From the Department of Pharmacology & Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755
Received for publication, July 24, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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The human prostacyclin receptor is a
seven-transmembrane Vascular smooth muscle relaxation and inhibition of
platelet aggregation are two key physiological processes mediated by
human prostacyclin. Dysfunctional prostacyclin activity has been
implicated in the development of a number of cardiovascular diseases
including thrombosis, myocardial infarction, stroke, myocardial
ischemia, atherosclerosis, and systemic and pulmonary hypertension (1). In contrast to other members of the rhodopsin-like G-protein coupled receptor (GPCR)1 subfamily
such as the adrenergic receptors or other members of the prostanoid
family, there are currently no high affinity selective antagonists for
the prostacyclin receptor. This finding suggests that the prostacyclin
receptor may possess a unique ligand-binding pocket.
Receptor activation is contingent upon ligand binding interactions,
which initiate a conformational change in protein structure that is
subsequently transmitted to the G-protein. Determining the exact nature
and location of receptor-ligand binding interactions at the molecular
level is essential for understanding the functions of prostanoid
receptor physiology. Moreover, such insights would lend to the
development of novel and highly specific modes of treatment for
prostanoid-related disorders. Based upon the position of the
chromophore (covalently bound 11-cis-retinal) within the binding pocket of rhodopsin along with the location of other ligands within similar rhodopsin-type GPCRs (2), the putative binding pocket
for GPCRs with small nonpeptide ligands is believed to be located
predominantly within the hydrophobic core of the transmembrane domain
in close proximity to the extracellular boundary of the receptor.
However, the crucial anchoring points that comprise the fundamental
structure of the binding pocket, securing important receptor-ligand
associations between prostacyclin and its receptor, have yet to be determined.
As is the case with all prostanoids, prostacyclin (PGI2) is
a derivative of the C20 unsaturated fatty acid arachidonic acid (5,8,11,14-eicosatetraenoic acid) (Fig. 1). The general structure of
prostanoid molecules consists of a centralized cyclopentane ring
(thromboxane has an oxane ring) flanked by two hydrocarbon chains, the
Recent studies have begun to identify generalized regions within the
prostacyclin receptor and other prostanoid receptors that appear
crucial for ligand-binding specificity and affinity. Studies using
chimeric combinations of mouse prostaglandin D and prostaglandin I
receptors have shown that protein segments within transmembrane domains
VI and VII (TMVI and TMVII) are involved in distinct binding
interactions with prostacyclin side chains, whereas TMI along with a
portion of the first extracellular loop confers broader binding
functions, incorporating recognition and interaction with the
cyclopentane ring (5, 6). Additionally, glycosylation at Asn-7 and
Asn-78 (7) and proline residues within the transmembrane domains (8)
have also been shown to be essential for proper binding and activation.
Although neither of the two recently identified naturally occurring
polymorphisms (i.e. V25M and R212H) have revealed inherent
effects on binding, R212H in the third intracellular loop has been
shown to exclusively effect activation and exhibits defective binding
only under acidic conditions (9).
Using site-directed mutagenesis, prostanoid ligand and receptor
comparisons, and a three-dimensional computer-generated homology model
of the hIP receptor derived from the recently published crystal
structure of bovine rhodopsin (10), four crucial points of interaction
between prostacyclin and the upper perimeter of the transmembrane
domain of the hIP were identified. These crucial points include
Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII),
which interact with the C1-COOH, oxalane ring and Materials--
Iloprost ligands, radiolabeled
[3H]iloprost (17.0 Ci/mmol) and non-radiolabeled
iloprost, were purchased from Amersham Biosciences. Oligonucleotide
primers were purchased from Sigma, whereas the hIP cDNA was a
generous gift from Dr. Mark Abramovitz (Merck Frosst, Quebec, Canada).
Approach to Elucidating the hIP-binding Pocket--
Initially,
criteria were established to identify potentially important residues
within the upper half of the transmembrane domain (i.e. the
proposed locale of the putative prostacyclin-binding pocket) whose
side-chain functional groups may interact with prostacyclin via
electrostatic interactions, hydrogen bonding, or hydrophobic associations. Thus, candidate hIP residues with probable implications on ligand binding were targeted for site-directed mutagenesis, initially changing each residue to alanine. A series of competition binding assays were performed using iloprost, a stable high affinity analogue of prostacyclin. For those mutations eliciting a notable change in binding affinity, further appropriate residue replacements (catering to more specific size and/or polarity changes) were made to
determine the specific characteristics of the amino acid with adverse
effects on ligand binding. The influence on binding affinity was
postulated to be the result of: 1) direct molecular interactions among
critical binding-related residues and ligand side chains within the
immediate binding pocket; 2) indirect molecular interactions among
receptor residues involved in local preservation of the proximal
binding domain; or 3) outlying interactions among Construction of Mutant Receptors--
Human IP cDNA was
cloned into the plasmid vector pMT4, and point mutations were generated
using conventional methods of PCR mutagenesis. Complementary
oligonucleotide primers were designed extending 10-12 nucleotides 3'
and 5' from the desired mutation site. The PCR reaction mixture
contained 1× Pfu reaction buffer, 200 ng of DNA construct,
150 ng of each primer (sense and antisense), 10 mM dNTPs,
and 2.5 units of Pfu DNA polymerase (Stratagene, Austin, TX)
and was heated and cooled at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 10 min for 16 cycles. The products were then
digested with DpnI restriction enzyme (Promega, Madison, WI)
for 3 h to remove any parental wild-type strands of DNA. Ten microliters of PCR product was used to transform competent DH5 Transfection of COS-1 Cells--
Transient transfections were
performed on COS-1 cells as follows: the initial wash of cells with
Cellgro® Dulbecco's modified Eagle's medium (DMEM, Mediatech, Inc.,
Herndon, VA) was followed by addition of mutant DNA (20 µg/plate) in
diethylaminoethyl-Dextran (DEAE-Dextran, Sigma) and DMEM. Cells were
then incubated at 37 °C with 5% CO2 for 6 h after
which 0.1 mM chloroquine solution was added. Cells were
subsequently incubated for 1 h, and chloroquine was removed
through washes with DMEM. Cells were harvested 72 h
post-transfection.
Membrane Preparations--
Preparations of COS-1 cell membranes
were carried out as follows: cells were washed in phosphate-buffered
saline and harvested using cell scrapers. Vortexing (providing shear
forces) for 3 min in sucrose (0.25 M) was followed by low
speed spin (~1260 × g) for 5 min, and the
supernatant collected. After a high speed centrifugation
(~30,000 × g for 15 min), the pellet was then washed twice in 1× HEM (20 mM Hepes, pH 7.4, 1.5 mM
EGTA, and 12.5 mM MgCl2) followed by
resuspension in 1× HEM containing 10% glycerol and stored at
Ligand Binding--
Ligand-binding characteristics for the
expressed receptors were determined through a series of competition
binding assays using the radiolabeled ligand
[3H]iloprost. An analysis involved the construction of
reaction mixtures (in duplicate wells) containing 50 µg of membrane
protein, HEM buffer, and 15 nM [3H]iloprost
along with 1 of 11 different concentrations of cold (non-radiolabeled)
iloprost ranging from 10 µM to 0.1 nM. After 1.5 h of incubation at 4 °C, reactions were stopped by the
addition of ice-cold 10 mM Tris/HCl buffer, pH 7.4, and
filtered onto Whatman® GF/C glass-fiber filters using a Brandel®
cell harvester. The filters were washed five times with ice-cold
Tris/HCl buffer, and radioactivity was measured in the presence of 5 ml
of EcoscintTM H scintillation fluid (National Diagnostics,
Atlanta, GA). Nonspecific binding was determined by the addition of a
500-fold excess of non-radiolabeled iloprost. The concentration of
[3H]iloprost was varied from 1 to 100 nM for
saturation binding studies. Data were analyzed using GraphPad Prism
software (GraphPad Software, Inc., San Diego, CA). IC50
values were converted to Ki using the Cheng-Prusoff
equation, and Ki values were expressed as a
mean ± S.E. An analysis of variance (ANOVA) and Student's
t tests were used to determine significance differences
(p < 0.05).
Western Blot Analysis--
The presence of mutant protein with
low affinity to iloprost was determined through Western blot analysis
using monoclonal antibodies targeting the 1D4 epitope tag (8). Thirty
microliters of membrane preparation containing 30 µg of membrane
protein was subjected to 10% SDS-polyacrylamide gel electrophoresis.
This was transferred to a nitrocellulose membrane, immunoblotted using a 1D4 monoclonal antibody and horseradish peroxidase-conjugated anti-mouse secondary antibody, and detected with enhanced
chemiluminescence reagents.
Modeling of Prostacyclin Ligand--
Because of the instability
of the native ligand (prostacyclin), a synthetic analogue (iloprost)
(Fig. 1) was used in all experimental assays. However, seeing that both structures are virtually identical and share the same reactive components that confer binding
specificity with the hIP (e.g. C1-COOH, C11-OH, C15-OH,
bicyclic ring, Modeling of hIP Receptor--
A theoretical three-dimensional
homology model of the seven-transmembrane The prostacyclin receptor serves important roles in vascular
smooth muscle relaxation, platelet aggregation, and inflammation (13-16). Prostacyclin agonists are now widely used for the treatment of pulmonary hypertension (17-20). More recently, it has also been suggested that prostacyclin may also be useful as a therapeutic agent
in treating lung (21) and colon cancers (22, 23). Our current knowledge
of the hIP receptor is very limited with only a handful of studies
addressing the structure-function characteristics of this important
receptor. Currently, there are no commercially available high affinity
antagonists and only a few stable high affinity agonists. An
understanding of the particular residues that constitute the
ligand-binding pocket would be useful in determining any unusual
features this receptor may contain and assist in the development of
more functionally specific hIP ligands, particularly selective antagonists.
11 of 29 Candidate Residues When Mutated to Alanine Had a
Significant Decrease in Binding Affinity for Iloprost--
Candidate
residues were first individually mutated to alanine (alanine scanning)
to determine those that had an affect on binding affinity (Table
I). These amino acids were selected based upon their potential to interact with prostacyclin side chains. To avoid preconceived bias with regard to the binding pocket position within the seven-transmembrane domains, all prospective binding pocket
residues such as charged, polar, and large side-chain amino acids
(e.g. phenylalanines and tyrosines) in the upper half of the
TM domain were mutated. The wild-type prostacyclin receptor expressed
well (1.8 pmol/mg membrane protein) with a binding affinity (Ki) for iloprost of 7.9 ± 1.7 nM
(n = 9) (Table I). The competition binding best fitted
to a one-site competition binding curve. In the absence of a high
affinity antagonist, the labeled agonist iloprost was used for
competition binding. Further studies were performed on the wild-type
receptor in the presence of Gpp(NH)p, a non-hydrolyzable form of GTP,
to effectively uncouple G-protein from the receptor. The presence of
100 µM Gpp(NH)p had no significant effect on binding
affinity for wild-type protein (7.9 ± 0.1 nM
(n = 3)), establishing that this affinity was not the
result of G-protein coupling. Of the 29 residues mutated, 11 had a
significant effect on binding (Table I) (Fig.
2, squares). These were
located in TMII (D60A, S68A, and Y75A), TMIII (F95A and F97A), TMIV
(F150A), TMV (S185A and Y188A), and TMVII (D274A, F278A, and R279A). In
the absence of a high affinity antagonist for ligand binding, only
those mutant receptors with significant differences of 0.5-1.7 log in
binding affinity (S68A, F95A, F97A, S185A, and F278A) compared with
wild-type receptor were able to yield a competition binding curve (Fig.
3). For those remaining mutants with
extremely low binding affinity (>500 nM) (D60A, Y75A, F150A, Y188A, D274A, and R279A), little iloprost binding was observed despite adequate amounts of protein being present. Both Western analysis (Fig. 4) and saturation binding
(Table I) showed detectable yet significantly reduced amounts of
protein expression for these severely affected mutant receptors.
Moreover, Western analysis also revealed the complex-glycosylated
states (multiple bands) that are typically observed with these
receptors (8).
Evidence from Sequence Alignments, Ligand Comparisons, and Modeling
Supporting Four Residues as Having Direct Agonist Receptor
Interactions--
Receptor configuration and TM helices were based
upon sequence homology and alignment with the crystal structure of the
rhodopsin receptor. Highly conserved ligand substituents on
prostaglandins are likely to interact with conserved amino acids on the
prostaglandin receptor. This principle has been used and confirmed in
the investigation of many GPCRs including the adrenergic receptors (4)
and rhodopsin (24). Independently, the computer-generated homology
model of the hIP transmembrane domain (minus ligand) was evaluated and visualized using the Swiss PDB Viewer program. Initial superimposition of the model with the crystal structure of rhodopsin yielded
significant structural similarity. To further determine the validity of
our hIP model, a Ramachandran plot analysis was employed. Of the 182 amino acids formulating the transmembrane helices, 180 (99%) were within the allowable region for a right-handed Arg-279 (TMVII) Forms Ionic Interaction with the C1-Carboxylate
Group--
The most significant binding pocket amino acid is the
highly conserved (100%) Arg-279 found within the seventh transmembrane domain (TMVII) of the hIP receptor. Upon mutation to alanine (R279A), a
significant decrease in agonist binding affinity was observed (Ki > 500 nM iloprost,
p < 0.001) (Table I) as compared with the hIP1D4
wild-type receptor (Ki = 7.9 ± 1.7 nM iloprost). In addition, R279A protein expression was
decreased greater than 3-fold in comparison with the wild-type
construct (R279A Bmax = 0.5 pmol/mg membrane
protein versus hIP1D4 Bmax = 1.8 pmol/mg membrane protein). Previous mutagenesis studies performed on
the EP3 (25, 26) and EP2 (27) receptors have highlighted the impact of
this residue in both ligand binding as well as receptor activation.
Moreover, it has been shown that this residue has the capacity to not
only form an ionic bond with the C1-carboxylate group of various
ligands but serves as a hydrogen donor for carbonyl groups as well. EP1
receptor studies have confirmed that the primary interaction between
this residue and ligand constituents is ionic (electrostatic) rather
than hydrogen bonding since modification to various esters resulted in
a greatly reduced affinity and potency (28). Thus, the complete level
of conservation of Arg-279 across all of the prostanoid receptors,
marked effect on ligand binding (when mutated to alanine), and complete
conservation of the C1-carboxylate among all of the native prostanoid
agonists strongly supports a direct ionic interaction between Arg-279
and the C1-carboxylate of prostacyclin (Fig.
5).
Phe-278 (TMVII) Provides Supplemental Hydrophobic Interaction with
Oxolane Ring and Tyr-75 (TMII) Exhibits Functional Duality with C11-Hydroxyl and
Cyclopentane Ring--
The Tyr-75 residue on TMII is conserved
throughout the majority of the prostanoid receptors with the exception
of the thromboxane receptors, which contain a histidine residue.
Interestingly, a C11-hydroxyl group is present on all native
prostanoids with the exception of thromboxane. In the prostaglandins,
this hydroxyl is attached to C11, a member of a cyclopentane ring;
however, in thromboxane, the equivalent oxygen is incorporated as part of a six-membered oxane ring (Fig. 1). Furthermore, from energy minimization, the C11-hydroxyl group on prostacyclin is in close proximity with the C1-carboxylate moiety. This is closely paralleled by
the close proximity between Arg-279 and Tyr-75, adding further support
for our model. Thus, our mutagenesis and modeling results predict a
direct interaction between Tyr-75 and the C11-hydroxyl group as binding
of agonist with the Y75A mutant revealed a greater than 50-fold
decrease in affinity (p < 0.001) with little specific binding (Ki > 500 nM iloprost) (Table
I) as compared with the hIP1D4 wild-type receptor
(Ki = 7.9 ± 1.7 nM iloprost). To
determine the precise structural features involved in this
residue-ligand interaction (i.e. the presence of a phenyl ring, a hydroxyl group, or both), further mutagenesis (Y75F and Y75S)
was performed. Unexpectedly, no significant defects in binding were
observed with either of the additional mutations with
Ki values comparable with wild type (Y75F
Ki = 10.5 ± 4.1 nM iloprost
(n = 5) and Y75S Ki = 13.3 ± 2.8 nM iloprost (n = 4)). Expression was
decreased to roughly 30% for all three mutations with
Bmax values of 0.5, 0.4, and 0.5 pmol/mg
membrane protein for Y75A, Y75F, and Y75S, respectively. From our
modeling, hydrogen bonding appears to be the major means for
interaction (Fig. 5); however spatial flexibility (by angstroms) in
either the ligand or the receptor may allow for the formation of
hydrophobic ring-ring interactions between the centralized
(non-oxolane) cyclopentane ring of prostacyclin and the Tyr-75-phenyl
ring. Thus, either interaction alone (i.e. Y75F hydrophobic
ring-ring association or Y75S hydrogen bond) is sufficient in
maintaining efficient receptor-ligand affinity. Possessing a high level
of conservation across all prostanoid receptors (apart from the
thromboxane receptor), shared common structural C11-hydroxyl groups
with ligands (apart from thromboxane), close proximity to the critical
Arg-279 residue, and reduced affinity upon mutagenesis to alanine
supports Tyr-75 as an anchoring point to the C11-hydroxyl of prostacyclin.
Phe-95 (TMIII) Is a Key Element in Accommodating the
Residues That Affect Binding but Are Predicted to Have Indirect
Ligand-Receptor Interactions--
Despite significant effects on
binding affinity, amino acid residues Asp-60, Ser-68, Phe-97,
Phe-150, Ser-185, Tyr-188, and Asp-274 are not predicted by our model
to be directly involved in receptor-ligand binding (Table I). Asp-60
(TMII), although highly conserved (100%) across all prostanoid
receptors, lies too far (12.3 Å) from our proposed ligand-binding
pocket. Similarly, Ser-68 in TMII, although moderately conserved
(~40%), is not predicted to be directly involved in binding. Phe-97
contributes to the largely hydrophobic region found in TMIII. Although
it is very close to its Phe-95 counterpart, Phe-97 is oriented away
from the receptor-bound prostacyclin molecule and thus has no direct impact on ligand binding. When mutated to alanine (F97A), a marked decrease in affinity suggests a potential role as a TMIII position stabilizer through interhelical interactions with the adjacent TMIV.
Phe-150 is also an important binding-related residue as indicated by
the marked reduction in binding affinity upon mutation to alanine
(Ki > 500 nM iloprost
(n = 4); p < 0.001) (Table I).
However, contributions to ligand binding are indirect due to the
increased distance (~ 8.5 Å) from the bound ligand as predicted by
our model. Ser-185 in TMV is found in only 29% of all prostanoid
receptors and has been shown to moderately affect ligand-binding
affinity upon mutation to alanine. According to our model, Ser-185 is
in close proximity to both Phe-150 (TMIV) and Tyr-188 (TMV). The
Tyr-188 position is conserved only in mass (across the prostanoid
receptors) with the majority of amino acids at this position being
phenylalanine. Unlike Tyr-75, the Tyr-188 residue is not predicted to
be directly involved in receptor-ligand interactions but rather may
serve as a structural contributor, participating in potential
hydrophobic (ring-ring) interactions with F146 (TMIV). Asp-274 in TMVII
is conserved in ~40% of all prostanoid receptors. Within our
proposed model, no direct residue-ligand interaction was evident;
however, the proximity to the ligand combined with a Model Based Identification of Additional Interactions and
Compensatory Mutations--
With the receptor-bound prostacyclin
molecule in place and the structure refined, the ability to probe for
other receptor-ligand interactions was now possible using the Swiss PDB
Viewer as an exploratory device. Two additional residues, namely Leu-67
(TMII) and Met-99 (TMIII), were initially identified as being potential binding pocket contributors, affording probable receptor-ligand associations within the L67A and L67W (TMII)--
A probable binding pocket contributor,
Leu-67, is found in only a small number of prostanoid receptors
including the hIP. Normally, such a small and comparatively unreactive
(non-polar) molecule would not be sought out as a direct contributor to
ligand binding as was corroborated by an L67A mutation, which exhibited wild-type-like affinity (Ki = 4.8 ± 1.1 nM iloprost (n = 3)) (Fig. 5). However,
when converted to a much larger amino acid (i.e. tryptophan,
L67W), a significant decrease in binding affinity was observed
(Ki > 500 nM iloprost;
p < 0.001 (n = 3)). A steric repulsion
between the larger L67W side chain and the M99L (TMIII)--
Another prospective binding pocket residue with
potential direct interaction with ligand constituents was Met-99
(TMIII), which is highly conserved and present in ~88% of all
prostanoid receptors. In reviewing our model (Fig. 5), it was our
belief that Met-99 may contribute to binding affinity through hydrogen bond formation with the C15-hydroxyl group of prostacyclin, which is a
highly preserved feature in all prostaglandin ligands. However, a
methionine-to-leucine change (M99L) at this position exhibited no
significant change in binding affinity (Ki = 2.7 ± 0.7 nM iloprost (n = 3)) as
compared with wild type. This is consistent with previous studies on
the EP2, EP3, and EP4 receptor subtypes, which showed that the
conserved C15-hydroxyl group may not play an important role in agonist
affinity (28, 32).
F95A (TMIII) in Combination with V71L or V71F (TMII)--
As both
the above results added marginal support to our model, we directed our
focus toward producing compensatory mutations that might help
counteract one of the more destructive binding pocket changes examined
earlier, namely the important Phe-95 residue (TMIII) that (upon
mutation to alanine) disrupted the hydrophobic interaction with the
Model-based Prediction of hIP Activation by Prostacyclin--
It
should be noted here that our model is a static image of the initial
binding of ligand to receptor. Such an interaction in reality is
dynamic with significant changes in both receptor and ligand
conformation. Being an agonist, prostacyclin and iloprost within the
binding pocket would result in conformational changes initiating
receptor activation. It has been observed for rhodopsin as well as
other GPCRs including our hIP studies that poor receptor expression
occurs upon mutation of residues critical for ligand binding (33, 34).
This observation strongly suggests that, in addition to binding, these
residues may also serve as important structural stabilizers in the
empty state (no ligand). Moreover, these binding pocket residues may
contribute to the constraining influence on receptors that when broken
by ligand (e.g. salt bridges) leads to receptor activation
(33). As a consequence of prostacyclin or iloprost binding, such
stabilizing factors may be disrupted, leading to both ligand and
receptor conformational changes. Given the position of prostacyclin in
the binding pocket, we would predict that TMIII and TMVII would rotate
and move apart upon agonist binding. There is precedence for such
movements as biochemical and EPR assays on rhodopsin upon
photoisomerization of 11-cis-retinal to
all-trans-retinal have shown movements in TMIII and TMVII
(29, 35-38). These major changes are most probably induced by ligand chain movements (e.g. The Unique IP Agonist-binding Pocket--
This study pinpoints
specific residues that comprise the fundamental structure of the
hIP-binding pocket, securing crucial receptor-ligand associations as
well as those amino acids in close proximity to the general binding
domain. Our findings support a structural model of receptor-bound
prostacyclin in which four distinct anchoring sites (comprised by seven
TM amino acids) link ligand to receptor. These observations were
somewhat unexpected because they placed the prostacyclin-binding
pocket at the same level but in an opposing direction to the ligands of
rhodopsin and the biogenic amine receptors, both of which also have
ligands consisting of a carbon ring with a hydrocarbon chain
(catecholamine and 11-cis-retinal, respectively). For
rhodopsin, the -helical G-protein coupled receptor, which plays
important roles in both vascular smooth muscle relaxation as well as
prevention of blood coagulation. The position of the native
ligand-binding pocket for prostacyclin as well as other derivatives of
the 20-carbon eicosanoid, arachidonic acid, has yet to be determined.
Through the use of prostanoid receptor sequence alignments,
site-directed mutagenesis, and the 2.8-Å x-ray crystallographic
structure of bovine rhodopsin, we have developed a three-dimensional
model of the agonist-binding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII
exhibited a marked decrease in agonist binding. Of this group, four
amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII),
and Phe-95 (TMIII), were identified (via receptor amino acid sequence
alignment, ligand structural comparison, and computer-assisted homology
modeling) as having direct molecular interactions with ligand
side-chain constituents. This binding pocket is distinct from that of
the biogenic amine receptors and rhodopsin where the native ligands
(also composed of a carbon ring and a carbon chain) are accommodated in
an opposing direction. These findings should assist in the development
of novel and highly specific ligands including selective antagonists
for further molecular pharmacogenetic studies of the human prostacyclin receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
- and
-chains, whose configuration and functional groups
determine further classification (Fig. 1). In particular, the
prostacyclin molecule contains an additional oxolane (cyclic ether)
ring fused to the cyclopentane ring as well as two hydroxyl groups
located at C11 and C15. A characteristic terminal carboxylate group is
present at the C1 position as well as carbon-carbon double bonds
linking C5 to C6 and C13 to C14. Similar molecular features can be seen in synthetic prostacyclin analogues such as iloprost, a
stable high affinity agonist that substitutes a secondary cyclopentane ring in place of the PGI2 oxolane ring, carries an
additional C16-methyl group and a
-chain triple bond (Fig. 1). Side
chains of certain amino acids have been shown in receptors to interact directly with substituents of ligands, conferring binding affinity (3,
4) through major forces such as hydrogen bonding, hydrophobic interactions, and ionic interactions. Thus, structural similarities and
differences between both prostanoid receptors and prostanoid ligands
play an important role in determining sites of interaction between
receptor and ligand. For example, conserved serine residues found in
TMV of the adrenergic receptors have been shown to interact with the
conserved hydroxyl groups extending from the catechol ring of the
biogenic amines (4).
-tail, C11-OH, and
-tail of prostacyclin, respectively. This agonist-binding pocket is
quite distinct from that of the biogenic amine receptors and rhodopsin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix-maintaining
amino acids. To further define the receptor amino acids that interacted
with ligand, sequence alignments for all prostanoid receptor
transmembrane domains were obtained from GPCRDB Prostanoid
(www.gpcr.org). Forty-two prostanoid receptor sequences including
multiple PE21, PE22, PE23, PE24, PF2R, TA2R, PD2R, and PI2R from
different species were internally compared with each other as well as
with rhodopsin. Similar principles were applied in comparing functional
groups of prostacyclin (as well as its analogues) to other
prostaglandin ligands. Conservation of amino acids at equivalent
positions on corresponding receptors along with the maintenance of
certain functional groups on other prostaglandins suggested probable
functional correlations and importance. Residues identified within the
putative binding pocket and exhibiting substantial influence on ligand
binding affinity were reconstructed on a computer-generated hIP model
based upon the crystal structure of rhodopsin. A structural-based
prostacyclin molecule was then inserted (bound) onto the model receptor
comparing multiple positions. Taking into account all of the
aforementioned features, a theoretical three-dimensional model of the
prostacyclin-binding pocket was developed.
Escherichia coli cells (~2 × 109 cells)
followed by DNA extraction from selected clones. Large plasmid
preparations were performed using Wizard® Plus Maxiprep kits (Promega), and all mutant constructs were confirmed via PCR DNA
dideoxynucleotide chain termination sequencing (Molecular Biology Core
Facility, Dartmouth Medical School, Hanover, NH).
70 °C. A Bradford protein assay was performed to quantitate
membrane proteins.
-chain, and
-chain) in addition to identical
binding affinities, it was our feeling that the endogenous prostacyclin
molecule would be more appropriate and biologically relevant within our
modeling system. Computer-assisted molecular modeling of the
prostacyclin ligand (native agonist to the hIP receptor) was performed
using Swiss PDB Viewer (GlaxoSmithKline, Geneva, Switzerland) (11). A
three-dimensional PGI2 molecule was modeled based upon the
known chemical structure as well as a previously predicted conformation of a receptor-associated PGI2 (12). Initial modeling
constraints required the adjustment of individual atoms such that they
would conform to the chemically acceptable limits of the ligand
structure. This included the adjustment of all covalent bonds to agree
with standard lengths and angles: sp3-hybridized C-C
single bond = 1.54 Å and 109.5°; sp2-hybridized C=C
double bond = 1.33 Å and 120.0°; sp3-hybridized
C-O single bond = 1.40 Å and 108.0°; and
sp2-hybridized C=O double bond = 1.20 Å and 120.0°.
Because of substantial torsional strain, all interior angles for both
five-membered rings were approximated at 108.0° with the exception
for the C-O-C bond angle of the oxane ring, which was set closer to
112.0°. The root mean square deviation for all bonds and angles was
calculated at 0.006 Å and 0.4°, respectively, compared with the
standard numbers referenced above. Furthermore, the two rings were
constructed to assume the common envelope configuration with the
characteristic four co-planar atoms combined with a fifth member out of
plane. The structural conformation of our receptor-bound prostacyclin molecule is similar to that of previous investigations in modeling conformations of receptor-associated PGI2 (12) where the
-chain is maintained in a bent conformation (back upon the two
centralized rings), whereas the long hydrophobic
-chain is in an
extended configuration. This configuration was independently confirmed using MacSpartan Pro software where the energy-minimized conformations (
7 to
10 kcal/mol) all exhibited such a ring and
-chain
conformation with the major variances being in position of the
-chain (data not shown).
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Fig. 1.
Structures of prostanoid precursor and
selected prostanoid ligands. An illustration comparing the
structural similarities and differences of some representative
prostanoid ligands is shown. A, arachidonic acid
(5,8,11,14-eicosatetraenoic acid) serves as the precursor molecule for
all native prostaglandin and thromboxane ligands. B, three
native prostanoids (PGI2, PGE1, and
TXA2) showing common features: the C1-COOH, the -chain;
the C11-OH, the
-chain; and the C15-OH. There are distinct variances
within the centralized ring structure with prostacyclin having two
rings. C, the structure of iloprost (stable, high affinity
synthetic PGI2 analogue) is shown. It has the four common
structural features described in B. However, a cyclopentane
ring replaces the oxalane ring, there is a C16-methyl substitution, and
there is a C18-triple bond.
-helices of the hIP
receptor was constructed using the internet-based protein-modeling
server Swiss Model (GlaxoSmithKline) (11). Amino acid sequences from
all prostanoid receptors were obtained from the GPCR data base (42 sequences in total, GPCRDB Prostanoid) and aligned with those of the
bovine rhodopsin receptor. Based upon this alignment, seven distinct
peptide segments (each containing 26 amino acids) corresponding to the
seven-transmembrane domains of the hIP were determined, and a homology
model was generated using the 2.8-Å resolution x-ray crystallographic
structure of the bovine rhodopsin receptor as the template (Protein
Data Bank code 1HZX). Receptor residues were tethered by harmonic
constraints to their corresponding rhodopsin transmembrane templates
and assembled into helical conformations by successive manipulations of
selected degrees of freedom (rigid body rotational/translational
followed by torsional). Once assembled, the transmembrane domains were energy-minimized utilizing the Gromos96 force field to improve the
stereochemistry of the model and remove unfavorable clashes (Swiss
Model). Visualization and evaluation of the model as well as insertion
of the prostacyclin ligand was performed using the Swiss PDB Viewer.
Additional amino acids were added to the extracellular transmembrane
domain region and allowed to adopt an
-helical conformation (where
appropriate) using the crystal structure of rhodopsin as a template.
From the crystal structure of the rhodopsin, some of the transmembrane
-helices extend beyond 26 amino acids (e.g. TMIII).
Furthermore, the binding of prostacyclin may extend into the
extracellular domain. The complete interhelical loops were
intentionally excluded from our model as they are known in rhodopsin to
be flexible with areas missing from the crystal structure. In addition,
they share no homology with the loops of the hIP. Thus, our study
focuses exclusively on the putative binding pocket within the
transmembrane domain of the hIP.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Competition binding and saturation binding experiments for the initial
29 transmembrane mutations to alanine
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Fig. 2.
Mutation sites on secondary structure of the
hIP receptor. Diagram highlighting the positions for all 31 (29 original plus 2 latter mutations) putative binding pocket residues
within the upper half of the seven-transmembrane helical domains
(shaded boxes) is shown. Shown in bold squares
are the residues that showed a significant difference in iloprost
binding when mutated to alanine as compared with the wild-type hIP1D4
protein. The circles are the residues that are unaffected by
such an alanine mutation. Extracellular, transmembrane, and cytoplasmic
regions are designated based on the structure of rhodopsin with
approximate membrane boundaries numbered. The conserved disulfide bond
found in the extracellular domain is indicated with a dashed
line as are the two palmitoylation sites located along the
cytoplasmic domain. The two glycosylation sites are indicated by the
small circles. The C terminus has been tagged with a 1D4
epitope (bold). A shaded box highlights the
region for the putative ligand-binding pocket.
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Fig. 3.
Competition binding for hIP1D4 and mutations
that exhibited a significant decrease in binding affinity.
Competition-binding curves (15 nM
[3H]iloprost versus 12 different
concentrations of non-radiolabeled iloprost 10 µM-0.1
nM) are shown. Shown are the mutant constructs (S68A, F95A,
F97A, S185A, and F278A) that had significantly reduced binding affinity
as compared with wild-type hIP1D4. All of them show a significant and
parallel shift to the right as compared with the wild-type curve.
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Fig. 4.
Western analysis for hIP1D4 and mutants with
very low binding affinity. Western analysis was performed as
described under "Experimental Procedures" using the high affinity
1D4 monoclonal antibody to the C-terminal epitope tag. Those mutations,
D60A, Y75A, F150A, Y188A, D274A, F278A, and R279A, resulting in
significant binding deficits were compared (all transfected in parallel
using 20 µg of DNA/15-cm plate). Thirty micrograms of
membrane-prepared protein was loaded/well with the exception of WT
(hIP1D4), which only contained 15 µg of membrane protein.
-helix, whereas 171 (94%) were within the preferred region. This corresponded well with
the expected seven-transmembrane
-helical secondary structure of the
hIP receptor. It was also determined (via Swiss PDB Viewer) that none
of the residues was involved in any intramolecular or intermolecular
clashes with the protein backbone or other side-chain constituents, nor
were there any signs of global inconsistencies. By inserting
prostacyclin into the model of the hIP, identification of distinct
receptor-ligand interactions was possible. The major determinant for
the final receptor-bound position of prostacyclin was based upon the
experimentally observed requirement for the C1-carboxylate to interact
with Arg-279.
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Fig. 5.
Molecular model of receptor-bound
prostacyclin and important binding pocket residues. Homology-based
model of hIP receptor transmembrane domains with bound prostacyclin
ligand is shown. An extracellular view of the critical contacts made
between receptor residues within transmembrane domains II, III, and
VII, and prostacyclin is shown. Green-dashed lines represent
interactions, either hydrogen bonding or electrostatic attractions. The
major sites of interactions are electrostatic interaction
(green-dashed lines) between Arg-279 and the negatively
charged C1-carboxylate group of prostacyclin; hydrophobic ring-ring
interaction between Phe-278 and the oxolane ring of prostacyclin; and
hydrogen bond (green-dashed line) and hydrophobic
interactions between Tyr-75 and the C11-hydroxyl group of
prostacyclin and hydrophobic stabilizations among Phe-95, Leu-67,
and the -chain of prostacyclin. Phe-97, although not directly
interacting with the ligand, forms an important interhelical
interaction with TMIV.
-Chain--
Directly adjacent to the crucial
Arg-279 residue is Phe-278, an exclusive residue found only in the IP
receptor at this position. The distinctiveness of this residue seems to
directly correlate with unique structural features found on IP receptor
ligands, in particular the additional oxolane (cyclic ether) ring found on the native prostacyclin ligand. Some synthetic IP ligands such as
iloprost and carbacyclin contain secondary cyclopentane rings at this
position, whereas other naturally occurring prostaglandins (e.g. PGE1) do not (Fig. 1). A drastic reduction
in binding affinity was observed in the F278A mutation with
Ki = 351.3 ± 88.9 nM
(n = 5) iloprost (p < 0.001) (Table
I). Expression was also diminished as indicated by the lowered
Bmax value of 0.8 pmol/mg membrane protein.
Unlike the positively charged Arg-279, the Phe-278 residue contains a
completely non-polar side chain. Thus, the most likely association
between this residue and prostacyclin involves a hydrophobic ring-ring
interaction between the oxolane ring of prostacyclin (cyclopentane ring
of iloprost) and the Phe-278 phenyl constituent. As observed with our
model (Fig. 5), there may be an additional interaction with the
-chain. It is our belief that both of these ring structures serve as
unique yet corresponding elements that are crucial for proper ligand
binding and help to supplement the main electrostatic bond at Arg-279.
The combination of Phe-278 and Arg-279 facilitate key anchoring points
at the C1-carboxylate chain and secondary oxolane ring of prostacyclin, accommodating the
-chain such that it remains in a "bent" state (Fig. 5) as observed with the energy-minimized configurations of prostacyclin.
-Chain--
The Phe-95 residue is part of a phenylalanine-rich area
within TM domain III. According to our model and binding analyses, Phe-95 plays a crucial role in accommodating the mainly hydrophobic
-chain of prostacyclin. A cluster of hydrophobic residues is present
in TMIII on all prostanoid receptors in the region of the putative
binding pocket. The binding affinity for the F95A mutation differed
significantly from the wild-type hIP1D4, revealing a considerable
binding deficit (F95A Ki = 143.6 ± 98 nM iloprost (n = 6); p < 0.05) (Table I). The Bmax value for F95A was 0.8 pmol/mg membrane protein. The Phe-95 side chain provides a planar
hydrophobic "sidewall" that helps to secure the freely rotating
-chain constituent (Fig. 5). Previous chimera studies have shown
this entire TM region to be interchangeable between mouse prostaglandin
I and mouse prostaglandin D receptors (6). Furthermore, TMIII is one of
two critical regions required for GPCR activation (29-31). We have
previously shown that this region is also likely to be similarly
important in the hIP (8). Thus, with attachments for the
-chain
carboxylate group (Arg-279, TMVII) and both centralized rings (Phe-278
(TMVII) and Tyr-75 (TMII)) accounted for, the large hydrophobic region
of TMIII, which includes Phe-95, comprises the fourth fundamental point
of attachment-interaction between receptor and ligand, accommodating
the
-chain.
-carbon
carboxylate group (negative charge) suggests that Asp-274 is an
essential structural contributor near the binding domain, possibly
through the formation of a salt bridge. Therefore, these indirect
binding-related residues are important in supporting the fundamental
binding pocket and anchoring points established for the receptor-bound
prostacyclin model (i.e. Arg-279, Phe-278, Tyr-75, and
Phe-95). Further model refinements were pursued.
-chain area of prostacyclin. An additional residue, Val-71, was targeted as a possible compensatory mutation candidate for the binding deficit created by F95A. The prognostic capability and succeeding results achieved with our working model lend
to both its internal validity and accuracy.
-chain of prostacyclin is
predicted by our model (Fig. 5). Therefore, with accommodating
position, size, and side-chain neutrality, Leu-67 seemingly complements
other hydrophobic residues in containing the
-chain of prostacyclin
as it is bound to the hIP receptor.
-chain. Upon examination of our model, Val-71 in TMII appeared to be
a good candidate for compensation of the F95A-induced binding deficit
when changed to phenylalanine (V71F) (Fig.
6). Thus, we investigated both V71L and
V71F mutations in conjunction with the original F95A mutation. The
combined F95A/V71L mutation exhibited a very poor binding affinity
(Ki > 500 nM (n = 3))
in comparison with F95A/V71F of 7.2 ± 3.0 nM
(n = 3). Despite this rescue in binding, no improvement
in expression was observed (0.2 pmol/mg membrane protein). The validity
of our model was supported by this compensatory mutation.
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Fig. 6.
Compensatory mutation of F95A with V71F.
Model of hIP shows proximity of both Phe-95 (TMIII) and Val-71 (TMII)
to the -chain of prostacyclin. The other points of ligand receptor
interactions (i.e. Arg-279, Phe-278, and Tyr-75) have been
removed for clarity. A, the wild-type protein with Phe-95
and Val-71 residues. Phe-95 interacts with the
-chain of
prostacyclin. B, mutating Phe-95 to alanine removes
hydrophobic interaction with the
-chain, leading to a drop in
binding affinity. C, in the presence of F95A, a co-mutation
of V71F restores binding affinity by substituting for the lost
hydrophobic interaction between Phe-95 and the
-chain.
-chain moves TMVII and
-chain
TMIII). Thus, the first piece of evidence substantiating this
hypothesis has now been provided; however, further studies are required
for definitive confirmation.
-ionone ring of 11-cis-retinal (20 carbons
in size) faces TMV and TMVI, and the carbon chain is covalently
attached via a Schiff base to TMVII (10). With the biogenic amines (9 carbons), hydroxyl groups from the catechol rings interact with serines
on TMV and the amine group with an acidic residue in TMIII (4, 39). Prostacyclin like 11-cis-retinal and the biogenic amines has
similarly important interactions with TMVII and TMIII; however, the
bicyclic rings face TMI and TMII rather than TMV and TMVI (Fig.
7). We hypothesize that this may be a
unique feature of the prostacyclin receptor that has reduced the
availability of high affinity selective ligands. This insight may
assist in the development of unique and highly specialized agents
including additional agonists and, more notably, selective antagonists
for the treatment and study of prostanoid-related disorders.
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Fig. 7.
Positions of three ligands:
11-cis-retinal, epinephrine, and prostacyclin in the
agonist binding pocket. Homology modeling was performed as
described under "Experimental Procedures" using the rhodopsin
crystal structure as the template. Extracellular view of the
homology-based prostacyclin receptor transmembrane -helices with
bound prostacyclin ligand (red) is shown. Also superimposed
in the IP receptor is the configuration of 11-cis-retinal
(green) in rhodopsin and epinephrine (yellow) in
the adrenergic receptors.
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ACKNOWLEDGEMENTS |
---|
We wish to thank Dr. Kathleen Martin (Dartmouth Medical School, Hanover, NH), Professor Robert Graham (Victor Chang Cardiac Research Institute, Sydney, Australia), and Dr. Peter Reik (Victor Chang Cardiac Research Institute, Sydney, Australia) for critically reviewing the paper. We thank Dr. Bernard Trumpower (Department of Biochemistry, Dartmouth Medical School) for assistance and advice in the energy minimization of prostacyclin.
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FOOTNOTES |
---|
* This work was supported by a start-up grant provided by the Department of Pharmacology & Toxicology and an American Heart Association Scientist Development Grant 0235260N.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.
To whom correspondence should be addressed: Dept. of Pharmacology
& Toxicology, 7650 Remsen, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1813; Fax: 603-650-1129; E-mail:
John.Hwa@Dartmouth.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M207420200
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ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G-protein coupled receptor; PGI2, prostacyclin; Gpp(NH)p, 5'-guanlyl-imidodiphosphate; ANOVA, analysis of variance; hIP, human prostacyclin receptor; TM, transmembrane.
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