The information on this page
was excerpted from:
- van Rhee, A.M.; Fischer, B.; Jacobson, K.A.
Modelling the P2Y purinoceptor using rhodopsin as template.
Drug Design & Discovery, 1995, 13: 133-154.
for a complete report, and more details, please use this publication as a
The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993)
FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate
residues tentatively involved in ligand recognition a molecular model of the
P2Y1 purinoceptor was constructed. The model was based on the
primary sequence and structural homology with the G protein-coupled
photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and
Weinstein ((1995) Meth. Neurosci. 25, 366-428).
New and Selective Ligands
The complete elucidation of P2 subtypes and mechanisms of action
have been impeded by the limited number of stable and selective agonists and
antagonists available. We have recently reported the synthesis of a variety of
ATP analogues modified at either the ribose or the purine, including a series
of functionalized congeners based on the potent P2Y purinoceptor
The 2-thio-ether derivatives were shown to be potent P2Y
purinergic ligands stimulating the production of inositol phosphates in turkey
erythrocyte membranes with K0.5-values ranging from 1.5 to 770 nM.
Moreover, N6-methyl-2-(5-hexenylthio)-ATP was shown to be a potent
and selective P2Y agonist (K0.5 = 26 ▒ 7 nM). Since
substitution at the N6 position of the adenine moiety seemed to
convey selectivity for a specific P2Y receptor
we now synthesized the 5'-mono- and triphosphates of
N6-(2-phenylethyl)-adenosine. Progress in the development of
antagonists has also been made recently.
The dendrogram above, composed for the sequences of
29 G protein-coupled receptors (including orphan receptors) and
bacteriorhodopsin shows that the known metabotropic P2 receptor
sequences (P2Y and P2U) are more closely related to
each other than to any other receptor. Comparison of the P2Y (chick)
and P2U (rat) receptor subtypes with other GPCR sequences showed
only low percentages of sequence identity (e.g., angiotensin II -
P2Y 27 %, angiotensin II - P2U 22 %, thrombin -
P2Y 25 %, thrombin - P2U 25 %; interleukin 8 -
P2Y 22 %; interleukin 8 - P2U 23 %).
The P2 receptors,
regardless of subtype, whether GPCR or ligand-gated ion channel, are often
regarded as related to the better characterized and more widely known adenosine
and most subsequent reviews]. From the dendrogram, however, it must be
concluded that, e.g., the A2A adenosine receptor is more
closely related to the biogenic amine receptors, than to any of the GPCR
P2 receptors. Both P2Y and P2U receptors
bear only a marginal sequence identity with the A1 adenosine
receptor (21 % for P2Y and less than 12 % for P2U
receptor). P2 receptors are more closely related to the PAF,
AT1A, and IL-8A receptors, and various orphan receptors, than
to any other GPCR subfamily.
A comparison of P2Y vs.
P2U receptors revealed 38.8 % identity and 58.6 % similarity.
In the transmembrane regions the identity percentage was even higher - 52 %.
Bacteriorhodopsin, included to facilitate comparison with other modelling
is clearly not related to any of the GPCR
subfamilies. The degree of relatedness between bacteriorhodopsin and GPCRs
shown in the dendrogram is probably an overestimate of the actual distance,
caused by the residue alignment procedure of the program.
Several residues that have been shown to be critical in ligand binding in
other GPCRs are
in the P2Y1 purinoceptor.
Potential binding sites for agonists have been explored by docking several
derivatives (including newly synthesized N6-derivatives) into the
model. The N6-phenylethyl substituent is tolerated
pharmacologically, and in our model this substituent occupies a region
predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and
F120 (TM3). The dimethylated analogue of ATP,
N6,N6-dimethyl-adenosine 5'-triphosphate, is less well
tolerated pharmacologically, and our model predicts that the attenuated activity
is due to interference with hydrogen bonding capacity to Q296 (TM7).
The also model suggests four basic residues (H121 in TM3, H266 and K269 in
TM6 and R299 in TM7) near the extracellular surface that might be involved in
ligand binding. These basic residues might be essential in coordinating the
triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP).
Figure 2: Transmembrane domain 5 thru 7, with ATP bound.
Figure 2 is an cut-away drawing of TMs 5-7, shown to contain multiple ligand
binding residues in the
The binding of the endogenous ligand ATP seems to occur
around the upper third to upper half of the helical bundle.
ATP is oriented in the plane of the lipid bilayer, almost perpendicular to
the TMs. Figure 2 also demonstrates the use of
near the bottom of TM5 and TM7, and the top of TM6.
Figure 3A: The phosphate binding domain in the P2Y1 purinoceptor.
Figures 3A and 3B focus on the ligand binding domain (BD) formed by TMs 5-7.
It appears that the binding of the triphosphate moiety is a major determinant
in binding of ligands to the P2Y1 purinergic receptor. Since this
part of the molecule contains multiple negative charges, one would expect to
find counterions in the BD.
Indeed, the P2Y1 receptor sequence
contains several positively charged residues. Our modelling study reveals that,
of these, Lys269 (TM6) and Arg299 (TM7) are likely candidates for this
function and are appropriately positioned within the helical bundle to execute
We propose that these two basic residues are assisted by two
histidine residues, His121 (TM3) and His266 (TM6), and one tyrosine residue,
Tyr125 (TM3)(Figure 3A). These residues tentatively coordinate the
alpha-phosphate (His121 and R299), the beta-phosphate (Y125, K269 and R299),
and the gamma-phosphate (His266, and K269).
Although adenosine 5'-monophosphate
analogues are widely regarded as inactive at P2 purinergic receptors,
Fischer et al
recently demonstrated that one monophosphate analogue in particular,
2-(5-hexenyl)thio-adenosine 5'-monophosphate, is a more potent
(K0.5 = 328 ▒ 43 nM; 8.5-fold over ATP) agonist at
P2Y receptors on turkey erythrocytes than ATP
(K0.5 = 2800 ▒ 700 nM).
In our model there is sufficient
coordination of the alpha-phosphate to warrant such an interaction, although
the number of stabilizing interactions, and hence the interaction energy and
affinity, will be lower than in the case of the corresponding triphosphate.
This is pointedly illustrated by the K0.5 values
of 2-(5-hexenylthio)-ATP (10 ▒ 4 nM) and 2-(5-hexenylthio)-AMP
(328 ▒ 43 nM).14 Since the interaction between
the receptor and a ligand monophosphate is much weaker than with a
triphosphate, the effect of substituents at distal sites, such as in
N6PEAMP (no effect at 10-4 M)
and N6diMeAMP (no effect at 10-4 M), increases and the
combined effect of deleting two phosphates and adding
N6-substituents proved detrimental to activity.
Although not directly involved in ligand binding, Pro218 in TM5 and Pro264 in
TM6 have a great impact on receptor structure, and therefore, the BD.
They are both located at the same distance from the membrane surface as the
ligand and, more importantly, Phe215 and Phe219 (Figure 3A, far right),
located at opposite sides of the discontinuity formed by Pro218, are in close
proximity of the terminal phosphate of ATP. This particular geometry is highly
suggestive of the much heralded, but as yet unproven conformational change
mechanism induced by agonists.
Figure 3B: The nucleoside binding domain in the P2Y1 purinoceptor.
Interestingly, Erb et al
showed that Lys289 (TM7) in the mouse P2U receptor,
corresponding to Gln296 (TM7)(Figure 3B, top left) in the chick P2Y
receptor, when mutated to an arginine, reversed the selectivity of the
triphosphates ATP and UTP to the corresponding diphosphates. In our proposed
model Gln296 (TM7) is not in the vicinity of the phosphate BD, and this
suggests that P2Y and P2U receptors display
significantly different modes of binding of ligands, as already implied by
the pharmacology-derived nomenclature of the receptors.
Two other residues were tentatively involved in coordinating the ribose moiety.
The side chain of Ser303 (TM7)(Figure 3B, left center) and the O2' are
separated by only 3.03 Å. The side chain of Ser306 (TM7)(Figure 3B,
bottom center) is within hydrogen bonding distance of O2' at 2.95
Å and O3' at 2.84 Å.
Ligand docking was initially performed with ATP, using a typical conformation
based on crystallographic data for protein-bound nucleotides. To avoid the
characteristically curled conformation of the triphosphate chain found in
several phosphate transferases (The structure in Figure 4 without explicit
hydrogens), we opted to use the nucleotide bound to the phosphate hydrolase
transducin (The structure in Figure 4 with explicit
even though it involved substituting the purine guanine with the purine
Figure 4A: Comparison of nucleotides bound to phosphate transferases or
(Alignment of the adenine moieties.)
The effect of the conformation of the triphosphate, becomes even more clear
when the triphosphate moieties are aligned (Figure 4B), rather than the
adenine moieties (Figure 4A).
Figure 4B: Comparison of nucleotides bound to phosphate transferases or
(Alignment of the triphosphate moieties.)
The orientation of the adenine
moiety relative to the ribose ring was anti (i.e., the dihedral angle
C9-N9-C1'-O4' was 30.18°). The ring puckering, defined by the dihedral
angle C1'-C2'-C3'-C4', was -3.85°, resulting in a 2'-exo, 3'-endo
conformation for the two hydroxyl groups.
We have sought to identify positively charged amino acid residues (Arg or Lys)
as anchoring points which could contribute major electrostatic interactions
with the phosphates of ATP. Such residues should be conserved within the
P2 GPCR family and should also be pointing towards the center of
the receptor cavity. Likewise, they should probably be located around the
middle or upper third of the transmembrane regions, where most of the
non-peptide GPCRs are thought to bind ligands. These requirements only yield
two possible anchoring points: Lys269 in TM6 and Arg299 in TM7.
in the A2A receptor
in the NK1 receptor,
[the equivalents of His266 (TM6) in the P2Y receptor and His262 in
the P2U receptor] were both shown to be important for
ligand binding. The same holds true for
(NK1) [the equivalents of Lys269 (TM6)],
(A2A) [the equivalents of Gln296 (TM7)],
(A2A) [the equivalents of Arg299 (TM7)],
(A2A) corresponding to Ser303 (TM7), and
(A2A) corresponding to Ser306 (TM7).
In contrast, Lys114 (TM3) in the P2Y and
(TM3) in the P2U receptor were not implicated in ligand
, whereas mutation of the equivalent residue,
in the rat m1 receptor resulted in loss of affinity.
This residue is located at the fringe of the transmembrane domain or
even in the first extracellular loop in our model. It is therefore likely
that the residue is involved in accessibility of the ligand binding domain
or in maintaining a specific structure in the loop.
Thus, we identified at least eight residues, Gln296 (TM7) in
the adenine binding domain,
Ser303 and Ser306 (both TM7) in the ribose binding domain,
His121 and Tyr125 (TM3), His266 and Lys269 (TM6),
and Arg299 (TM7) in the triphosphate binding domain,
that are involved in ligand binding according to this model.
Furthermore, we have shown that our model is consistent with the current
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