(Received for publication, April 21, 1995; and in revised form, August 21, 1995)
From the
To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-Å deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.
The neurohypophysial hormones arginine vasopressin (AVP) ()and oxytocin (OT) are two closely related nonapeptides
that exhibit a high degree of functional diversity through interaction
with specific receptors. Four different receptor subtypes have been
characterized and possess different pharmacological and G protein
coupling properties(1) . V2 vasopressin receptors are coupled
to adenylyl cyclase and are responsible for the antidiuretic effect of
AVP. V1a and V1b vasopressin receptors are both coupled to
phospholipase C. V1a receptors are involved in blood pressure control
and in all other known functions of AVP, except for the stimulation of
corticotropin secretion by the adenohypophysis which is mediated via
V1b receptor subtype. OT receptors are coupled to phospholipase C and
are responsible for the galactobolic and uterotonic effects. Recently,
cDNAs for rat, human, and pig V2 receptors (2, 3, 4) , rat and human V1a
receptors(5, 6) , pig and human OT receptors (4, 7) , fish arginine vasotocin (AVT)
receptor(8) , and human V1b receptor (9) have been
cloned and sequenced. This confirmed that these receptors belong to the
G protein-coupled receptor (GPCR) family, characterized by seven
hydrophobic putative
-helical transmembrane regions. All subtypes
cloned so far are similar to each other in both identity and size. The
level of conservation is remarkable, not only in the transmembrane
domains but also in the first and second extracellular loops. Despite
this high degree of receptor identity and a strong structural homology
between the two hormones, the receptor subtypes are distinguished on
the basis of different pharmacological and functional profiles. Many
potent selective agonists as well as peptidic and nonpeptidic
antagonists have been developed and used as pharmacological probes for
the characterization of AVP and OT receptor subtypes(10) .
Previous molecular modeling studies of GPCR, based on the bacteriorhodopsin structure(11) , suggest that a similar binding pocket is present within all these receptors(12, 13, 14) . This site has already been explored in detail in the case of the cationic neurotransmitter receptors(12, 15) . Concerning GPCR for neuropeptide ligands, a model in atomic detail of the thyrotropin-releasing hormone within its receptor has been developed(16) . In this case, the tripeptide interacts with transmembrane domains (TM) of the receptor. At present, for peptides with a size much larger than three residues, the docking of these naturally occurring hormones and neurotransmitters has not yet been defined. The AVP and OT receptor family could constitute an interesting system to study hormone-receptor interactions in combining three-dimensional modeling and mutagenesis approaches. The present study aims at delineating residues in the vasopressin receptors responsible for the high affinity binding of AVP. Therefore, we have defined a computer-generated 3D model of the rat V1a vasopressin receptor subtype which allowed us to propose residues likely to be involved in agonist binding. Among these residues, several were selected for site-directed mutagenesis on the basis of putative direct interactions with bound ligands. Our experimental results validated the 3D model of the ligand-receptor interactions. This model proposes a major anchoring of the hormone within transmembrane regions of the receptor. Large polar residues such as glutamine and lysine, common to all members of AVP and OT receptors, could be involved in hormone binding. Preliminary results supporting our conclusions have been presented in 1994 at the 76th Annual Meeting of Endocrine Society in Anaheim (California) and at the XIIth International Congress of Pharmacology in Montreal (Quebec, CANADA).
Recently, a low resolution map of bovine
rhodopsin has been published (17) . These data confirmed the
seven helix topology of GPCR, but the relative positioning and tilt
angles of the helices seemed to be somewhat different from the ones in
bacteriorhodopsin. Therefore, the existing V1a model was refined by
repositioning the seven TM helices. An electron density map at 9
Å was calculated from the refined V1a receptor model with the
program Xplor and compared with experimentally derived footprint of
bovine rhodopsin. The refined model showed a good match with the bovine
rhodopsin projection map. Finally, the extracellular and intracellular
loops were built, and the disulfide bridge between Cys and Cys
was added. Side chain conformations were
altered manually in order to optimize inter-residue interactions
between the side chains. The model was energy minimized with the
``AMBER All Atom'' parameter set installed in SYBYL 6.04 to
relax the structure and to remove bad steric contacts.
In the next
step, AVP was docked into the proposed ligand-binding site. A first
attempt to use a structure of AVP derived from the crystallographic
structure of deamino-oxytocin (18) by simple side chain
exchange and energy minimization did not lead to satisfying
receptor-ligand interactions. Therefore, the minimized coordinates of
the vasopressin structure were used as a starting point for a 300-ps
molecular dynamic simulation run using the ``AMBER All Atom''
parameter set installed in SYBYL. The shake algorithm was used with an
integration step of 1 fs and with a residue based ``cutoff''
of 8 Å. In the first 10 ps, the system was heated from 10 to 300
K with 30 K rise per 1000 steps by randomly assigning velocities from
the Bolzmann distribution. The system was then allowed to equilibrate
for 50 ps and the simulation continued for another 240 ps. The results
from the last 240 ps were collected every 5 ps. A set of 48 low energy
conformations of AVP was generated from molecular dynamic simulation
and subsequent energy minimization and was used in further docking
experiments. AVP was manually docked into the V1a receptor in order to
optimize the structural and physicochemical complementarity. The
criteria which were applied to choose among the different docking
possibilities were the following: 1) the side chain of Arg had to lay in the neighborhood of the first extracellular loop of
the receptor in order to account for the photoaffinity labeling
data(19) ; 2) the hydrophobic side of the cyclic part of AVP
(Cys
, Cys
, Tyr
, and
Phe
) had to fit into a hydrophobic pocket while the more
polar side (Gln
and Asn
) had to fit into a
polar zone of the receptor; 3) the ligand-receptor hydrogen bonding
network had to be optimal. The docking procedure was repeated several
times with different initial orientations of the ligand and of the side
chains. Only one way to fullfill the above criteria was found (see
``Results''). This ligand-receptor complex was finally energy
minimized for 2000 steps with a dielectric constant of 5.
In the
final interaction complex, AVP possesses a hydrogen bonding pattern
which is different from the x-ray structure in the related
deamino-oxytocin. In this conformation, the carbonyl oxygen of
Tyr forms a hydrogen bond with the amide proton of
Cys
. Furthermore, Phe
, Gln
, and
Asn
form a
-turn with a hydrogen bond from the
carbonyl oxygen of Phe
to the amide proton of
Asn
. A similar conformation has been found by NMR studies
of [Lys
]-vasopressin in
Me
SO(20) . In view of the limited accuracy of the
model of the receptor and the limited number of AVP conformations
investigated, the possibility that AVP might bind in a different
conformation/orientation to the receptor cannot be ruled out.
In the final model, the extracellular loops surround the entrance of
a central cavity in the transmembrane bundle, which appears as a
15-20-Å deep cleft defined essentially by helices 2-7 (Fig. 1). The bottom of the cleft is mainly hydrophobic in
nature, closed by aromatic and hydrophobic residues: Val,
Ala
, and Met
on TM3, Phe
on
TM5, Tyr
, Trp
, Phe
, and
Phe
on TM6, Ala
and Ala
on
TM7 (see legend of Table 1for the numbering of the residues). In
contrast, the entrance of the TM region and the cleft itself contain
predominantly hydrophilic residues: Gln
and Gln
on TM2, Lys
and Gln
on TM3,
Ser
and Gln
on TM4, Thr
and
Thr
on TM5, Cys
and Gln
on
TM6, and Ser
on TM7. Interestingly, this dual polarity is
also found back in the endogenous ligand AVP: the exocyclic tripeptide
Pro
-Arg
-Gly
and one side of the
hormone cycle (Gln
, Asn
) are mainly
hydrophilic, while the other part of the cycle (Cys
,
Cys
, Tyr
, and Phe
) is essentially
hydrophobic in nature. We attempted to dock AVP in the receptor cavity
taking into account this dual polarity in both the ligand and the
receptor cleft. The final docking result is given in Fig. 1. In
this model, the cyclic part of AVP is embedded within the TM region of
the receptor, filling the whole of the cleft up to the extracellular
loops. The presence of the Arg
side chain close to the
first extracellular loop is in agreement with photoaffinity labeling
studies by Kojro et al.(19) . This hypothetical model
allowed specific residues involved in hormone binding to be proposed.
In Table 1, a review of putative interactions including a series
of aromatic/aromatic, hydrogen bonding and ionic interactions likely to
stabilize the receptor-hormone complex are listed. We concentrated on
amino acid residues in the TM regions of the V1a receptor likely to be
involved in ionic and hydrogen bonding interactions with AVP. Based on
the 3D model, some of these amino acids were selected and mutated into
alanine. All the residues chosen for site-directed mutagenesis, except
Thr
, are conserved in all members of AVP and OT receptor
family cloned so far (Fig. 2).
Figure 1:
Vasopressin docked into the rat V1a
vasopressin receptor. A, view of the complex from the
extracellular surface of the receptor, in a direction perpendicular to
the membrane. B and C, side views from a direction
parallel to the cell membrane surface. B shows in detail the
interactions between the receptor and the hormone. C shows
that the binding pocket for the neuropeptide is localized in the upper
part (first third) of the transmembrane regions: white arrows indicate upper and lower limits of the lipidic bilayer. In all
panels, the positioning of transmembrane domains 1-7 is
anticlockwise. The C-chain trace of the transmembrane
regions is displayed in magenta. The vasopressin C backbone is
displayed in green. The carbon skeleton of the side chains of
residues chosen for site-directed mutagenesis are displayed in white. Oxygen atoms are in red, nitrogen atoms are in dark blue, and sulfur atoms are in yellow. Only
hydrogen atoms likely to be involved in hydrogen bonds are displayed (light blue). Except for tyrosine 115 (receptor primary
sequence numbering) located in the first extracellular loop, residues
are labeled according to the following code: the left digit indicates
the number of the transmembrane
-helix, the next two digits
indicate the rank of the residue in this transmembrane region as
numbered in the alignment in Fig. 2(e.g. Lys
is the 8th residue of helix 3).
Figure 2: Alignment of the seven putative TM domains of vasopressin and oxytocin receptors. The considered receptors are as follow: pV2, pig V2; hV2, human V2; rV2, rat V2; rV1a, rat V1a; hV1a, human V1a; hOT, human OT; pOT, pig OT, fAVT, fish AVT; and hV1b, human V1b. Residues selected for site-directed mutagenesis in the rat V1a receptor and conserved in other members of the receptor subfamily are highlighted in black.
For each
mutated receptor, except for T505A, the substitution resulted in a
substantial reduction in the affinity for the three agonists tested (Table 2). Substituting glutamine 214 and 218 by alanine resulted
in 6-fold (Q214A) and 290-fold (Q218A) reduction in AVP affinity. For
two other mutants, K308A and Q311A, loss of AVP affinity reached 60-
and 40-fold, respectively. Most of the point mutants described here
retained normal binding affinity for I-HO-LVA,
d(CH
)
[Tyr(Me)
]AVP, and
SR49059, suggesting that the chosen residues are not critical
determinants for the antagonist affinities. By contrast, one mutant
(Q413A) showed a substantial reduction in affinity for the antagonists
(100-fold for
d(CH
)
[Tyr(Me)
]AVP,
10-fold for HO-LVA and SR49059), which may indicate a perturbation of
the antagonist/receptor interaction or a general perturbation of the
receptor structure. The decrease in AVP affinity found with this mutant
was the most pronounced (1220-fold). In contrast, substitution of
threonine 505 in helix 5 (T505A) did neither affect the agonists nor
the antagonists binding, suggesting that this residue does not play a
critical role in ligand binding. The last substitution introduced in
the V1a receptor concerns glutamine 620 situated in helix 6 (Q620A).
This substitution resulted in a small decrease in affinity for the
agonists tested (8-fold). For all the mutated receptors, the loss in
affinity observed for AVP was accompanied by a similar decrease in
affinity for the two other agonists Phe
Orn
VT
and OT. The experimental results are in very good agreement with the
proposed agonist-binding site in the 3D model and strongly support our
AVP/V1a receptor interactions hypothesis. Moreover, as antagonist
binding is in general not affected, this may indicate that agonist- and
antagonist-binding sites of V1a vasopressin receptor are different.
To
verify whether the substitutions had any effect on the coupling
properties of the receptors, AVP-induced inositol phosphates
accumulation was also studied in COS-7 cells transfected with mutant
receptors. Table 3shows that for each mutant the K is shifted to higher concentrations of AVP,
except for T505A where the value is comparable to that of the
wild-type. The loss in K
value observed for each
mutant receptor paralleled and confirmed the loss in AVP affinity
observed in binding studies. Values for K
/K
ratios (AVP) calculated
for wild-type and mutant receptors are constant (6.1 < K
/K
< 10.9), except for
Q214A (ratio = 1.1), thus demonstrating that mutant receptors
are coupled to phospholipase C with properties equivalent to that of
the wild-type V1a receptor (Table 3). Q214 is located two turns
above the Asp
, a residue which plays a major role in
coupling properties of the V1a receptor (see below). This proximity
could explain a partial impairment of the coupling properties of mutant
Q214A. In our 3D model, D207 has not been proposed to directly interact
with AVP. However, since this residue is known to play a role in signal
transduction in other GPCR(27, 28) , we decided to
replace it by alanine. Results obtained with mutant receptor D207A are
characteristic of an uncoupled receptor. Indeed, even with a measurable
affinity for D207A (Table 2, K
AVP =
338 ± 61 nM), AVP was not able to stimulate inositol
phosphates production, even with high concentrations such as
10
M (basal accumulation, 4862 ± 486
disintegrations/min (n = 3), maximal concentration,
5443 ± 1700 disintegrations/min (n = 3)). In
fact, this aspartic acid residue in the second transmembrane domain is
highly conserved among GPCR. It is functionally important in biogenic
amine receptors such as
2-adrenergic receptor (27) and in
peptide receptors such as AT1a receptor (28) where its
substitution modifies ligand binding affinity and G protein coupling,
respectively. It has also been implicated in the allosteric regulation
of muscarinic receptors by intracellular sodium(29) . For the
V1a vasopressin receptor, this aspartic acid also seems to play a
crucial role in coupling to phospholipase C.
We have constructed a three-dimensional model of the rat V1a
vasopressin receptor subtype and docked the endogenous ligand AVP. As
in the previous cases
studied(12, 13, 14, 15) , the
complementarity in amino acid composition and physicochemical
properties of the cleft in the receptor and the AVP structure is
striking and represents a working hypothesis that we have
experimentally verified. According to the model, the C-terminal
carboxamide of AVP might form a strong hydrogen bond with Gln on TM2. Gln
might also contribute to AVP binding by
hydrogen bonding to the amino group of Cys
at the N
terminus of AVP and to the carboxamide at the C terminus. The mutation
of Gln
and Gln
lead to a decreased affinity
for agonists. Structure-activity relationship studies show that the AVP
terminal glycinamide is absolutely required for biological activity of
the two hormones AVP and OT(30) . These data suggest that
Gln
and Gln
could significantly contribute
to the V1a receptor agonist binding. In contrast, both mutations Q218A
and Q214A leave antagonists binding unaffected. These data are in
agreement with the observation that in antagonists the carboxamide
terminus of the peptides is not required for high affinity
binding(31) .
The interaction between a close analogue of
AVP and the bovine V2 receptor subtype has been recently studied by
photoaffinity labeling (19) . These results suggest that the
exocyclic residues of AVP are located in the vicinity of the first
extracellular loop. In our 3D model of the V1a receptor-hormone
complex, the hormone Pro-Arg
-Gly
backbone is located within the V1a receptor transmembrane domain, but
the side chain of Arg
is in the direct neighborhood of
tyrosine 225 located in the first extracellular loop (see Table 1; Tyr
in primary sequence). This tyrosine
has been shown to represent a key position responsible for agonist
binding and selectivity(32) . Indeed, when this Tyr is replaced
by the residues naturally occurring in the V2 or the OT receptor
subtypes, the agonist selectivity of the V1a receptor switches
accordingly.
The Q311A mutation leads to a decrease in AVP binding
affinity while antagonist binding is again not significantly affected.
Gln could play a role in stabilizing AVP in its active
conformation and in positioning correctly AVP, OT, and structurally
related agonists in the binding site by doing a hydrogen bond with the
backbone carbonyl of Arg
in AVP. Gln
could
form additional hydrogen bonds with the amide carbonyl proton of
Cys
and the amide proton of Gln
in AVP. It
should be noted that Gln
in AVP/OT receptors corresponds
in sequence alignment to the Asp on TM3 which is highly conserved in
cationic neurotransmitter receptors and plays a key role in their
binding(27) . A similar structural role might be played by
Lys
which is located one helix turn above
Gln
. The substitutions of Lys
and
Gln
by alanine probably make the hormone/receptor
interactions less efficient with a consequent decrease in agonist
binding. The observation that the affinity of peptidic antagonists is
not changed by these mutations suggests different interactions of
antagonists on V1a receptor.
The mutation Q413A is of special
interest since it strongly influences agonist and antagonist binding.
Furthermore, agonist and antagonist binding affinities are not affected
to the same extent. Docking of AVP highlighted a hydrogen bond between
Gln in the hormone and Gln
on TM4 in the V1a
receptor. An additional hydrogen bond can be formed between Gln
side chain and the carbonyl oxygen of Phe
in AVP.
However, the decrease in AVP affinity is too high to be accounted for
by the loss of two hydrogen bonds. One should note that Gln
is adjacent in the receptor sequence to a proline residue. As
discussed previously (12) , proline behaves as a hinge residue
in a transmembrane
-helix. This allows such helices to wobble
around an equilibrium position. Gln
can stabilize the
helix conformation by forming an intramolecular hydrogen bond via its
side chain to the helix backbone. One might think that this local
phenomenon plays a key role in the dynamics of the receptor, ligand
binding, and functional coupling to the G protein. The dramatic
decrease in AVP affinity observed in the Q413A mutant might be
explained by a perturbation in both helix 4 dynamics and hydrogen bonds
formation.
In our model, the carboxamide of Gln side
chain in the hormone makes an additional hydrogen bond with
Thr
, located on TM5. This possible interaction is of
particular interest since this threonine residue corresponds to a
serine residue conserved in catecholamine receptors. This serine has
been shown to contribute to catecholamine binding(33) .
However, the binding and efficacy of agonists and antagonists on this
T505A mutant are not affected in V1a receptor. It seems that in V1a
receptor this residue plays a much less important role in the
recognition process. Moreover, this threonine 505 on TM5 is not
conserved in V2 vasopressin receptors. Another possibility is that T505
forms a hydrogen bond with the ligand in the V1a receptor but, due to
the large total number of interactions subsites, the net contribution
of binding energy of this hydrogen bond is negligible.
With the
exception of the D207A and Q214A mutations, there is a linear
correlation between binding (K) and efficacy (K
) constants for all mutants tested. This
suggests that the hydrophilic residues studied are specifically
involved in the ligand binding process and do not modulate
intrinsically the efficacy of the functional response.
Results reported in this paper clearly indicate that the binding sites for agonists and antagonists considered do not coincide in the V1a receptor. Three different classes of antagonists, cyclic and linear peptides, as well as nonpeptide, have been tested. None of the mutations in this study significantly affected antagonist binding except Q413A and K308A. These data show that the structure of cyclic and linear peptide antagonists is not totally superimposed with the structure of agonists although these ligands have similar amino acid composition. Similar results have shown that binding sites for agonists and antagonists are different on other GPCR such as neurokinins receptors(34) .
Taken together, the experimental data reported here indicate that large, cyclic neuropeptides such as AVP and OT most probably penetrate and bind in a pocket located in the first third of the transmembrane region of the receptor, extending from the extracellular loops up to 15-20 Å deep in the transmembrane region. In the case of cationic neurotransmitter receptors, this similar binding cleft has been explored in details using site-directed mutagenesis and contains residues important for both agonist and antagonist binding and receptor activation(35) . Based both on a striking complementarity between residues in this cleft and the corresponding ligands, it was proposed that this region corresponds to the activation site in most GPCR(12, 14) .
Other peptide receptors share common properties with AVP and OT receptors. Although experimental data indicate that transmembrane regions are involved in ligand-receptor interactions in angiotensin (36) , endothelin(37, 38) , neuromedin(39) , neurokinin(40) , thyrotropin-releasing hormone(16) , and somatostatin (41) receptors, it appears that most peptides interact with residues widely distributed throughout the surface of their receptors(42) . In addition, in receptors for the neurokinins (peptides of size comparable to that of vasopressin), thrombin and chemiotactic peptide fMet-Leu-Phe, the extracellular domains are of crucial importance in the binding of either agonists or antagonists analogs(40, 43, 44) .
The
docking of AVP in V1a receptor allowed us to identify functional
domains underlying the binding of the hormone by introducing single
amino acid substitutions in the receptor sequence. Site-directed
mutagenesis was restricted to the rat V1a vasopressin receptor.
Interestingly (see Fig. 2), all residues supposed to have a key
role in agonist binding (glutamines 214, 218, 311, 413, and 620, lysine
308, and even amino acids which have not been mutated such as
Trp and Phe
) are highly conserved in all
the AVP and OT receptors cloned so far. Therefore, we propose that the
agonist binding pocket is common to all the different subtypes of this
receptor family. Moreover, as predicted from early modeling studies, it
appears that GPCR agonists with very different chemical structures such
as cationic, low molecular weight neurotransmitters, linear peptides,
cyclic peptides, retinal, and even glycoproteins have in common the
property to interact with a similar region in their respective
receptors, resulting from the transmembrane helix topology. However, as
we already demonstrated(32) , part of the agonist selectivity
of AVP/OT receptor family is not localized in the TM regions of these
receptors but in the first extracellular loop.