From Institut de Recherche Interdisciplinaire en
Biologie Humaine et Nucléaire, Université Libre de
Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles,
Belgium, ** Laboratori de Medicina Computacional, Unitat de
Bioestadística, Facultat de Medicina, Universitat
Autònoma de Barcelona, 08193 Bellaterra, Spain,
§ Service de Conformation des Macromolécules
Biologiques, Université Libre de Bruxelles, CP 160/16,
Avenue F. Roosevelt, 1050 Bruxelles, Belgium, and
Novasite Pharmaceuticals Inc.,
San Diego, California 92121
Received for publication, December 26, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CCR5 is a G-protein-coupled
receptor activated by the chemokines RANTES (regulated on activation
normal T cell expressed and secreted), macrophage inflammatory protein
1 Chemokine receptors are currently one of the most extensively
studied subfamilies of G-protein-coupled receptors
(GPCRs).1 This is due to
their key role in the immune response, where they act as attractors and
stimulators of specific leukocyte populations (1), and their essential
role in HIV infection. In particular, the chemokine receptor CCR5 is
the main co-receptor for macrophage-tropic HIV-1 strains, which are
responsible for disease transmission and predominate during the
asymptomatic phase of the disease (2, 3). It hence appears as one of
the crucial targets for developing new therapeutic strategies against
HIV.
CCR5 is activated by the chemokines RANTES, MIP-1 The mechanisms by which chemokines bind their receptors and induce
activation are currently unclear. Mutagenesis studies of chemokines
suggest that a major role in binding is played by receptor interactions
with their compact domain, while the flexible NH2 terminus
is required mainly for receptor activation (5, 6). NH2-terminally truncated chemokines usually bind their
receptors with wild type affinities but elicit a severely impaired
functional response (6, 7).
On the receptor side, several studies have shown that its extracellular
domains play an essential role in chemokine binding (8, 9). In
particular, the NH2-terminal domain of the receptor was
shown to be mandatory for chemokine binding, with several charged and
aromatic residues playing a crucial role (10, 11). On the other hand,
some of us have shown that most of the ligand specificity is encoded in
the second extracellular loop of CCR5 (12).
Clearly belonging to the rhodopsin-like family of GPCRs, chemokine
receptors share all of the highly conserved sequence motifs characteristic of this family. The overwhelming majority of these sequence motifs are located in the transmembrane region, suggesting the
conservation of a common fold for this region throughout the entire
rhodopsin-like family. The existence of a conserved fold may in turn
imply similar mechanisms in receptor activation involving the
membrane-embedded portion of the proteins.
A detailed atomic model representing this common fold has at long last
become available with the recent determination of the high resolution
x-ray structure of bovine rhodopsin (13). This structure confirms the
well documented seven-transmembrane It has been established that for many receptors, which are activated by
small ligands like neurotransmitters, agonist binding and subsequent
triggering of activation involves a water-accessible pocket centrally
located within the transmembrane helix bundle. This pocket corresponds
roughly to the retinal binding site in rhodopsin (15-17). The strong
similarity of the transmembrane regions of chemokine receptors to those
of other rhodopsin-like GPCRs suggests that these proteins undergo
ligand-induced activation processes, involving analogous conformational
changes. Some of these changes have been monitored for various GPCRs,
using different techniques (for a review, see Ref. 17). In particular,
transmembrane helix 6 (TM6) was reported to rotate its cytosolic end
away from TM3 in several receptors (18-24).
This crucial rigid body motion of a part of TM6 is thought to be
enabled by the presence of a highly conserved proline in the middle of
the helix (25), which introduces a local break in the helix structure.
Such a break, denoted a proline kink (PK), is likely to impart the
backbone flexibility (16, 26-28) required for the conformational
change associated with the activation process. Mutations of the
conserved Pro in TM6 in several receptors were indeed shown to produce
phenotypes ranging from severely impaired expression of the receptor
(29) to reduced functional coupling (30) or even constitutive
activation (31). Mutations of conserved prolines in other helices,
notably TM5 (32) and TM7 (33, 34), were also found to cause significant
perturbations. For instance, proline mutations in the conserved
NPXXY motif in TM7 often produce particularly strong
phenotypes, including impaired activity (33, 35, 36).
However, although some of the structural rearrangements associated with
activation are likely to be conserved throughout the rhodopsin-like
receptor family, the extraordinary diversity of ligand types, ranging
from small size neurotransmitters to large glycoprotein hormones (17),
suggests that receptor subfamilies have presumably evolved specific
binding modes with activation mechanisms probably requiring somewhat
different structural adaptations.
This study investigates such subfamily-specific properties in the
chemokine receptors. All chemokine receptors are shown here to share a
proline in TM2. Analysis of their aligned sequences also reveals the
presence of a conserved threonine residue 2 positions upstream of this
Pro forming the TXP motif. Considering that threonine residues have been observed to induce small distortions in This hypothesis is investigated using an approach, which combines
theoretical and experimental procedures. The theoretical procedures are
aimed at characterizing the effect of the TXP motif on the
intrinsic conformational properties of the transmembrane helix and on
its putative interactions with other helices in the bundle. To this
end, we have performed molecular dynamics (MD) simulations of an
isolated polyalanine helix comprising a TXP motif and
several variants thereof in which the Thr residue is replaced by other
side chains, Ser, Cys, Val, and Ala, respectively. In addition, using
the recently determined three-dimensional structure of rhodopsin as a
template, the structural role of this motif in the context of the
seven-helix bundle is assessed. This allows us to formulate hypotheses
on how the conformational states of the TXP motif-containing
helix might act to produce structural changes in the helix bundle.
The experimental procedures involve site-directed mutagenesis, in which
the Thr of the TXP motif of TM2 in CCR5 is replaced by the
same side chains as in the simulation analysis and where Ala is
substituted for the Pro in order to abrogate the PK. The different CCR5
mutants are then tested in order to determine their ligand binding and
activation properties.
Our results reveal a significant correspondence between the modulating
effect on the Pro kink angle and helix conformational flexibility by
Thr versus other residues in the TXP motif and the activation properties measured experimentally for the corresponding mutants in CCR5. The implications of these findings for
chemokine-receptor interactions and chemokine-induced activation are discussed.
Numbering Scheme of GPCRs--
In this work, we use a general
numbering scheme to identify residues in the transmembrane segments of
different receptors (28). Each residue is numbered according to the
helix (helix 1 through 7) in which it is located and according to the
position relative to the most conserved residue in that helix,
arbitrarily assigned to 50. For instance, Pro-2.58 is the
proline in the transmembrane helix 2 (TM2), 8 residues following the
highly conserved aspartic acid Asp-2.50.
Survey of Helices Containing a TXP Motif in Known Protein
Structures--
Since stable structural motifs are likely to recur in
proteins of known structures (40), we also surveyed the recent release of the Protein Data Bank (Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ) for Molecular Dynamics Simulations of Transmembrane Helices--
To
study the conformational properties of an
In a hydrophobic environment, the side chains of Ser, Thr, and Cys are
most likely to form hydrogen bonds with other polar groups of the
protein or of the ligand whenever present. Surveys of known protein
structures (41, 42) show that in
For Thr and Ser, such bonds can form only in the g+ or
g
Starting structures were placed in a rectangular box (59 × 37 × 38 Å) containing methane molecules at a density approaching half that of hydrocarbons in lipid bilayer, in order to mimic the
plasma membrane environment. The peptide-methane systems were subjected
to 500 iterations of energy minimization and then heated to 300 K in 15 ps. This was followed by an equilibration period (15-500 ps) and a
production run (500-1500 ps). The simulations were carried out at
constant volume and constant temperature (300 K), with the latter
maintained through coupling to a heat bath. The particle mesh Ewald
method was employed to compute electrostatic interactions (43).
Structures were collected for analysis every 10 ps during the last 1000 ps of simulation. The molecular dynamics simulations were run with the
Sander module of AMBER5 (44), using an all atom force field (45), the
SHAKE bond constraints on all bonds, and a 2-fs integration time step.
Bending of the Pro containing peptides was measured as described above
using backbone atoms of helical segments comprising residues 2-11
(before the Pro) and 16-24 (after the Pro). One-way analysis of the
variance plus a posteriori one-sided Dunnett's t tests were performed to determine if the bend angles of
the helices containing the TAP, SAP, CAP, and VAP motifs are greater than the bend angle of the helix containing the AAP motif, taken as
reference. To choose representative structures for each trajectory, the
structures saved during the production run were clustered on the basis
of their relative backbone root mean square deviation using the
NMRCLUST program (46) with a cut-off of 3 Å.
CCR5 Mutants--
Plasmids encoding the CCR5 mutants studied
here were constructed by site-directed mutagenesis using the
QuickChange method (Stratagene). Following sequencing of the
constructs, the mutated coding sequences were subcloned into the
bicistronic expression vector pEFIN3 as previously described for
generation of stable cell lines (12). All constructs were verified by
sequencing prior to transfection.
Expression of Mutant Receptors in CHO-K1 Cells--
CHO-K1 cells
were cultured in Ham's F-12 medium supplemented with 10% fetal calf
serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). Constructs encoding wild type
or mutant CCR5 in the pEFIN3 bicistronic vector were transfected using
Fugene 6 (Roche Molecular Biochemicals) in a CHO-K1 cell line
expressing an apoaequorin variant targeted to mitochondria (47).
Selection of transfected cells was made for 14 days with 400 µg/ml
G418 (Life Technologies) and 250 µg/ml zeocin (Invitrogen; for
maintenance of the apoaequorin-encoding plasmid), and the
population of mixed cell clones expressing wild type or mutant
receptors was used for binding and functional studies. Cell surface
expression of the receptor variants was measured by flow cytometry
using monoclonal antibodies recognizing different CCR5 epitopes; 2D7
(phycoerythrin-conjugated; Pharmingen), MC-1, MC-4, MC-5, and MC-6
(kindly provided by Mathias Mack, Medizische Poliklinik,
Ludwig-Maximilians, University of Munich, Munich, Germany) were
detected by anti-mouse IgG phycoerythrin-coupled secondary
antibody (Sigma).
125I-RANTES Binding Assays--
CHO-K1 cells
expressing wild type or mutant CCR5 were collected from plates with
Ca2+- and Mg2+-free phosphate-buffered saline
supplemented with 5 mM EDTA, gently pelleted for 2 min at
1000 × g, and resuspended in binding buffer (50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin).
Competition binding assays were performed in Minisorb tubes (Nunc) with
40,000 cells in a final volume of 0.1 ml. The mixture contained 0.05 nM 125I-RANTES (2000 Ci/mmol; Amersham
Pharmacia Biotech) as tracer and variable concentrations of competitors
(R & D Systems). Total binding was measured in the absence of
competitor, and nonspecific binding was measured with a 100-fold excess
of unlabeled ligand. Samples were incubated for 90 min at 27 °C, and
then bound tracer was separated by filtration through GF/B filters
presoaked in 0.5% polyethylenimine (Sigma) for
125I-RANTES. Filters were counted in a Functional Assays--
Functional response to chemokines was
analyzed by measuring the luminescence of aequorin as described (48,
49). Cells were collected from plates with Ca2+- and
Mg2+-free Dulbecco's modified Eagle's medium supplemented
with 5 mM EDTA. They were then pelleted for 2 min at
1000 × g, resuspended in Dulbecco's modified Eagle's
medium at a density of 5 × 106 cells/ml, and
incubated for 2 h in the dark in the presence of 5 µM coelenterazine H (Molecular Probes, Inc., Eugene, OR).
Cells were diluted 5-fold before use. Agonists in a volume of 50 µl of Dulbecco's modified Eagle's medium were added to 50 µl of cell suspension (50,000 cells), and luminescence was measured for 30 s
in a Berthold luminometer.
A Conserved TXP Motif in TM2 of Chemokine Receptors--
Multiple
sequence alignments of the second transmembrane helix of 55 mammalian
chemokine receptors were performed. Fig.
1 shows alignment of the human and mouse
sequences, together with TM2 of bovine rhodopsin. Inspection of the
aligned sequences reveals a highly conserved TXP sequence
motif in TM2, where X represents a variable hydrophobic
residue. Pro, at position 842.58 (84 is the residue number in
the CCR5 sequence, and 2.58 is the corresponding number in
the general numbering scheme), is completely conserved across all
chemokine receptors. The Thr residue is also highly conserved, present
in 47 sequences out of 55, while Ser is found in four receptors. The
last four receptors have an Ile or Leu in position 2.56.
A survey of the 1200 rhodopsin-like GPCRs present in the G-protein
Coupled Receptors Database (50) reveals that this motif is also
found at the equivalent position in the sequences of about 50 non-chemokine receptors, comprising essentially peptidergic, such as
angiotensin and opioid, receptors.
Fig. 1 also shows that the primary structure of bovine rhodopsin TM2 is
highly similar to that of chemokine receptors from its NH2
terminus (cytosolic border) to the TXP motif but strongly diverges between the TXP motif and its COOH terminus
(beginning of ECL1). This suggests a structural and functional
conservation in the cytosolic half of this transmembrane segment.
Influence of the TXP Motif on the Conformation of a Transmembrane
Helix--
To assess the influence of the TXP motif on the
conformation of a transmembrane helix 2, complementary approaches were
used. A first approach consisted in surveying known protein structures in the Protein Data Bank for
A second approach was therefore undertaken. This involved performing
molecular dynamics simulations on polyalanine helices, 25 residues
long, embedded in a nonpolar solvent, and containing the AAP, TAP, SAP,
CAP, and VAP motifs, respectively, in their midst. Table
II lists the average helix kink angles in
conformations along the MD trajectories. Representative structures from
the different trajectories are displayed in Fig.
2, A and B.
We find that the presence of a single proline (AAP) or of the VAP motif
produces helix bend angles of about 20°. For the simulation of the
AAP containing peptide, our results agree with those of earlier
simulation studies on Pro-containing polyalanine (51).
Significantly larger bending angles (27-35°) are observed when Thr,
Ser, or Cys is introduced 2 positions before the Pro. Detailed analysis
of the conformations in the trajectories show, as expected, that the
side chain hydroxyl (or SH) groups of these residues form hydrogen
bonds with the carbonyl group of residue i-4, in a significant
proportion of the conformations (85-100%). Such hydrogen bonds are
formed with Thr in the g+ or g
On the other hand, Ser in g
The results of our simulation analysis hence suggest that in a nonpolar
environment, the nature of the residue located at position i-2 relative
to the proline modulates the magnitude and direction of the PK through
the formation of a hydrogen bond between the side chain and the
backbone carbonyl group at position i-3 or i-4. In particular, the
presence of Thr, Ser, and Cys side chains at position i-2 relative to
the Pro increases the average helix bend angle by about 10°, whereas
that of Val does not.
Accommodating a Kinked TM2 Helix in the Receptor Three-dimensional
Structure--
To obtain a rough idea on the possible consequences
that the presence of TXP motif might have on the structure
of the receptor, and more particularly on the TM region, we performed a
molecular modeling exercise using the recently determined
three-dimensional structure of bovine rhodopsin as the template. As
shown in the alignment (Fig. 1), the sequence of TM2 is strongly
conserved (~50% sequence identity) between chemokine receptors and
rhodopsin between the cytosolic border and the TXP motif.
This leaves no ambiguity in aligning the rhodopsin and CCR5 sequences
in this region and allowed us to readily position representative
structures from the simulations of the AAP and TAP containing model
peptides into the TM bundle of rhodopsin. In particular, the backbone
atoms of the two helical turns preceding the PK in our model peptides were superimposed on those of the two turns preceding the equivalent residue (in CCR5, the PK starts at 2.54, four residues
before Pro-2.58) in rhodopsin, respecting the correspondence
of the sequence alignment. Interestingly, rhodopsin has two successive
glycines in positions 2.56 and 2.57 (with a Phe
and not a Pro at 2.58, forming a GGF motif). Most probably
as a result of the conjunction of these two flexible residues, its TM2
is strongly distorted, so that its extracellular part leans toward TM1.
Fig. 2 (C and D) shows the result of
superimposing the representative structures from the TAP g+
(red) and AAP (yellow) simulations on TM2 of
rhodopsin. Strikingly, the kink induced by Pro-2.58 in AAP
(yellow) orients the extracellular moiety of TM2 toward TM3
and away from TM1 (Fig. 2C). The presence of Thr in TAP,
which, as shown above, increases the helix bend angle by about 10°,
causes the extracellular side of TM2 to lean even more toward
TM3 and slightly toward the center of the bundle (Fig. 2D).
The differences in the amino acid sequences of the TM2 in the opsin
(GGF) versus the chemokine (TXP) families may
thus be related to structural differences in this region. In
particular, in the chemokine receptors, the extracellular sides of TM2
and TM3 would come into close contact. It is noteworthy that chemokine
receptors have a cluster of aromatic residues at the extracellular end
of TM2 and TM3. In other GPCRs, helix-helix interactions mediated by
aromatic clusters are believed to play a role in ligand-induced
receptor activation (52).
Effects of Mutations in the Conserved TXP Motif on CCR5 Expression
and Function--
To investigate the possible role of the
TXP motif in CCR5 expression and function, several mutants
were generated in the corresponding positions. Mutant P842.58A
was built in order to completely eliminate the PK, while mutants T822.56S, T822.56C, T822.56V, and
T822.56A were aimed at investigating the kink modulation
effects produced by the same residues as those studied in the model
peptide MD simulations.
Cell Surface Expression of the CCR5 Mutants--
Cell surface
expression of the CCR5 mutants was measured by fluorescence-activated
cell sorting analysis using a set of monoclonal antibodies, recognizing
various epitopes of the receptor, ranging from well defined linear
epitopes in the NH2-terminal domain (MC-5) to complex
conformational epitopes spanning multiple domains (MC-6). As shown in
Fig. 3, all mutants were properly
expressed at the cell surface, as compared with the WT receptor.
With the exception of P84A, the mutant receptors were recognized as
similar levels by all monoclonals, suggesting that the mutations did
not alter significantly the folding of the extracellular
domain.
The pattern observed for the P84A mutant seems to indicate a deeper
conformational modification for this mutant, which could in turn cause
the alteration of the extracellular domain conformation and eventually
affect conformational epitopes. Nevertheless, the antibodies
recognizing the amino-terminal part of the receptor did detect the P84A
mutant at the cell surface at levels similar to those observed for WT CCR5.
Chemokine Binding Properties of WT CCR5 and Mutant
Receptors--
The ability of the different CCR5 mutants to bind the
four high affinity CCR5 agonists, the chemokines RANTES, MIP-1
As shown earlier, RANTES appears as the strongest ligand for WT CCR5
(4), with an IC50 of 0.28 nM (Table
III). The IC50 values for
MIP-1
P84A is able to bind RANTES with WT affinity (IC50 = 0.25 nM), while MIP-1
RANTES displays an unaffected affinity for the CCR5 mutants T82S, T82C,
T82A, and T82V, with IC50 values ranging from 0.2 to 0.3 nM. The three other ligands show affinities that are about 10 times lower than RANTES for all Thr-82 mutants, as already observed
for WT CCR5. MIP-1
In summary, we find that all of the analyzed mutants bind the agonists
RANTES, MIP-1 Functional Activation of the Mutants by CCR5 Agonists--
A third
and crucial set of tests was performed in order to investigate the
ability of the five CCR5 mutants to be activated by the same four
agonists. This was done using a sensitive assay based on the use of
apoaequorin as a reporter system for intracellular calcium release.
Activation of chemokine receptors, including CCR5, is known to result
in calcium signaling. Control stimulation of the cell lines was
achieved with a saturating concentration of ATP, which activates
endogenously expressed P2Y2 receptors and
generates a strong luminescent signal. We measured the cell response to
ATP in all experiments and normalized the results as a percentage of
this signal.
Fig. 5 shows typical activation curves
obtained for the six WT and mutant receptors using the four agonists.
In agreement with previous observations (4), we find that RANTES is the most potent agonist of WT CCR5, with an EC50 of 3.5 nM (Table III), whereas the EC50 of MIP-1
However, unlike for the binding assays, in which all mutants except
P84A displayed a WT-like binding behavior, the functional assay
demonstrated various degrees of impairment for the different mutants.
The CCR5 mutants with the most impaired function are P84A and T82V.
P84A is activated by RANTES, with an average EC50 right-shifted by about 1 order of magnitude (one log unit in Fig. 5)
and an Emax reaching only 20% of the ATP
signal. But strikingly, none of the other three agonists elicits any
detectable signal in this functional assay. This is not too surprising
for MIP-1
The strongly reduced functional response of the T82V mutant is somewhat
surprising, especially in light of the milder functional impairment
observed for T82A (Fig. 5). Indeed, the substitution of Thr by Val is
isosteric. It preserves the
The T82A mutant comes next in the degree of functional impairment.
RANTES remains the best agonist for this mutant, followed by MIP-1
The mutants whose activities are least affected are T82S and T82C. T82S
is stimulated by RANTES to a similar degree as WT CCR5, with an
EC50 of 4.5 nM and an
Emax of 60%. MIP-1
These results taken together provide evidence on the important role
played by Thr-82 and Pro-84 in CCR5 activation. The extent to which the
activation of these mutants is affected by the series of
agonists, measured under similar conditions, leads to the
following ranking in terms of impairment of receptor activation:
P84A > T82V > T82A > T82C > T82S.
In this study, we identified a sequence motif TXP, in
TM2 of chemokine receptors, which is conserved throughout this
important subfamily of GPCRs. We made the hypothesis that it plays an
important role in receptor function and investigated this putative role by using a combination of theoretical and experimental techniques. Here, the findings from the two types of techniques are brought together and rationalized in light of our current knowledge of the CCR5
receptor structural and functional properties. This leads to the
proposal of a mechanism for the implication of the TXP motif
in CCR5 activation.
The TXP Motif Is a Structural Determinant in Chemokine
Receptors--
The molecular dynamics simulations, whose aim was to
investigate how the side chain at position i-2 from the Pro residue
affects the intrinsic conformational properties of a Pro-containing
Our modeling exercise, using the high resolution three-dimensional
structure of rhodopsin, suggests that the presence of the TXP motif in TM2 would require a rearrangement of the TM
helix bundle interactions in the chemokine receptors relative to rhodopsin.
In particular, Pro in position 2.58 would orient the
extracellular part of TM2 toward TM3 and not close to TM1 as in
rhodopsin; the addition of a Thr at i-2, as in the TXP
motif, furthermore directs this part of TM2 toward the center of the
bundle (Fig. 2, C and D). Our modeling study also
suggests that in chemokine receptors, the extracellular region of TM2
would interact with TM3 and possibly with TM7, which is not feasible in
the rhodopsin structure.
Our results thus lead us to suggest that the TXP motif in
TM2 is a structural determinant, in chemokine receptors, by virtue of
significant local effects on the helix conformation, which propagate
through a lever action to the extracellular parts of the TM bundle. It
is noteworthy that there is ample experimental evidence that this
region of GPCRs is involved in their functional properties (16), which
suggests in turn that mutants modifying these local conformational
effects should also modify receptor function.
Role of the TXP Motif in Ligand-induced Activation of
CCR5--
Our functional studies of TXP mutants of CCR5
were aimed at verifying this suggestion. The P842.58A mutant
displayed a significantly affected pattern of binding for some ligands.
We hypothesize that the profound structural perturbation in this
mutant, caused by the abrogation of the PK, might change the
conformation of some part of the extracellular domain, thus modifying
the interactions with ligands. In agreement with this view, this
putative conformational change could also be responsible for the
observed decrease in recognition of the P842.58A mutant by
monoclonal antibodies directed at conformational epitopes. This
suggests that this PK is mandatory for the structural integrity of the
protein. Alanine replacement of Pro-842.58 abolished the
functional response of CCR5 to any of its agonists with the exception
of RANTES, which induced minute activity, demonstrating the central
role of this PK in the activation process.
All tested chemokines bound the various Thr-822.56 mutants with
unchanged affinity, indicating that this residue is not involved in
direct interaction with the ligands. In contrast to the binding properties, the chemokine-induced activation of CCR5 was quite sensitive to mutations of Thr-822.56, showing a gradation of
the effects corresponding to that observed in the simulation.
Activation profiles of T822.56S showed mild differences with
those of WT CCR5. However, this mutant had a reduced
Emax, when stimulated with MIP-1
This could occur if the equilibrium between the active and inactive
forms of T822.56S is somewhat shifted toward the inactive one.
The fact that RANTES activates this mutant normally could mean that it
stabilizes more efficiently the active forms. The results of our MD
simulations on the SAP-containing peptide suggest a molecular
explanation to this shift in equilibrium. They show that this peptide
features a similar
The isosteric mutant T822.56V was even more impaired in the
functional tests than T822.56A. T822.56V required high
chemokine concentrations to trigger very modest activities, although
the binding properties were not significantly affected for any of the
ligands tested. We cannot provide a simple explanation for the
difference of phenotype observed between these two mutants, but
considering the observations made in the MD simulations, the hydrogen
bonding capacity of the side chain at position 2.56 seems to
be a more important feature for activation than the
Our results show that, in chemokine receptors, the Pro in TM2 is
crucial for proper receptor activation and that the conserved Thr
located 2 positions before the Pro modulates the function of the
receptor. The experiments therefore confirm the theoretical hypothesis
stating that the TXP motif plays a key structural role in
chemokine receptors, the PK constituting the main element and the Thr
acting as a modulator of this PK. Moreover, this motif is mainly
involved in receptor activation but plays little role in ligand
binding. These results also demonstrate that high affinity binding of
chemokines by CCR5 is not dependent on the coupling state with
G-proteins. A previous description of other non-functional CCR5 mutants
characterized by unimpaired affinities for their chemokine ligands (10)
supports this hypothesis. Along the same line of evidence, it is well
established that NH2-terminally truncated chemokines often
keep their high affinities for their cognate receptors while becoming
antagonists or weak partial agonists.
New Insights into Chemokine-induced Activation--
Surprisingly,
although the TXP motif is present in all chemokine
receptors, functional alteration observed in the Thr-822.56
mutants is strongly chemokine-dependent. RANTES is the
least affected agonist, MCP-2 being strongly sensitive to all
mutations, while MIP-1
Interestingly, RANTES differs structurally from the other CCR5 agonists
in its amino terminus, with 9 residues before the first conserved
cysteine for RANTES and 10 residues for the three other ligands. Also,
the sequence of the MCP-2 NH2 terminus is somewhat
different from that of the other agonists, whereas those of MIP-1
This leads us to formulate the hypothesis that chemokine induced
activation involves interactions of the ligand NH2 terminus with the portion of transmembrane domain whose conformation may be
modulated by the TXP motif. Thus, the differences in
behavior among ligands could result from differences in these
interactions. This hypothesis could be investigated by testing mutant
receptor activation by chimeric chemokines exchanging the
NH2 terminus or the core region.
As mentioned above, chemokines truncated in their NH2
terminus often still elicit some functional responses at high
concentrations and hence behave as partial agonists. This parallels the
effects of full-size chemokines on our Thr-822.56 mutant series
and could result from the binding of the chemokine core region to the
extracellular loops of the receptor. It was proposed that this
interaction would induce partial signal transduction, characterized by
inhibition of cAMP, but would not trigger calcium influx or chemotaxis
(6). Thus, it does not contradict our proposal that full activation
involves interactions with the NH2-terminal region of the chemokine.
Based on chimeras between CCR2b and CCR1, it was recently proposed that
activation of chemokine receptors involves the first extracellular loop
ECL1 (53). Having shown here that TM2 in this receptor family extends
to position 2.67, the effects of these chimeras are most
probably due to the exchange of variable residues at the extracellular
portion of this helix, rather than to the swap of ECL1, which is
well conserved in these receptors. Interestingly, these changes
produced mutant receptors with good ligand affinity but impaired
chemokine-induced functional response. These data parallel our present
finding and hence agree with our hypothesis on the importance of the
extracellular part of TM2 in receptor activation.
Other recent studies have described low molecular weight chemical
compounds acting as antagonists (54-56) with promising applications in
inflammatory diseases and HIV infection. Interestingly, the binding
sites of these antagonists have been located within the transmembrane
bundle of the receptors (57, 58). Insights provided here on the role of
structural determinants in this region may therefore be helpful in
further elucidating the action of these compounds and in designing
effective drugs. The availability of the high resolution structure of
rhodopsin will greatly facilitate this task and allow more detailed
analyses of the type described here to be extended to other
rhodopsin-like GPCRs.
and 1
, and monocyte chemotactic protein 2 and is the
main co-receptor for the macrophage-tropic human immunodeficiency virus
strains. We have identified a sequence motif (TXP) in the
second transmembrane helix of chemokine receptors and investigated its
role by theoretical and experimental approaches. Molecular dynamics
simulations of model
-helices in a nonpolar environment were used to
show that a TXP motif strongly bends these helices, due to
the coordinated action of the proline, which kinks the helix, and of
the threonine, which further accentuates this structural deformation.
Site-directed mutagenesis of the corresponding Pro and Thr residues in
CCR5 allowed us to probe the consequences of these structural findings
in the context of the whole receptor. The P84A mutation leads to a
decreased binding affinity for chemokines and nearly abolishes the
functional response of the receptor. In contrast, mutation of
Thr-822.56 into Val, Ala, Cys, or Ser
does not affect chemokine binding. However, the functional response was
found to depend strongly on the nature of the substituted side chain.
The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment
parallels the bending of the
-helix observed in the molecular
simulation study.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, MIP-1
, and
MCP-2 and binds a natural chemokine antagonist, MCP-3 (4). Chemokines
are small globular proteins, 60-100 residues long, comprising a well
structured domain, and a flexible NH2 terminus, with a
Cys-Cys or Cys-X-Cys motif (where X represents a
variable residue) marking the limit between the two parts (1).
-helix bundle topology, proposed
on the basis of lower resolution structural studies (14). It
furthermore provides a detailed atomic picture of the interactions
between the transmembrane helices, particularly those involving the
conserved GPCR sequence motifs.
-helices (37, 38), we hypothesize here that the conjunction of these two
conserved and structurally relevant residues in chemokine receptors
might constitute a key motif required for proper receptor function. The
influence of Thr in Pro-containing helices had been identified earlier
for other integral membrane proteins (39).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical segments featuring a TXP, SXP, or CXP
motif with no other Thr, Ser, or Cys within the PK and no other Pro
anywhere in the segment. Since measuring the bending angle requires at
least one helical turn prior to the PK and a helical turn following it,
we selected helical segments of at least 12 residues. We performed our
search in a nonredundant set of protein structures with resolutions of 3 Å or better, identifying 16 helical segments (Protein Data Bank numbers 1AR1, 1B7E, 1B94, 1BDB, 1BPO, 1FCB, 1FIY, 1FVK, 1OCC, 1PJC,
1REQ, 1RVE, 1TCO, 1VHB, 2AK3, and 2GST). Detailed analysis of the
corresponding structures showed that most of these helices are exposed
to solvent, with water molecules often interacting with the backbone at
the level of the PK. Since such interactions are not likely to occur in
a membrane-embedded helix, structures displaying these interactions were rejected, finally yielding only seven structures (see Table I).
The bending angle of these structures is defined as the angle between
the axes computed as the least square lines through the backbone atoms
(N, C
, C) of the
-helical part before and after the
Pro kink (using the InsightII software, MSI, San Diego).
-helix containing a
TXP motif, we performed molecular dynamics simulations of the model peptides
Ace-Ala11-XXX-Ala11-NMe, where
XXX is either AAP, TAP, SAP, CAP, or VAP. These
25-residue peptides were built in the standard
-helical conformation
(
,
=58°,
47°).
-helices, these side chains
hydrogen-bond primarily the carbonyl group in the preceding turn of the
helix (residue i-4 or i-3).
side chain conformations, whereas for Cys, they can be
formed only in the g+ conformation. The g
conformation of Cys is energetically unfavorable because of the steric
clash between the S
atom and the carbonyl oxygen of
residue i-3 (42). For these side chains, the t cannot form
such hydrogen bonds as it points the OH group away from the backbone.
The model peptides were hence built with the Thr and Ser side chains in
either g+ or g
and with the Cys side chain in
g+. The hydrophobic Val side chain was built in
the t conformation.
-scintillation
counter. Binding parameters were determined with the Prism software
(GraphPad Software) using nonlinear regression applied to a
one-site competition model.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (82K):
[in a new window]
Fig. 1.
Alignment of TM2 sequences from chemokine
receptors and bovine rhodopsin. For the sake of clarity, only
human and murine sequences are shown. The generalized numbering scheme
(see "Experimental Procedures") is used to label the alignment. The
TXP (or SXP) motif is indicated in
boldface characters, and its conserved residues
are highlighted. The sequence of TM2 of bovine rhodopsin is
also aligned, showing the high homology between CCR5 and bovine
rhodopsin in the cytosolic part of TM2, up to the TXP motif.
Note that we make the assumption that, in chemokine receptors, TM2
extends to position 2.67 (included). This is based on the
observation that TM2 extends to this residue in the three-dimensional
structure of rhodopsin (13) and on the suggestion that position
2.67 terminates TM2 in the dopamine D2 receptor, on the
basis of the substituted cysteine accessibility method (59).
-helices containing either a
TXP, SXP, or CXP motif, using the
criteria specified under "Experimental Procedures." This resulted
in identifying only seven structures, all of which displayed a strong
bend, with angles ranging from 25 to almost 50° as shown in Table
I. It is noteworthy that the reported
average bend angle of
-helices containing Pro is about 25° (26).
The helices identified here thus seem to be as strongly bent as, if not
more strongly bent than, the average PK, but the very small number of
observations precludes drawing a reliable conclusion.
Bending angles for TXP motifs found in the Protein Data Bank (PDB)
Bending angles measured in the different simulations
View larger version (34K):
[in a new window]
Fig. 2.
Effect of AAP and TAP g+
motif on the conformation of -helices.
A, representative structures for AAP
(yellow), TAP g+ (red), and an
ideal
-helix (white). Backbones are represented as
ribbons, and side chains of Pro and Thr are shown as
solid sticks (with polar hydrogen in
white) as well as the hydrogen bonding carbonyl, 4 residues
before the Thr. Hydrogen bonds are indicated by dotted
white lines. B, representative
structures for AAP, TAP g+, SAP g
(orange), and
an ideal
-helix. This view is rotated by 90° along the helical
axis relative to A. In the g
rotamer, the Ser
hydrogen bonds with carbonyl situated 3 residues upstream, which
induces clearly a different orientation of the PK. C, the
representative structure of the AAP (yellow
ribbon) and TAP g+ (red
ribbon) motifs are positioned in the rhodopsin template,
respecting the homology between CCR5 and rhodopsin. The two
representative helices were superimposed on the cytoplasmic end of TM2
in the rhodopsin structure (Protein Data Bank number 1F88), using
backbone atoms up to position 2.54. Rhodopsin
(turquoise) helices are shown as cylinders,
except for the extracellular part of TM2, which is shown as a
ribbon. This panel is viewed from the side of the protein.
D, same representation as in C but viewed from
the extracellular side. The influence of the Thr on the PK is clearly
visible, as it bends the helix inside the bundle.
conformations, Ser in g+, and Cys in g+.
achieves and maintains
hydrogen bonding with the carbonyl of residue i-3 position. This
seemingly minor alteration in the hydrogen bonding pattern appears,
however, to induce a dramatic modification in the conformation of the
helix, as illustrated in Fig. 2B. Not only is the helix kink
angle increased significantly, but the COOH-terminal moiety of the
helix points to a completely different direction in space.
View larger version (41K):
[in a new window]
Fig. 3.
Level of expression of the receptors.
Cell surface expression of WT CCR5 and the different mutants was
measured by fluorescence-activated cell sorting using five different
monoclonal antibodies. The data are representative of three different
experiments. The 2D7 antibody recognizes a conformational epitope
centered on ECL2. We have recently identified the epitopes of the other
antibodies tested here (C. Blanpain, M. Mack, J.-M.
Vanderwinden, V. Wittamer, E. Le Poul, G. Vassart, and M. Parmentier,
manuscript in preparation); MC-1 and MC-6 recognize multidomain
conformational epitopes, while MC-4 targets a conformational
NH2-terminal epitope and MC-5 a linear epitope also located
in the amino-terminal domain of CCR5. Values represent mean cell
fluorescence normalized by the value obtained for CCR5 (100%)
separately for each antibody.
,
MIP-1
, and MCP-2, was tested by competition binding assays, using
125I-RANTES as tracer. Fig. 4
shows the competition curves for the various constructs.
View larger version (27K):
[in a new window]
Fig. 4.
Binding properties of the WT and mutant
receptors. Competition binding assays were performed on
CHO-K1 cell lines expressing WT CCR5 and the different mutants using
125I-RANTES as tracer. The data are representative of at
least two experiments. Results were analyzed by the GraphPad Prism
software, using a single-site model, and the data were normalized for
nonspecific (0%) and specific binding in the absence of competitor
(100%). All points were run in triplicate (error
bars represent S.E.). Unlabeled ligands are as follows:
RANTES ( ), MIP-1
(
), MIP-1
(
), and MCP2 (
).
(3.7 nM), MIP-1
(1.3 nM), and MCP-2
(2.4 nM) are shifted to slightly higher values as compared
with those obtained previously with 125I-MIP-1
as
tracer, but the order of potencies is conserved (Table III). Changes in
apparent affinities as a function of the tracer used have been observed
previously for chemokine and other receptors (9).
Binding and functional properties of WT CCR5 and mutant receptors
and MCP-2 show significantly decreased
binding, with IC50 values right-shifted by 3 orders of
magnitude (Fig. 4 and Table III). MIP-1
does not compete for the
bound tracer, even at the highest concentration tested (1 µM).
, MIP-1
, and MCP-2 display roughly WT
affinities for all four Thr-82 mutants, with only mild variations according to the mutant. Their IC50 values are all in the
range of 1.5-5.7 nM, confirming that none of the Thr-82
mutants have significantly affected binding properties. The largest
change is a 3-fold increase in average IC50 for MIP-1
binding to T82V.
, MIP-1
, and MCP-2 with WT affinities. One exception
is the P84A mutant. Although it binds RANTES as well as the WT
receptor, its affinity for MIP-1
and MCP-2 is reduced, and it does
not bind MIP-1
.
is
twice as large on average (7.2 nM), and MCP-2 and MIP-1
are somewhat less efficient with average EC50 values of
23.4 and 25.0 nM, respectively.
View larger version (24K):
[in a new window]
Fig. 5.
Activation of the different receptors by the
four CCR5 agonists. Shown is the functional response to RANTES
( ), MIP-1
(
), MIP-1
(
), and MCP-2 (
) of CHO-K1 cells
expressing WT CCR5 or the various mutants using the aequorin assay. All
points were run in triplicate (error bars
represent S.E.). The displayed curves represent a typical experiment
out of at least three performed independently. Results were analyzed by
nonlinear regression using the GraphPad Prism software. Data were
normalized to maximal cell line stimulation by a saturating
concentration of ATP. Note that the vertical
scales of the graphs have been adapted to the
maximal responses obtained for each line.
, considering its severely decreased affinity for this
mutant (IC50 >1000 nM). MIP-1
and MCP-2,
however, did not activate P84A, even at 300 nM (MCP-2) or
1000 nM (MIP-1
) concentrations (data not shown), despite
full 125I-RANTES displacement at these concentrations in
the binding assay.
-branched character of the side chain
and hence presents a less drastic change than a substitution by Ala,
which truncates the side chain beyond the C
atom. T82V
displays a dramatically reduced response to RANTES, with the
activation curve for this ligand shifted to the right by more than 1 order of magnitude. With MIP-1
, high concentrations are required
(EC50 = 136 nM) to achieve moderate signals,
whereas MIP-1
is unable to elicit detectable signals from this
mutant in most experiments, although in some cases, a very weak
response is measured at the highest concentrations. MCP-2 induces no
response in T82V, although a normal binding affinity is measured for
this chemokine, as shown above.
(EC50 = 52 nM), and both ligands display a
reduced Emax (~30% of maximum response).
MIP-1
stimulates T82A with an EC50 of 63 nM
and MCP-2 with an EC50 averaging 147 nM, both
with a very low efficacy (Emax values below
15%; Fig. 5, Table III).
, MIP-1
, and MCP-2 show
similar activation profiles with this mutant, characterized by a strong
functional response, with an EC50 moderately displaced to
the right and an Emax somewhat lower than for WT
CCR5. RANTES and MIP-1
stimulate the T82C mutant with WT potencies
but reduced Emax. MIP-1
elicits a poor
response on this mutant, while MCP-2 is almost inactive.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix, lead to the following main conclusions: the presence of Thr,
Ser, and Cys side chains at position i-2 increases the average PK angle by about 10°, whereas Val at that position has a negligible effect. The fact that the Val side chain is nonpolar and the observation that
the other analyzed side chains, which were all polar, formed hydrogen
bonds with the helix backbone during the simulations suggests that the
observed effect on the bending angle arises from local deformations in
helix geometry induced by these bonds.
, MIP-1
,
and MCP-2. This behavior is reminiscent of that observed with partial
agonists on WT receptors and hence suggests that these chemokines
cannot fully activate this mutant.
-helix kink geometry as in the TAP-containing
peptide when Ser is in the g+ conformation but adopts a very
different orientation of the PK when this side chain is in
g
(see Fig. 2B). This latter conformation may
stabilize the inactive form of mutant receptors. This effect of Ser
might possibly also explain the paucity of Ser residues at position
2.56 in the chemokine receptors (only 4 of 55 members have it).
-branched character of the side chain.
and MIP-1
behave as partial agonists for
the various mutants. How can we explain these observations?
and MIP-1
are quite similar to each other. Hence, there seems to be
a relation between the sensitivity of receptor activation by the
different chemokines to the mutations in TXP and the
differences between the NH2-terminal regions of these
molecules. It is significant that these regions were shown to be
important in receptor activation by mutation and deletion experiments
(6, 7).
![]() |
ACKNOWLEDGEMENTS |
---|
Expert technical assistance was provided by M. J. Simons, and Jean Richelle is gratefully acknowledged for valuable help with the computer systems. We thank Mathias Mack for kindly providing monoclonal antibodies and Harel Weinstein for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Actions de Recherche Concertées of the Communauté Française de Belgique, the French Agence Nationale de Recherche sur le SIDA, the Center de Recherche Inter-universitaire en Vaccinologie, the Belgian program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming, BIOMED and BIOTECH programs of the European Community Grants BIO4-CT98-0543 and BMH4-CT98-2343, the Fonds de la Recherche Scientifique Médicale of Belgium, Télévie, and the Fondation Médicale Reine Elisabeth (to M. P). This work was also supported in part by Comisión Interministerial de Ciencia y Tecnología, Spain Grant SAF99-073, Fundació La Marató TV3 Grant 0014/97, and Improving Human Potential of the European Community Grant HPRI-CT-1999-00071. Computer facilities were provided by the Center de Computació i Comunicacions de Catalunya.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.
¶ These two authors contributed equally to this work.
Aspirant of the Belgian Fonds National de la Recherche Scientifique.
§§ To whom correspondence should be addressed. Tel.: 32 2 555 41 71; Fax: 32 2 555 46 55; E-mail: mparment@ulb.ac.be.
¶¶ Fellow of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011670200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G-protein-coupled receptor; HIV, human immunodeficiency virus; TM, transmembrane helix; PK, proline kink; MD, molecular dynamics; MIP, macrophage inflammatory protein; MCP, monocyte chemotactic protein; WT, wild type; RANTES, regulated on activation normal T cell expressed and secreted.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Baggiolini, M. (1998) Nature 392, 565-568[CrossRef][Medline] [Order article via Infotrieve] |
2. | Littman, D. R. (1998) Cell 93, 677-680[Medline] [Order article via Infotrieve] |
3. | Samson, M., Libert, F., Doranz, B. J., Rucker, J., Liesnard, C., Farber, C. M., Saragosti, S., Lapoumeroulie, C., Cognaux, J., Forceille, C., Muyldermans, G., Verhofstede, C., Burtonboy, G., Georges, M., Imai, T., Rana, S., Yi, Y., Smyth, R. J., Collman, R. G., Doms, R. W., Vassart, G., and Parmentier, M. (1996) Nature 382, 722-725[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Blanpain, C.,
Migeotte, I.,
Lee, B.,
Vakili, J.,
Doranz, B. J.,
Govaerts, C.,
Vassart, G.,
Doms, R. W.,
and Parmentier, M.
(1999)
Blood
94,
1899-1905 |
5. | Hemmerich, S., Paavola, C., Bloom, A., Bhakta, S., Freedman, R., Grunberger, D., Krstenansky, J., Lee, S., McCarley, D., Mulkins, M., Wong, B., Pease, J., Mizoue, L., Mirzadegan, T., Polsky, I., Thompson, K., Handel, T. M., and Jarnagin, K. (1999) Biochemistry 38, 13013-13025[CrossRef][Medline] [Order article via Infotrieve] |
6. | Jarnagin, K., Grunberger, D., Mulkins, M., Wong, B., Hemmerich, S., Paavola, C., Bloom, A., Bhakta, S., Diehl, F., Freedman, R., McCarley, D., Polsky, I., Ping-Tsou, A., Kosaka, A., and Handel, T. M. (1999) Biochemistry 38, 16167-16177[CrossRef][Medline] [Order article via Infotrieve] |
7. | Gong, J. H., and Clark-Lewis, I. (1995) J. Exp. Med. 181, 631-640[Abstract] |
8. |
LaRosa, G. J.,
Thomas, K. M.,
Kaufmann, M. E.,
Mark, R.,
White, M.,
Taylor, L.,
Gray, G.,
Witt, D.,
and Navarro, J.
(1992)
J. Biol. Chem.
267,
25402-25406 |
9. |
Ahuja, S. K.,
and Murphy, P. M.
(1996)
J. Biol. Chem.
271,
20545-20550 |
10. |
Farzan, M.,
Choe, H.,
Martin, K. A.,
Sun, Y.,
Sidelko, M.,
Mackay, C. R.,
Gerard, N. P.,
Sodroski, J.,
and Gerard, C.
(1997)
J. Biol. Chem.
272,
6854-6857 |
11. |
Blanpain, C.,
Doranz, B. J.,
Vakili, J.,
Rucker, J.,
Govaerts, C.,
Baik, S. S.,
Lorthioir, O.,
Migeotte, I.,
Libert, F.,
Baleux, F.,
Vassart, G.,
Doms, R. W.,
and Parmentier, M.
(1999)
J. Biol. Chem.
274,
34719-34727 |
12. |
Samson, M.,
LaRosa, G.,
Libert, F.,
Paindavoine, P.,
Detheux, M.,
Vassart, G.,
and Parmentier, M.
(1997)
J. Biol. Chem.
272,
24934-24941 |
13. |
Palczewski, K.,
Kumasaka, T.,
Hori, T.,
Behnke, C. A.,
Motoshima, H.,
Fox, B. A.,
Le, T., I,
Teller, D. C.,
Okada, T.,
Stenkamp, R. E.,
Yamamoto, M.,
and Miyano, M.
(2000)
Science
289,
739-745 |
14. | Unger, V. M., Hargrave, P. A., Baldwin, J. M., and Schertler, G. F. (1997) Nature 389, 203-206[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Strader, C. D.,
Fong, T. M.,
Graziano, M. P.,
and Tota, M. R.
(1995)
FASEB. J.
9,
745-754 |
16. |
Ji, T. H.,
Grossmann, M.,
and Ji, I.
(1998)
J. Biol. Chem.
273,
17299-17302 |
17. |
Gether, U.,
and Kobilka, B. K.
(1998)
J. Biol. Chem.
273,
17979-17982 |
18. | Elling, C. E., Nielsen, S. M., and Schwartz, T. W. (1995) Nature 374, 74-77[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Sheikh, S. P.,
Vilardarga, J. P.,
Baranski, T. J.,
Lichtarge, O.,
Iiri, T.,
Meng, E. C.,
Nissenson, R. A.,
and Bourne, H. R.
(1999)
J. Biol. Chem.
274,
17033-17041 |
20. | Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Farrens, D. L.,
Altenbach, C.,
Yang, K.,
Hubbell, W. L.,
and Khorana, H. G.
(1996)
Science
274,
768-770 |
22. |
Dunham, T. D.,
and Farrens, D. L.
(1999)
J. Biol. Chem.
274,
1683-1690 |
23. |
Javitch, J. A.,
Fu, D.,
Liapakis, G.,
and Chen, J.
(1997)
J. Biol. Chem.
272,
18546-18549 |
24. |
Rasmussen, S. G.,
Jensen, A. D.,
Liapakis, G.,
Ghanouni, P.,
Javitch, J. A.,
and Gether, U.
(1999)
Mol. Pharmacol.
56,
175-184 |
25. |
Gether, U.,
Lin, S.,
Ghanouni, P.,
Ballesteros, J. A.,
Weinstein, H.,
and Kobilka, B. K.
(1997)
EMBO J.
16,
6737-6747 |
26. | Barlow, D. J., and Thornton, J. M. (1988) J. Mol. Biol. 201, 601-619[Medline] [Order article via Infotrieve] |
27. | Woolfson, D. N., and Williams, D. H. (1990) FEBS Lett. 277, 185-188[CrossRef][Medline] [Order article via Infotrieve] |
28. | Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25, 366-428 |
29. |
Kolakowski, L. F., Jr.,
Lu, B.,
Gerard, C.,
and Gerard, N. P.
(1995)
J. Biol. Chem.
270,
18077-18082 |
30. |
Nakayama, T. A.,
and Khorana, H. G.
(1991)
J. Biol. Chem.
266,
4269-4275 |
31. |
Tonacchera, M.,
Chiovato, L.,
Pinchera, A.,
Agretti, P.,
Fiore, E.,
Cetani, F.,
Rocchi, R.,
Viacava, P.,
Miccoli, P.,
and Vitti, P.
(1998)
J. Clin. Endocrinol. Metab.
83,
492-498 |
32. | Javitch, J. A., Fu, D., and Chen, J. (1995) Biochemistry 34, 16433-16439[Medline] [Order article via Infotrieve] |
33. | Wess, J., Nanavati, S., Vogel, Z., and Maggio, R. (1993) EMBO J. 12, 331-338[Abstract] |
34. |
Hong, S.,
Ryu, K. S.,
Oh, M. S.,
Ji, I.,
and Ji, T. H.
(1997)
J. Biol. Chem.
272,
4166-4171 |
35. |
Vichi, P.,
Whelchel, A.,
and Posada, J.
(1999)
J. Biol. Chem.
274,
10331-10338 |
36. | Barak, L. S., Menard, L., Ferguson, S. S., Colapietro, A. M., and Caron, M. G. (1995) Biochemistry 34, 15407-15414[Medline] [Order article via Infotrieve] |
37. | Blundell, T., Barlow, D., Borkakoti, N., and Thornton, J. (1983) Nature 306, 281-283[Medline] [Order article via Infotrieve] |
38. |
Ballesteros, J.,
Deupi, X.,
Olivella, M.,
Haaksma, E.,
and Pardo, L.
(2000)
Biophys. J.
79,
2754-2760 |
39. |
Ri, Y.,
Ballesteros, J. A.,
Abrams, C. K.,
Oh, S.,
Verselis, V. K.,
Weinstein, H.,
and Bargiello, T. A.
(1999)
Biophys. J.
76,
2887-2898 |
40. | Wintjens, R. T., Rooman, M. J., and Wodak, S. J. (1996) J. Mol. Biol. 255, 235-253[CrossRef][Medline] [Order article via Infotrieve] |
41. | Gray, T. M., and Matthews, B. W. (1984) J. Mol. Biol. 175, 75-81[Medline] [Order article via Infotrieve] |
42. | McGregor, M. J., Islam, S. A., and Sternberg, M. J. (1987) J. Mol. Biol. 198, 295-310[Medline] [Order article via Infotrieve] |
43. | Darden, T., York, D., and Pedersen, L. (1993) J. Chem. Phys. 98, 10089-10092[CrossRef] |
44. | Case, D. A., Pearlman, D. A., Caldwell, J. W., Cheatham, T. E., Ross, W. S., Simmerling, C. L., Darden, T. A., Merz, K. M., Stanton, R. V., Cheng, A. L., Vincent, J., Crowley, M., Ferguson, D. M., Radmer, R. J., Seibel, G. L., Singh, U. C., Weiner, P. K., and Kollman, P. A. (1997) AMBER5 , University of California, San Francisco |
45. | Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Jr., F., D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. (1995) J. Am. Chem. Soc. 117, 5179-5197 |
46. | Kelley, L. A., Gardner, S. P., and Sutcliffe, M. J. (1996) Protein Eng. 9, 1063-1065[Medline] [Order article via Infotrieve] |
47. |
Rizzuto, R.,
Pinton, P.,
Carrington, W.,
Fay, F. S.,
Fogarty, K. E.,
Lifshitz, L. M.,
Tuft, R. A.,
and Pozzan, T.
(1998)
Science
280,
1763-1766 |
48. | Stables, J., Green, A., Marshall, F., Fraser, N., Knight, E., Sautel, M., Milligan, G., Lee, M., and Rees, S. (1997) Anal. Biochem. 252, 115-126[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Blanpain, C.,
Lee, B.,
Vakili, J.,
Doranz, B. J.,
Govaerts, C.,
Migeotte, I.,
Sharron, M.,
Dupriez, V.,
Vassart, G.,
Doms, R. W.,
and Parmentier, M.
(1999)
J. Biol. Chem.
274,
18902-18908 |
50. |
Horn, F.,
Weare, J.,
Beukers, M. W.,
Horsch, S.,
Bairoch, A.,
Chen, W.,
Edvardsen, O.,
Campagne, F.,
and Vriend, G.
(1998)
Nucleic Acids Res.
26,
275-279 |
51. | Yun, R. H., Anderson, A., and Hermans, J. (1991) Proteins 10, 219-228[Medline] [Order article via Infotrieve] |
52. | Javitch, J. A., Ballesteros, J. A., Weinstein, H., and Chen, J. (1998) Biochemistry 37, 998-1006[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Han, K. H.,
Green, S. R.,
Tangirala, R. K.,
Tanaka, S.,
and Quehenberger, O.
(1999)
J. Biol. Chem.
274,
32055-32062 |
54. | Donzella, G. A., Schols, D., Lin, S. W., Este, J. A., Nagashima, K. A., Maddon, P. J., Allaway, G. P., Sakmar, T. P., Henson, G., De Clercq, E., and Moore, J. P. (1998) Nat. Med. 4, 72-77[Medline] [Order article via Infotrieve] |
55. |
Baba, M.,
Nishimura, O.,
Kanzaki, N.,
Okamoto, M.,
Sawada, H.,
Iizawa, Y.,
Shiraishi, M.,
Aramaki, Y.,
Okonogi, K.,
Ogawa, Y.,
Meguro, K.,
and Fujino, M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5698-5703 |
56. |
Hesselgesser, J.,
Ng, H. P.,
Liang, M.,
Zheng, W.,
May, K.,
Bauman, J. G.,
Monahan, S.,
Islam, I.,
Wei, G. P.,
Ghannam, A.,
Taub, D. D.,
Rosser, M.,
Snider, R. M.,
Morrissey, M. M.,
Perez, H. D.,
and Horuk, R.
(1998)
J. Biol. Chem.
273,
15687-15692 |
57. |
Dragic, T.,
Trkola, A.,
Thompson, D. A.,
Cormier, E. G.,
Kajumo, F. A.,
Maxwell, E.,
Lin, S. W.,
Ying, W.,
Smith, S. O.,
Sakmar, T. P.,
and Moore, J. P.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5639-5644 |
58. |
Mirzadegan, T.,
Diehl, F.,
Ebi, B.,
Bhakta, S.,
Polsky, I.,
McCarley, D.,
Mulkins, M.,
Weatherhead, G. S.,
Lapierre, J. M.,
Dankwardt, J.,
Morgans, D., Jr.,
Wilhelm, R.,
and Jarnagin, K.
(2000)
J. Biol. Chem.
275,
25562-25571 |
59. | Javitch, J. A., Ballesteros, J. A., Chen, J., Chiappa, V., and Simpson, M. M. (1999) Biochemistry 38, 7961-7968[CrossRef][Medline] [Order article via Infotrieve] |