(Received for publication, November 9, 1994; and in revised form, December 23, 1994)
From the
Two subtypes of G protein-coupled receptors for nucleotides
(P and P
purinoreceptors) contain several
conserved positively charged amino acids in the third, sixth, and
seventh putative transmembrane helices (TMHs). Since the fully ionized
form of nucleotides has been shown to be an activating ligand for both
P
and P
purinoceptors (P
R and
P
R), we postulated that some of these positively charged
amino acids are involved in binding of the negatively charged phosphate
groups of nucleotides. To investigate the role of the conserved
positively charged amino acids in purinoceptor function, a series of
mutant P
R cDNAs were constructed so that lysine 107 and
arginine 110 in TMH 3, histidine 262 and arginine 265 in TMH 6, and
arginine 292 in TMH 7 were changed to the neutral amino acid leucine or
isoleucine. The mutated P
R cDNAs were stably expressed in
1321N1 astrocytoma cells and receptor activity was monitored by
quantitating changes in the concentration of intracellular
Ca
upon stimulation with full (ATP, UTP) or partial
(ADP, UDP) P
R agonists. Neutralization of
His
, Arg
, or Arg
caused a
100-850-fold decrease in the potency of ATP and UTP relative to
the unmutated P
R and rendered ADP and UDP ineffective. In
contrast, neutralization of Lys
or Arg
did
not alter the agonist potency or specificity of the P
R.
Neutralization of Lys
in the P
R, which is
expressed as a glutamine residue in the P
subtype, did not
alter receptor activity; however, a conservative change from lysine to
arginine at this position altered the rank order of agonist potency so
that ADP and UDP were approximately 100-fold more potent than ATP and
UTP. A three-dimensional model of the P
R indicates the
feasibility of His
, Arg
, and Arg
interactions with the phosphate groups of nucleotides.
The cDNAs encoding two subtypes of G protein-coupled receptors
(GPCRs) ()for extracellular nucleotides have been isolated (1, 2, 3, 4) and, based upon the
similarities of the predicted amino acid sequences, these nucleotide
receptors apparently form a new subset within the GPCR
superfamily(5) . These receptors, known as P
purinoceptors and recently renamed P2Y purinoceptors (6) , are linked predominantly via the G
/G
and G
/G
families of G proteins to the
inhibition of adenylyl cyclase or the mobilization of intracellular
calcium(7, 8) . Physiological processes involving G
protein-coupled P
purinoreceptors include platelet
aggregation and wound healing(9) , insulin
secretion(10) , mitogenesis(11) ,
vasodilation(12, 13) , and transepithelial ion
transport(14, 15) , making these receptors an
intriguing target for pharmacotherapy. For example, clinical studies in
cystic fibrosis patients indicate that local delivery of ATP or UTP to
lung epithelia stimulates chloride secretion and promotes mucohydration (16) , presumably by activating the P
subtype of
purinoceptors. Unfortunately, the lack of stable, selective agonists
and antagonists for the various P
purinoreceptor subtypes
has hindered further development of therapeutic applications centered
around these receptor molecules. The work described in this paper used
site-directed mutagenesis to investigate amino acid residues involved
in determining the agonist potency and specificity of the
P
R, an important step in the process of rational drug
design.
The ligand binding sites of most GPCRs, with the possible
exception of the glycoprotein and peptide hormone receptors, appear to
be located in the transmembrane -helical
regions(22, 23) . With this in mind and since the
fully ionized form of nucleotides has been shown to be an agonist for
both P
and P
purinoceptors(19, 24, 25) , we
postulated that several of the positively charged amino acids in the
putative transmembrane helices of G protein-coupled purinoceptors that
are conserved between the P
and P
subtypes
may be responsible for binding the negatively charged phosphate groups
of the nucleotide ligands.
Sequence alignment of the murine
PR, human P
R, and chick P
R
indicated that five positively charged amino acids (Lys
,
Arg
, His
, Arg
, and
Arg
) near the plasma membrane-extracellular interface of
TMH 3, TMH 6, and TMH 7 were conserved with only a modest change (from
Arg
to Lys) occurring in the P
subtype (Fig. 1). The adenosine A
receptor lacks these
lysine and arginine residues (Fig. 1), although histidine
residues in TMH 6 and TMH 7 of this receptor are involved in ligand
binding(26) . The conserved positively charged amino acids in
TMH 6 and TMH 7 are close (within 1-5 amino acids) to prolines or
glycines that have been implicated in forming hinges within
transmembrane
-helices as well as kinks and
turns(27, 28) . These perturbations of the
-helix
caused by prolines or glycines could serve to create a ligand binding
pocket involving juxtaposing residues(29) .
Figure 1:
Sequence alignment of the murine
P, human P
, chick P
, and bovine
A
adenosine receptor. Amino acids that form the third,
sixth, and seventh TMHs are shown. Altered amino acids are underlined, and the position within the murine receptor
sequence is indicated above each mutagenized residue. Hydropathicity
analysis and two-dimensional modeling of the P
R indicated
that the altered amino acids are located near the plasma
membrane-extracellular interface.
To investigate
the role of the conserved positively charged amino acids in nucleotide
receptor function, we constructed a series of mutant cDNAs that encode
PR proteins with single amino acid substitutions.
Concentration response curves generated for the wild type and mutant
P
Rs expressed in 1321N1 cells indicate that
His
, Arg
, and Arg
are
important for establishing the agonist potency and specificity of the
P
R. Neutralization of His
,
Arg
, or Arg
by substitution with the
uncharged amino acid leucine in each case caused a 100-850-fold
decrease in the potency of ATP and UTP without decreasing the efficacy
of these ligands relative to the wild type receptor (Fig. 2).
ADP and UDP, which are partial agonists of the wild type
P
R (Fig. 2), were ineffective agonists of the
His
, Arg
, and Arg
mutants at
concentrations as high as 3 mM (Fig. 2). Neutralization
of a nonconserved positively charged residue in TMH 7 (Lys
Ile) as well as the 2 conserved positively charged
residues in TMH 3 (Lys
Ile and Arg
Leu) had little effect on the agonist potency or
specificity of the P
R (Fig. 2), indicating that
these residues are not critical for receptor activation.
Figure 2:
Agonist stimulation of intracellular
calcium mobilization in 1321N1 cells transfected with wild type or
mutant PR constructs. Cells stably transfected with wild
type or mutant P
R cDNAs were assayed for P
R
activity by quantitating changes in the concentration of cytoplasmic
free calcium ([Ca
]
) in
response to the indicated concentration of ATP, UTP, ADP, or UDP. Data
are expressed as a percentage of the maximal increase in
[Ca
]
elicited by the
most effective ligand for each receptor construct and represent the
mean ± S.E. of three experiments. The maximal increase in
[Ca
]
obtained for the
wild type receptor and each mutant construct ranged from 200-500
nM, except for mutant Lys
Arg, which
exhibited a maximal increase of 90 nM. The average resting
[Ca
]
was 80-150
nM for all constructs.
Previous
studies with the PR in murine macrophages suggest that
this receptor is activated by both ATP
and
MgATP
but that ATP
is 10 times
more potent than MgATP
(apparent K
values were 0.65 µM and 6.5 µM,
respectively)(30) . Therefore, it was possible that the
rightward shift in the concentration response curves observed for the
His
Leu, Arg
Leu, and
Arg
Leu mutants was caused by a selective decrease
in receptor affinity for the fully ionized nucleotide. Activation of
these mutant P
Rs in the absence of extracellular divalent
cations, however, indicated that ATP
is still the
most potent form of agonist and that the potency of
ATP
, relative to total ATP, may be enhanced at these
mutant receptors (Table 1).
Although neutralization of
Lys by substitution with isoleucine had little effect on
the agonist potency or specificity of the P
R, a
conservative substitution of Lys
with arginine shifted
the agonist potency so that ADP and UDP were more effective agonists
than ATP and UTP (Fig. 2). Relative to the wild type
P
R, the potencies of ADP and UDP were increased by 4- and
7-fold, respectively, whereas the potencies of ATP and UTP were
decreased by 300- and 26-fold, respectively, for the Lys
Arg mutant. The efficacy of all four ligands, however, was
reduced compared to the wild type receptor (Fig. 2), which could
be due to a change in the receptor's tertiary structure
introduced by the Lys
Arg mutation or a low
expression level of this mutant receptor. Nonetheless, the altered rank
order of agonist potency of the Lys
Arg mutant
cannot be explained by a low level of receptor expression and strongly
suggests that this conservative mutation affects ligand binding and not
a subsequent step in the signaling pathway.
Interestingly,
Lys, which is conserved in the human and murine
P
R homologs, is instead the neutral amino acid glutamine
in the chick P
R (Fig. 1). Assuming that the
phosphate docking site in these nucleotide receptor subtypes is the
same, this could explain why neutralization of Lys
in the
P
R did not alter receptor activity and suggests that the
Lys
residue is not directly involved in ligand binding.
However, the finding that the Lys
Arg mutation
altered the rank order of agonist potency of the P
R could
suggest that Lys
is positioned close enough to the ligand
binding pocket so that substitution of this residue with the slightly
larger arginine residue is able to interfere with normal ligand
binding.
Molecular modeling of the PR sixth and seventh
transmembrane
-helical regions indicated the feasibility of
His
, Arg
, and Arg
interactions with the negatively charged phosphate residues of
ATP (Fig. 3). Although ligand binding could not be directly
demonstrated in this paper, due to the lack of a reliable assay, the
finding that neutralization of His
, Arg
,
and Arg
decreased the potency but not the efficacy of ATP
and UTP suggests that ligand binding is being affected. Furthermore,
the observation that the agonist potency of both ATP and UTP were
affected equally by these mutations supports the hypothesis that
His
, Arg
, and Arg
interact
with the phosphate residues rather than the purine or pyrimidine group
of these nucleotide ligands. Unlike the P
purinoceptor
subtype, the P
subtype is not activated by the pyrimidine
nucleotides UTP or UDP. Further mutagenesis studies as well as the
creation of P
/P
chimeric receptors should
help to delineate which regions of these nucleotide receptors are
responsible for selectivity of the nucleoside group.
Figure 3:
Molecular model of the PR
sixth and seventh TMHs showing the putative ATP binding site. Shown is
a space-filling model of amino acid residues (highlighted in fuchsia) that are thought to interact with the negatively
charged phosphate residues of ATP. The viewing direction is in the
plane of the plasma membrane and within the central cleft of the
receptor. ATP is colored by atom types: white, carbon; red, oxygen; orange, phosphorus; darkblue, nitrogen; lightblue,
hydrogen.