinations.
To examine potential coupling of the cloned P receptor
to adenylate cyclase, the effects of nucleotides on cellular cyclic AMP
contents were examined in C6-15 cells transfected with vector
alone or the cloned P
receptor-expression vector (Table 2). Forskolin increases cyclic AMP contents
3.5-4.2-fold in cells transfected with vector alone or the cloned
P
-expression vector. Either ADP, ATP, or UTP at 100
µM does not change cyclic AMP contents in
forskolin-stimulated cells transfected with either construct. The
results indicate that the cloned P
receptor is not coupled
positively or negatively to adenylate cyclase in C6-15 cells.
The results of the nucleotide selectivity of the cloned P receptor described above in the present study indicate that the
pharmacological feature of the cloned P
receptor is
distinct from that of either mouse and human P
receptor or
chicken P
receptor. We further tried to characterize the
pharmacological property of the cloned P
receptor by
examining the susceptibility of the cloned P
receptor to
known P
receptor antagonists(38, 39) . As
shown in Fig. 7, suramin (100 µM), an antagonist
for P
, P
, and P
receptors,
slightly (20%) but significantly inhibited ADP-induced inositol
phosphate production in cells expressing the cloned P
receptor. Reactive blue-2 (100 µM), an antagonist
for P
and P
receptors, more strongly (77%)
inhibited ADP-induced inositol phosphate production. Thus, the cloned
P
receptor is sensitive to known P
receptor
antagonists.
Figure 7:
Inhibition of ADP-induced inositol
phosphate production by suramin and reactive blue-2 (RB2) in
cells expressing cloned P receptor. Cells prelabeled with
myo-[
-H]inositol were preincubated with or
without 100 µM suramin or reactive blue-2 and then
stimulated with 100 µM ADP for 60 min in the presence of
10 mM LiCl. Total inositol phosphates were separated and
counted as described under ``Materials and Methods.'' Values
are the means ± S.D. of three
determinations.
Based on their pharmacological properties and signaling
mechanisms, mammalian P receptors have been classified into
five subtypes, P
, P
, P
,
P
, and P
(1, 2) . Among them,
P
subtype is expressed in very limited cell types
including platelets(1, 2) . P
and
P
have the properties of ligand-gated ion channels.
P
receptor cDNAs have recently been cloned (40, 41) and found to have only two transmembrane
domains and a pore-forming motif. On the other hand, P
and
P
are a metabotropic type of P
receptors and
widely expressed in various tissues. Recent molecular cloning of mouse,
human, and rat P
and chicken P
receptors
revealed that they belong to G protein-coupled receptors and are linked
to intracellular Ca
mobilization (16, 17, 18, 19) . In the present
study we demonstrate the isolation of a cDNA clone encoding a novel
member of P
receptors. Evaluation of functional properties
of the new receptor has revealed that it is not categorized into any of
the classical P
receptor subtypes mentioned above.
Sequence analysis of the cloned P receptor demonstrates
that the receptor possesses structural properties characteristic of the
G protein-coupled receptor superfamily and has a considerable homology
to P
and P
receptors (44 and 38% amino acid
identity, respectively) (Fig. 2). Functional analysis of the
cloned P
receptor shows that this receptor is coupled to
phospholipase C and Ca
mobilization (Fig. 5A), like P
and P
receptors. However, the agonist selectivity of the cloned P
receptor clearly differs from that of P
and
P
receptors; the rank order of agonist potency for the
cloned P
receptor is UTP > 2-methylthioATP = ADP
> ADP
S > ATP = ATP
S (Fig. 5B),
whereas for the mouse P
receptor UTP = ATP >
ATP
S 2-methylthioATP = ADP, and for the chicken
P
receptor 2-methylthioATP > ATP > ADP UTP. Thus,
the cloned P
receptor resembles the P
receptor
in that it can respond to the pyrimidine UTP with a relatively high
sensitivity (Fig. 5B). However, the cloned P
receptor also share some properties with classical P
receptors in that the cloned P
and classical P
receptors respond to ADP and 2-methylthioATP with a higher
sensitivity than the P
receptor and that responses
mediated by both cloned P
and classical P
receptors are inhibited by the receptor antagonist, reactive
blue-2 (37) (Fig. 7). All these results indicate that
the cloned P
receptor is a novel metabotropic P
receptor, which, together with the recently cloned P
and P
receptors, constitutes a distinct family of G
protein-coupled P
receptors.
Barnard et al.(42) and Fredholm et al.(31) in their
recent reviews have recommended reclassifying cloned P receptors into the two major families, the G protein-coupled
receptor P
and the intrinsic ion channel type receptor
P
. They proposed terming newly discovered G
protein-coupled P
receptors as P
,
P
, P
, etc., by consecutively numbering
them. They designated the chicken brain P
and its rat
equivalent as P
, mouse and human P
as
P
, and the third G protein-coupled P
receptor
with a strong preference for ADP that they have recently cloned but not
published yet as P
(42) . Our P
receptor clearly belongs to the G protein-coupled P
receptor family and should in future be named in a systematic way
as proposed(31, 42) .
Detection of a strong signal
of our cloned P receptor transcript in rat aorta (Fig. 3) implicates its physiological role in vascular function.
Because the cloned P
receptor transcript is detected in
cultured rat aortic smooth muscle cells, the cloned P
receptor is likely expressed in the smooth muscle layer of the
vascular wall, where it may be involved in the regulation of vascular
tone. The cloned P
receptor transcript is also detected in
the mesentery, indicating the expression of the cloned P
receptor in both large capacitance vessels and smaller resistance
vessels (Fig. 3). Because activation of the cloned P
receptor stimulates phospholipase C to induce an increase in the
[Ca
]
( Fig. 5and 6), the
cloned P
receptor on vascular smooth muscle cells most
likely mediates vascular smooth muscle contractile response. In
addition, it is an interesting possibility that the cloned P
receptor on vascular smooth muscle cells may also be involved in
promoting vascular smooth muscle cell growth under physiological or
pathological conditions, because it was shown that nucleotides induce
stimulation of DNA synthesis in cultured rat vascular smooth muscle
cells(43, 44) . The moderate levels of the cloned
P
receptor transcript are detected in smooth muscle organs,
including stomach and intestine, suggesting that the cloned P
receptor is involved in the functional regulation of nonvascular
smooth muscle as well.
A number of recent reports demonstrated that
the pyrimidine UTP causes potent vasoconstriction in a variety of
vascular beds(8, 9, 10, 11) . The
present study may suggest that the cloned P receptor
mediates UTP-induced vasoconstriction. We found that the P
transcript is also detected in rat aorta, mesentery, and cultured
RASM cells (Fig. 4). Therefore, both our novel P
and
P
receptors may be involved in the reported UTP-induced
vascular contraction. A previous study (45) demonstrated that
the rank order of potency for nucleotide-induced Ca
mobilization in RASM cells is more consistent with that of
P
receptors(16, 17) . This may suggest
that P
rather than our cloned P
receptor
functionally predominates in RASM cells. However, it is possible that
yet another P
subtype different from our cloned P
or P
(P
) are also involved in
nucleotide-induced vasoconstriction, because some studies suggested the
involvement of a P
subtype with pharmacological properties
distinct from either our P
or P
in
nucleotide-induced vascular contraction (10, 12) .
Identification of appropriate radioactive ligands and selective
antagonists for each P
receptor subtype will undoubtedly
greatly help to promote understanding the physiology of P
receptors.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D63665[GenBank].