From the 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,
** Euroscreen s.a., B-1070 Brussels, Belgium, and the
Service de Conformation des
Macromolécules Biologiques, Université Libre de Bruxelles,
CP 160/16, Avenue F. Roosevelt,
1050 Bruxelles, Belgium
Received for publication, June 8, 2002, and in revised form, October 24, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CCR5 is a G protein-coupled receptor
responding to four natural agonists, the chemokines RANTES
(regulated on activation normal T cell expressed and secreted),
macrophage inflammatory protein (MIP)-1 Chemokine receptors have been dragging more and more attention
since the cloning of the first member of the family a decade ago. Not
only are chemokines and their receptors now considered as the main
organizers of leukocyte trafficking, they have also been associated to
an ever increasing number of physiopathological disorders (for review,
see Refs. 1 and 2). In particular, some chemokine receptors are used by
the human immunodeficiency virus
(HIV)1 as coreceptors (in
addition to CD4) to infect target cells (reviewed in Ref. 3). Among
these, CCR5 has been shown to be essential for HIV pathogenesis, as
individuals homozygous for the CCR5 Our current understanding of the activation mechanisms of GPCRs is
rapidly evolving, thanks to the availability of the crystal structure
of the inactive state of one of its members, rhodopsin, and to the
growing amount of biochemical and physicochemical data that can help in
identifying the key aspects of this process (for review, see Refs. 5
and 6). It is now well accepted that the transition from inactive to
active states requires the reorganization of the transmembrane bundle
made of seven imperfect A striking feature within the rhodopsin-like GPCR family is that,
despite a strong sequence conservation of the transmembrane helices,
there is a wide structural diversity among extracellular ligands,
ranging from small neurotransmitters to large glycoproteins. The
structural adaptation of a receptor to its cognate ligand is expected
to involve, in most cases, sequence specificity in the extracellular
domains (for which sequence conservation is much lower across the
family), but also in the transmembrane region, which holds the binding
pocket of small ligands (for reviews, see Refs. 6 and 12). Therefore,
each receptor must have evolved specific structural characteristics to
link the specific recognition of its cognate ligand to what is believed
to constitute a common activation process (e.g. motion of
TM6). Thus, for each receptor subfamily, specific structural features
needed for the activation by agonists are expected to be found.
In the case of chemokine receptors, we have recently identified a
structural motif in TM2, which is central for chemokine-induced activation (13). This motif, which consists of a proline preceded by a
threonine two residues ahead (TXP), is found only in
chemokine receptors and a few related peptide receptors. Our study
indicated that the extracellular part of TM2, whose conformation is
governed by the TXP motif, is clearly involved in the
activation process, as specific mutations of the motif led to
unaffected chemokine binding but strong impairment of receptor
activation (13). Moreover, modeling studies performed on this region
suggested that, because of the action of the proline, the extracellular
part of TM2 would strongly interact with TM3. This organization is
structurally different from that of bovine rhodopsin, in which a
TM2-TM1 interaction is found.
As a follow-up of these observations, we have now investigated the
possible role of the TM2-TM3 interface in the activation process of
chemokine receptors. A sequence alignment of chemokine receptors (Fig.
1) reveals that the extracellular parts of TM2 and TM3 contain many
aromatic residues. Within the rhodopsin template, these residues are
located at relatively short distances, suggesting that they might form
an aromatic cluster within the three-dimensional structure of the
receptors. Aromatic residues have been proposed to be involved in the
activation mechanism in various GPCRs (14-22). Among other examples, a
role was attributed to such residues in the ligand selectivity and
ligand-induced activation of the D2 and D4 dopamine receptors (20, 23).
The high density of aromatic residues at the top of TM2 and TM3 in
chemokine receptors suggested that aromatic side chains could mediate
interactions between these helices.
In the present study, we have mutated the aromatic residues of
CCR5 TM2 and TM3 into their CCR2 counterparts, either individually or
in combination, and the mutants were tested for cell-surface expression, receptor conformation, ligand binding, and functional response. Molecular modeling of the transmembrane region of CCR5 has
been performed, providing a structural framework to interpret these
data. Integration of the experimental and molecular modeling data
indicates that aromatic residues at the TM2-TM3 interface are crucial
to the mechanism of receptor activation and suggests that this aromatic
cluster plays a key role in the conformational changes of CCR5, leading
from ligand recognition to receptor activation.
Numbering Scheme of GPCRs--
In this work, we use a general
numbering scheme to identify residues in the transmembrane segments of
different receptors (24). Each residue is numbered according to the
helix (1 through 7) in which it is located and 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), eight residues following the highly
conserved aspartic acid Asp-2.50. For the sake of clarity,
generalized numbers are italicized. When both numbers are given, the
general numbering is put as superscript.
Survey of Transmembrane Helices Containing a FWXXY Motif in Known
Membrane Protein Structures--
We surveyed the atomic coordinates of
the membrane proteins bacteriorhodopsin (PDB access number 1c3w,
1.55 Å resolution), aa3 (1occ, 2.8 Å) and ba3 (1ehk, 2.4 Å)
cytochrome c oxidases, photosynthetic reaction center (1prc,
2.3 Å), potassium channel (1bl8, 3.2 Å), mechanosensitive ion channel
(1msl, 3.5 Å), rhodopsin (1f88, 2.8 Å), halorhodopsin (1el2, 1.8 Å),
sensory rhodopsin (1h68, 2.1 Å), light harvesting complex (1lgh, 2.4 Å), photosystem I (1jbo, 2.5 Å), AQP1 (1hwo, 3.7 Å) and GlpF (1fx8,
2.2 Å) channels, P-type ATPase (1eul, 2.6 Å), and fumarate reductase
respiratory complex (1qla, 2.2 Å) for Molecular Dynamics Simulations of Transmembrane Helices--
The
model peptides
Ace-Ala11-Thr-Ala-Pro-Ala11-Nme and
Ace-Ala7-Thr-Gly-Ala4-Gly-Ala2-Ser-Gly-Ala15-Nme
were built in the standard Molecular Dynamics Simulations of the CCR5 Receptor and Mutant
Receptors--
The three-dimensional model of transmembrane helices 1 and 4-7 of CCR5 was constructed by computer-aided model building
techniques from the transmembrane domain of bovine rhodopsin, as
determined by Palczewski et al. (27). The following
conserved residues were employed in the alignment of rhodopsin and
human CCR5 transmembrane sequences: Asn-55 (55 being the residue number
in the 1F88 PDB file of rhodopsin) and
Asn-481.50 (48 is the residue number in the CCR5
sequence, 1.50 in the standardized numbering); Trp-161 and
Trp-1534.50; Pro-215 and
Pro-2065.50; Pro-267 and
Pro-2506.50; and Pro-303 and
Pro-2947.50. Representative structures of
transmembrane helices 2 and 3, selected by automatically clustering the
geometries obtained during the molecular dynamics trajectories into
conformationally related subfamilies with the program NMRCLUST (28),
were included into the model (see "Molecular Dynamics Simulations of
Transmembrane Helices" above). SCWRL-2.1 was employed to add the side
chains of the non-conserved residues based on a
backbone-dependent rotamer library (29). All ionizable
residues in the helices were considered uncharged with the exception of
Asp-2.40, Asp-2.50, Asp-3.49, Arg-3.50, Lys-5.50, Arg-6.30,
Arg-6.32, and Glu-7.39. To relieve residual
strain resulting from suboptimal positioning of the side chains and the
TM2-TM3 interface at the extracellular part, this resulting initial
structure was placed in a rectangular box containing methane molecules,
energy-minimized (500 steps), heated (from 0 to 300 K in 15 ps), and
equilibrated (from 15 to 100 ps). During these processes, the
C
Molecular models of the helix bundles for the mutant receptors
containing the F852.59L,
L1043.28F,
F852.59L/L1043.28F,
F1093.33H, F1123.36Y, and
F1093.33H/F1123.36Y
substitutions were constructed from the previously obtained structure
of CCR5, by changing the atoms implicated in the amino acid
substitutions by interactive computer graphics. Subsequently, wild-type
and mutant receptors were placed in a rectangular box (~71 Å × 60 Å × 50 Å in size) containing methane molecules (~2850 molecules in
addition to the transmembrane domain) to mimic the hydrophobic
environment of the transmembrane helices. The density of 0.4-0.5 g
cm CCR5 Mutants--
Plasmids encoding the CCR5 mutants studied
here were constructed by site-directed mutagenesis using the QuikChange
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 (35). 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 (Invitrogen), 100 units/ml penicillin, and 100 µg/ml
streptomycin (Invitrogen). 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 (36).
Selection of transfected cells was made for 14 days with 400 µg/ml
G418 (Invitrogen) 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, Munich, Germany). Unlabeled monoclonal antibodies were
detected by an anti-mouse IgG phycoerythrin-coupled secondary antibody (Sigma).
Binding Assays--
CHO-K1 cells expressing wild-type or mutant
CCR5 were collected from plates with
Ca2+/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
Biosciences) or 0.1 nM 125I-MIP-1 Aequorin Assay--
Functional response to chemokines was
analyzed by measuring the luminescence of aequorin as described (37,
38). Cells were collected from plates with
Ca2+/Mg2+-free DMEM supplemented with 5 mM EDTA. They were then pelleted for 2 min at 1000 × g, resuspended in DMEM 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).
Cells were diluted 5-fold before use. Agonists in a volume of 50 µl
DMEM were added to 50 µl of cell suspension (50,000 cells), and
luminescence was measured for 30 s in a Berthold Luminometer.
GTP MAP Kinase Assay--
Cells, serum-starved for 24 h, were
collected and resuspended in serum-free DMEM. After 3 min of
stimulation with various concentrations of RANTES and MCP-2, cells were
collected by centrifugation and heated to 100 °C for 5 min in lysis
buffer (100 mM Tris-HCl, pH 6.8, 4 mM EDTA, 4%
SDS, 20% glycerol, and 0.02% To investigate the possible role of aromatic residues at the
TM2-TM3 interface in defining the structure and function of the CCR5
receptor, we have compared the amino acid sequences of CCR2 and CCR5
(Fig. 1B). These receptors are
strongly related, sharing ~85% sequence identity within their TM
helices. However, their extracellular domains are much more divergent,
which certainly contributes to their strong selectivity toward their
respective ligands (35). The TM2-TM3 aromatic cluster was found to be
quite divergent between the two receptors. This could suggest that
these positions are not important for the structure and/or function of
the receptors and therefore highly tolerant to variability. Alternatively, these positions could be functionally important although
specific to each receptor, and the various substitutions would be
expected in this case to be correlated. To study the functional
consequences of the differences at aromatic positions observed between
CCR5 and CCR2 (Fig. 1B), we engineered CCR5 mutants in which
aromatic residues were substituted by the corresponding amino acids in
CCR2. The F852.59L and
Y892.63S mutants affected TM2,
L1043.28F; F1093.33H and
F1123.36Y involved TM3. We also combined these
point mutations either within TM2 (F85L/Y89S double mutant), within TM3
(F109H/F112Y, L104F/F109H/F112Y), or across both helices (F85L/L104F,
Y89S/L104F, F85L/Y89S/L104F).
, MIP-1
, and monocyte
chemotactic protein (MCP)-2, and is the main co-receptor for the
macrophage-tropic human immunodeficiency virus strains. We have
previously identified a structural motif in the second transmembrane
helix of CCR5, which plays a crucial role in the mechanism of receptor
activation. We now report the specific role of aromatic residues in
helices 2 and 3 of CCR5 in this mechanism. Using site-directed
mutagenesis and molecular modeling in a combined approach, we
demonstrate that a cluster of aromatic residues at the extracellular
border of these two helices are involved in chemokine-induced
activation. These aromatic residues are involved in interhelical
interactions that are key for the conformation of the helices and
govern the functional response to chemokines in a ligand-specific
manner. We therefore suggest that transmembrane helices 2 and 3 contain
important structural elements for the activation mechanism of
chemokine receptors, and possibly other related receptors as well.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32 mutation, which results in the
synthesis of a non-functional receptor, are highly (although not fully)
resistant to HIV infection. Chemokine receptors belong to the
rhodopsin-like family (family A) of G protein-coupled receptors (GPCR).
CCR5 binds and responds to four natural chemokines, RANTES, MIP-1
,
MIP-1
, and MCP-2, with nanomolar affinities (4).
-helices. In the rhodopsin-like family,
motions of transmembrane helix 3 (TM3) and TM6 during the activation
process have been identified (7-9). Rigid-body movements have also
been proposed for TM5 and TM7 (10, 11). Most of these conformational
changes have been observed in a diverse set of receptors (mainly
rhodopsin and the
2-adrenergic receptor), and it is
believed that they constitute a common conformational path in the
activation process, ultimately leading to the release of GDP in the
bound G protein and its exchange for GTP.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical segments featuring
the FWXXY motif.
-helical conformation (
,
=
58°,
47°). The amino acid side chains of Ser and Thr were set
to the g+ conformation. Molecular dynamics simulations of
these model peptides aim to explore the conformation of TM2 in CCR5
triggered by the Thr-2.56-X-Pro-2.58 motif and TM3 triggered by the presence of Thr-3.29,
Gly-3.30, Gly-3.35, Ser-3.38, and
Gly-3.39. A similar approach was recently used to model the
conformation of TM3 in the 5HT1A serotonin receptor (25).
Ser and Thr residues induce a small bending angle in TM because of the
additional hydrogen bond formed between the O
atom of Ser or Thr and
the i-3 or i-4 peptide carbonyl oxygen (26).
Moreover, the additional flexibility provided by the adjacent Pro
(because of the absence of the hydrogen bond with the carbonyl oxygen
in the preceding turn of the helix) or Gly (because of the lack of the
side chain) reinforces this effect. The obtained structures were placed
in a rectangular box containing methane molecules to mimic the
hydrophobic environment of the TM bundle. 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) at constant volume
using the particle mesh Ewald method to evaluate electrostatic
interactions. Structures were collected for analysis every 10 ps during
the production run (100 structures/simulation). To obtain a rough idea
of the possible consequences that the presence of these residues in TM2
and TM3 might have on the structure of the receptor, we performed a
molecular modeling exercise using the three-dimensional structure of
rhodopsin as the template. The backbone of one helical turn preceding
the highly conserved Asp-2.50 in TM2 and
(D/E)R3.50Y motif in TM3 superimposed the helix bundle
of rhodopsin with the computed structures.
atoms were kept fixed at their positions in the
rhodopsin crystal structure, with the exception of the residues forming
the TM2-TM3 interface (from 2.58 to 3.29). The optimized TM2-TM3 interface accomplishes: (i) the distance between the
top (C terminus) of TM2 and the top (N terminus) of TM3 is in the
10-11-Å range to allow the first extracellular loop (ECL1) of 4 residues to be shaped; (ii) Phe-852.59 interacts
with Leu-1043.28 and
Tyr-892.63 interacts with
Thr-993.23, in a similar manner to the
cytochrome c oxidase structure of helices III and VII in
subunit III (see "Results"); and (iii) there are not steric clashes
between helices. The interactions of the side chain of
Leu-1043.28 with the side chains of
Phe-852.59 and Trp-862.60
were further characterized by ab initio quantum mechanical
calculations at the MP2/6-31G* level of theory, which is capable of
describing the proposed C-H··
interactions (30).
3 of the methane box is approximately half of the
density observed in the hydrophobic core of the membrane. This is a
result of the different equilibrium distance between carbons in the
methane box and in the polycarbon chain of the lipid. However, it has been shown that this procedure reproduces several important structural characteristics of membrane embedded proteins (31). The
receptor-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 (15-100 ps) and a production run (100-250 ps) at
constant volume using the particle mesh Ewald method to evaluate
electrostatic interactions (32). Structures were collected for analysis
every 10 ps during the production run (15 structures per simulation). The molecular dynamics simulations were run with the Sander module of
AMBER 5 (33), the all-atom force field (34), SHAKE bond constraints in
all bonds, a 2-fs integration time step, and constant temperature of
300 K coupled to a heat bath.
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 unlabelled ligand. Samples were
incubated for 90 min at 27 °C, then bound tracer was separated by
filtration through GF/B filters pre-soaked in 0.5% polyethyleneimine
(Sigma) for 125I-RANTES or in 0.1% bovine serum albumin
(Sigma) for 125I-MIP-1
. Filters were counted in a
-scintillation counter. Binding parameters were determined with the
Prism software (GraphPad Software) using non-linear regression applied
to a one site competition model.
S Binding Assay--
The measurement of
chemokine-stimulated GTP
S binding to membranes of cells expressing
wt-CCR5 or the Y108A mutant were performed as described (39, 40).
Briefly, membranes (10 or 20 µg) from wt-CCR5 or Y108A cells were
incubated for 15 min at room temperature in GTP
S binding buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 3 µM GDP, 10 µg/ml saponin)
containing different concentrations of chemokines, in 96-well
microplates (Basic FlashPlates, PerkinElmer Life Sciences).
[35S]GTP
S (0.1 nM, Amersham Biosciences)
was added, microplates were shaken for 1 min and further incubated at
30 °C for 30 min. The incubation was stopped by centrifugation of
the microplate for 10 min, at 800 × g and 4 °C, and
aspiration of the supernatant. Microplates were counted in a TopCount
(Packard, Downers, IL) for 1 min/well. Functional parameters were
determined with the PRISM software (GraphPad Software) using nonlinear
regression applied to a sigmoidal dose-response model.
-mercaptoethanol). For Western blot
analysis, solubilized proteins corresponding to ~5 × 105 cells were loaded onto 10% SDS-polyacrylamide gels in
a Tricine buffer system (41). After transfer to nitrocellulose
membranes, proteins were probed with mouse anti-phospho-p42/p44
(1:1000) (Cell Signal) or rabbit anti-total p38 (1:2000) antibodies
(Santa Cruz).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (63K):
[in a new window]
Fig. 1.
Alignment of TM2 and TM3 sequences from
chemokine receptors. A, alignment comprising TM2 and TM3 for
the different chemokine receptors. The generalized numbering scheme
(see "Experimental Procedures") is used to label the alignment.
Aromatic residues are highlighted in bold
characters, whereas the TXP motif is
boxed. The limits of the helices were defined as those
observed for bovine rhodopsin (27). B, subset of the
alignment focusing on the sequence differences between CCR5 and CCR2.
Aromatic residues differing between the two receptors are in
bold and marked by arrows. The CCR5 numbering for
these positions is given above the sequence.
Cell Surface Expression of the Mutant Receptors
We determined cell surface expression of the CCR5 mutants by fluorescence-activated cell sorting analysis, using five well characterized monoclonal antibodies (40, 42). The epitopes recognized by these monoclonal antibodies have been mapped to the N-terminal domain of the receptor (MC-5 and 3A9), the second extracellular loop (2D7), or a combination of extracellular domains (523 and MC6).
Fig. 2 illustrates the average surface
expression of the different mutants following normalization to
wild-type CCR5 expression level. The TM2 mutant F85L exhibited a
moderate but significantly reduced expression, reaching ~50% of the
wt signal. Although the Y89S mutant was well expressed, the combination
of both substitutions (F85L/Y89S) led to a reduced expression similar
to that of F85L. Interestingly, although the single mutation L104F (in
TM3) did not affect cell surface expression, its combination with F85L restored to normal the low expression observed for F85L alone (mutant
F85L/L104F). Strikingly, the triple mutant F85L/Y89S/L104F was not
found at the cell surface, indicating that, although single and double
mutants are expressed, the combination of all three substitutions is
not tolerated. This mutant will therefore not be considered further in
the following experimental settings. Mutants Y89S/L104F, F109H, F112Y,
F109H/F112Y, and L104F/F109H/F112Y are all expressed above 50% of the
wt signal with a rather uniform pattern of recognition by the different
monoclonal antibodies, suggesting that these mutations do not alter the
conformation of CCR5 extracellular domains.
|
Binding and Functional Properties of the Mutant Receptors
The functional consequences of the mutations and their
combinations were characterized in terms of binding and
intracellular responses, using the four natural agonists of CCR5:
RANTES, MIP-1, MIP-1
, and MCP-2. The binding affinities for
the various ligands were determined by competition binding assays using
125I-RANTES or 125I-MIP-1
as labeled tracer,
whereas functional responses were monitored by using the aequorin-based
assay as described previously (13). Representative binding curves are
shown in Fig. 3, functional concentration-action curves are shown in Fig.
4, and the data are summarized in
Table I.
|
|
|
We first focused on residues Phe-85, Tyr-89, and Leu-104, located at
the extracellular ends of TM2 and TM3. The F85L mutant was clearly
impaired both at the level of binding, and in its functional response
to chemokines. Although binding of RANTES was almost unaffected, the
apparent affinities for MIP-1 and MCP-2 were significantly lower
than those observed for wt-CCR5 (see Table I and Fig. 3), and MIP-1
binding was undetectable. The functional responses of F85L were mild
(Fig. 4) and grossly consistent with the binding data. The potency of
RANTES was moderately affected on this mutant, but its efficacy was
reduced by 4-fold; MIP-1
and MCP-2 displayed a strong impairment of
both their potencies and efficacies, whereas MIP-1
was almost
inactive. The phenotype of the L104F mutant was grossly similar in
terms of functional responses (with slightly better efficacies) despite
binding parameters much closer to the wild-type levels. Interestingly,
the addition of the L104F substitution to the F85L mutant (F85L/L104F)
allowed partial restoration of the function of this mutant. Indeed, on the double mutant, MIP-1
was characterized by a binding affinity close to wt, and the functional response was significantly improved. The functional properties of MCP-2 appeared somewhat more affected by
the double mutation, whereas RANTES and MIP-1
showed similar behaviors on the double mutant and on both single mutants.
Mutating Y89S affected mildly the activity of RANTES and MIP-1
(conserved potency, 2-fold reduction of Emax)
decreased moderately the Emax and potency of
MIP-1
, but affected strongly the functional parameters of MCP-2,
despite the normal affinity of this mutant in binding assays. Combining
F85L and Y89S substitutions led to a severe impairment of functional
responses, particularly for MIP-1
and MCP-2 (no response), and to a
lower extent for MIP-1
, whereas RANTES was moderately affected.
These effects appeared more than simply additive as compared with the
single mutants. The Y89S/L104F mutant was well expressed at the cell
surface, but no specific binding could be detected by competition
binding assays, and this receptor was barely functional. Only RANTES
could elicit a small signal at high concentrations, with a strongly reduced Emax but a decent potency (Table I).
Modeling the TM2-TM3 Interface
The experimental data presented above demonstrate the importance of the TM2-TM3 aromatic cluster in the activation of CCR5. The fact that adding the L104F substitution to the F85L background partly restores the impaired function of this mutant (cell-surface expression and binding) might suggest a direct interaction between the two residues involved. Moreover, Tyr-89 might contribute to this interaction, as mutating this residue, although well tolerated by itself, is highly disruptive in the context of F85L, Y89S, or F85L/Y89S. Fig. 6A shows the location of the side chains of these residues in a CCR5 model using strictly the rhodopsin crystal as a template (27). The orientation of these side chains toward the lipidic environment is in apparent contradiction with the hypothesis that these residues are important for the structure and the activation mechanism of CCR5.
We have, however, suggested previously that TM2 of CCR5 would adopt, in its outer half, a conformation that is different from that of bovine rhodopsin (13). In CCR5 and other chemokine receptors, the extracellular side of TM2 is predicted to be in close contact with TM3 (and not with TM1 as in rhodopsin) as a result of the structural action of a conserved Thr2.56-X-Pro2.58 motif (13). We now propose that this region of TM2 would be part of a structural and functional motif involving the neighboring part of TM3 (aromatic cluster and surrounding residues).
To provide a structural framework allowing to understand better the experimental data presented above, we adopted a modeling procedure (detailed under "Experimental Procedures") based on the following scheme.
Independent Exploration of the Conformation of TM2 and TM3 by Molecular Dynamics Simulations-- Fig. 6 (B and C) shows the computed structures of TM2 (green) and TM3 (yellow). The bending of TM2 toward TM3, in its outer half, is tolerated in the context of the CCR5 helical bundle as the result of the relocation of TM3 toward TM5. It is important to note that these energetically available structures of TM2 and TM3 were obtained separately. Thus, the conformational spaces explored by these helices are the consequence of the amino acid sequence of TM2 and TM3 and not of steric hindrance between helices.
The TM2-TM3 Interface Was Further Optimized by MD Simulations of the Seven-helix Bundle in an Apolar Environment-- Fig. 6 (D and E) shows the result of superimposing the representative structure of the MD simulation and the rhodopsin template. The specific residues in TM2 and TM3 of CCR5 generate structural differences in the extracellular part of the receptor, without modifying its more compact cytoplasmic surface.
Membrane Protein Data Base Search--
Because stable
structural motifs are likely to recur in proteins of known structure,
we surveyed the data base of the structure of membrane proteins (see
"Experimental Procedures") for -helix segments containing the
aromatic cluster of TM2: the FWXXY motif. This motif is also
found in subunit III of the bovine cytochrome c oxidase (PDB
identification code 1occ), where transmembrane helix III of the enzyme
interacts with the neighboring helix VII. Inspection of this motif in
the cytochrome c oxidase structure reveals that the Phe-98
side chain in helix III interacts with Leu-252 residue in helix VII,
and that the Tyr-102 side chain in helix III hydrogen bonds Ser-255 in
helix VII. We propose a similar pattern to describe the interactions
between TM2 and TM3 of CCR5; Phe-85 would interact with Leu-104, and
Tyr-89 with Thr-99. It is important to note that Phe-85, Tyr-89,
Thr-99, and Leu-104 are found specifically in CCR5, but not in CCR2,
and contribute therefore to a CCR5-specific motif important for the
receptor structure.
Fig. 7A shows a detailed view of the TM2-TM3
interface in the model resulting from MD simulation. In this model,
Leu-104 is located in an aromatic pocket formed by the side chains of
Phe-85 and Trp-86, and the electron-poor C-H hydrogens of L104 interact with the electron-rich clouds of the aromatic rings. This type of
C-H··
interaction plays a significant role in stabilizing local
three-dimensional structures of proteins (43). Moreover, Phe-85
aromatic ring is located between Leu-103 and Leu-104 side chains. Thus,
there is a significant interaction between the aromatic residues
(Phe-85 and Trp-86) in TM2 and the hydrophobic residues (Leu-103 and
Leu-104) in TM3. In addition, the TM2-TM3 interface is stabilized by a
hydrogen bond between Tyr-89 and Thr-99. To evaluate the magnitude of
the TM2-TM3 interaction that might be attributed to the
Phe-85··Leu-104 and Trp-86··Leu-104 interactions, we performed
ab initio quantum mechanical calculations on minimal recognition models consisting of the functional groups of the intervening side chains (see "Experimental Procedures"). The
energies of interaction of Phe-85 and Trp-86 with the multiple C-H
hydrogens of Leu-104 are
2.4 and
2.6 kcal/mol, respectively.
Structural and Functional Role of Aromatic Residues in TM3
Residues Phe-109 and Phe-112 are located in TM3 within the outer
third of the membrane and are predicted to face toward the center of
the helix bundle, as inferred from the molecular model of CCR5 (see
above) or the rhodopsin template itself. The F109H mutation had little
effect on the receptor function. Both the binding properties and the
functional response of the mutant were closely similar to those of
wt-CCR5 for all four ligands (Table I, Figs. 3 and 4). In contrast, the
conservative F112Y substitution influenced strongly the activation of
the receptor by its agonists. The potencies of RANTES, MIP-1, and
MIP-1
were relatively preserved, whereas that of MCP-2 was decreased
by ~1 log. The efficacies of all ligands were however severely
affected, with Emax values ranging from 10 to
25% of the ATP response (Table I). Remarkably, combining these two
substitutions in the F109H/F112Y mutant significantly restored the
functional response of the receptor, with Emax
values ranging from 54 to 90% of the maximal cell response and
improved of potencies for all four ligands. The L104F/F109H/F112Y
change, which combine the three mutations of TM3, produced a receptor phenotypically close to that of the single L104F. Although binding affinities were wt-like, activation was affected differentially for the
different agonists; RANTES was almost not affected, MCP-2 severely
impaired, with MIP-1
and MIP-1
showing intermediate behaviors.
This suggest an addition of effects of the L104F single mutant
(significantly affected) and the F109H/F112Y double mutant (mild
effect). This is not surprising considering that these motifs are
located in distant part of the structure, as, in our model, Leu-104
interacts with TM2, whereas Phe-109 and Phe-112 face TM5 and TM6,
respectively (see "Discussion").
Mutant Y1083.32A
Considering the selective effect on receptor function observed
after mutation of transmembrane residues in TM2 and TM3, it appeared
also interesting to test the putative role of residue Tyr-108. This
locus, referred as position 3.32 in the generalized numbering scheme of Ballesteros (see "Experimental Procedures"), is
known as an important binding site in a wide variety of GPCRs (reviewed
in Refs. 6, 12, and 24). Site-directed mutagenesis has been used to
demonstrate its central role in different receptors for
neurotransmitters and peptides. The Y108A mutant was well recognized at
the cell surface by antibodies targeting the N terminus of the receptor
(mean fluorescence above 60% of signal for wt-CCR5), whereas
fluorescence was significantly decreased for antibodies recognizing
either the second loop or a combination of extracellular domains,
possibly underlying a conformational modification of the extracellular
loops of the receptor. This mutant exhibited a rather wild-type
behavior following stimulation by RANTES, MIP-1, or MIP-1
, with
slightly reduced efficacies, but was completely unreactive to MCP-2. We
could, however, measure high affinity binding of this ligand by
competition binding assay (Fig. 3 and Table I). As for several other
mutants described above, this mutant became unresponsive to MCP-2
without affecting the affinity for this ligand, whereas other CCR5
agonists (e.g. RANTES) were still able to simulate it. It is
known that GPCRs, including CCR5 (40), can adopt multiple active states
that trigger different intracellular cascades and are differentially
stabilized by various agonists. In this line, one could hypothesize
that MCP-2 would not be able to induce Ca2+ increase
through this mutant receptor, while being able to trigger other
intracellular cascades. As shown in Fig.
5A, the four natural agonists
could trigger GTP
S binding through the wt receptor. In this assay,
RANTES was the most potent agonist, whereas MCP-2 appeared more potent
than MIP-1
and MIP-1
. As observed with the aequorin assay, MCP-2
was unable to stimulate the Y108A mutant in the GTP
S assay. It has
been shown that chemokine-induced activation of CCR5 can lead to
activation of p42/p44 (44, 45). As shown in Fig. 5B,
stimulation of CCR5 by RANTES or MCP-2 led to the dose-dependant
phosphorylation of p42/p44 MAP kinases. In this assay, RANTES
stimulated the Y108A mutant, although with a reduced efficiency as
compared with wt-CCR5, and MCP-2 was almost inactive, except for a weak
response observed at 100 nM.
|
The specific alteration of the mutant response to MCP-2 is reminiscent
of the behavior observed in our previous study, in which mutations of
the TXP motif also affected preferentially the biological
response to MCP-2, without significantly affecting the binding of this
chemokine. In the wt model, the Tyr-108 side chain is positioned in the
face-to-edge orientation (T-shaped) with the indole ring of Trp-86 (see
Fig. 7A). This type of -
aromatic-aromatic interaction
has been described as stabilizing a protein structure (46). It is
expected that the conformational changes induced in TM2 by mutations
affecting the TXP motif would relocate the side chain of
Trp-86, located four residues apart, and as a consequence affect its
interaction with Tyr-108.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The three-dimensional model of CCR5 based on the rhodopsin crystal
structure (including the specific modeling of the TM2-TM3 interface as
described above; Fig. 6) provides a
coherent framework to interpret the mutagenesis data detailed under
"Results." We propose that the residues varying between CCR5 and
CCR2 at the level of the TM2-TM3 aromatic cluster are forming a
specific interaction motif. In the wt receptor,
Phe-852.59 would interact with
Leu-1043.28, whereas
Tyr-892.63 would H-bond with
Thr-993.23, and we suggest that the
substitutions observed in the CCR2 sequence are indeed correlated. It
is important to note that this modeled TM2-TM3 interface provides a
hydrophobic environment for Thr-822.56 of the
TXP motif in TM2, essentially through the side chain of Leu-104 (Fig. 7A). Thus,
Thr-82 orients the C atom toward this hydrophobic
environment, and the O
atom toward the polar peptide
bond of the backbone. The additional hydrogen bond between the
O
atom of Thr-82 and the carbonyl group increases the magnitude of the Pro-kink and this TXP motif is a structural
determinant involved in chemokine-induced activation (13).
|
|
To analyze in this structural framework the consequences of the amino acid substitutions explored experimentally, several of the mutations were introduced in our model, and we specifically studied the structural and dynamical properties of the extracellular part of the TM2-TM3 interface using MD simulations (see "Experimental Procedures"). This modeling procedure allows to propose a description of the TM interface for the mutants and suggests the nature of the structural changes that might lead to alterations of the receptor function (Fig. 7).
Structural Interpretation of Mutations Located at the Extracellular End of TM2 and TM3-- The F85L mutation weakens the interaction between the side chain of this TM2 residue and Leu-103 and Leu-104 in TM3, as a Leu-Leu interaction is not of the same magnitude as a Leu-Phe interaction. As a consequence, in the simulation, Leu-85 side chain reorients away from TM3 (Fig. 7B).
Replacing Leu-104 by Phe dramatically modifies the TM2-TM3 interface.
The aromatic side chain of Phe-104 would optimally interact with the
other aromatic side chains in a face to edge configuration (46). Thus,
in the molecular dynamic simulations, the side chain of Phe-104 tends
to achieve this interaction with the aromatic side chains of both
Phe-85 and Trp-86 (Fig. 7C). However, the side chain of
Phe-104 in this conformation is bulkier than that of Leu, resulting in
a significant reorientation of Phe-85 side chain toward the periphery
of the TM bundle, and in a weakening of the TM2-TM3 interface. It is
important to note that the L104F mutation increases the polarity of the
Thr-82 side chain environment. Thus, the presence of the electron-rich
clouds of Phe-104 aromatic ring might facilitate hydrogen bonding to
the O atom of Thr-82, in a manner similar to that
proposed for hydrogen bonding between benzene and water (47). This
would contrast with the wild-type receptor, in which a hydrogen bond is
formed with the polar peptide bond of the backbone. As already
discussed, the additional hydrogen bond of the Thr to the peptide bond
has a significant influence on the conformation of the helix (13,
26).
In the double F85L/L104F mutant, the aromatic side chain of Phe-104 is now located between Leu-85 and Leu-103 (Fig. 7D). Thus, the electron-rich clouds of the aromatic ring interact with the electron-poor C-H hydrogens of both Leu-85 and Leu-104. Notably, this mode of interaction of Phe-104 places Leu-85 in the proximity of Thr-82, mimicking the hydrophobic environment of Thr-82 in the wild-type receptor (see above). Moreover, the aromatic side chain of Tyr-89 is interacting in a face to edge configuration with Phe-F104. Thus, it appears from this simulation that the F85L/L104F mutant would partly restores the packing of the TM2-TM3 interface.
This could explain the improvement in cell-surface expression and
binding affinity for MIP-1 and MIP-1
, between the F85L and
F85L/L104F mutants (see Table I). As the conformation of the short ECL1
(four residues) is likely influenced by the packing of the TM2-TM3
interface, we would suggest that point mutations (like F85L) could
modify the conformation of the EC domain and affect the binding of ligands.
The triple mutant F85L/L104F/Y89S is intriguing. Intuitively, one would expect that, having replaced all differing aromatic positions into the CCR2 corresponding residues, the resulting mutant would show a better functional phenotype than the single or double mutant (being closer to the functional CCR2 receptor). However, it appeared that this triple mutant is not expressed at the cell surface (see Fig. 2), suggesting severe misfolding of the protein. Our modeling suggests that the addition of the Y89S substitution to the double F85L/L104F mutant would strongly modify the TM2-TM3 interface, because the shorter and non-aromatic side chain of Ser-89 cannot fulfill the interaction with both Thr-99 and Phe-104 (Fig. 7D). We hypothesize that this major structural difference between this mutant interface and the wt structure could be a factor responsible for the weak expression, either through a perturbation of the folding process, or through destabilization of the folded protein, leading to rapid internalization and/or degradation. In the single Y89S mutant, this modification of the packing of TM2 and TM3 would not happen as the Phe-85-Leu-104 interaction maintains a proper distance between the two helices. Moreover, Thr-993.23 would tend to interact with the side chain of Gln-932.67 (Fig. 7C), changing moderately the interface. Notably, the Y89S mutant shows a normal level of expression and is moderately affected in the functional tests, suggesting that the Tyr-89-Thr-99 interaction proposed here is necessary for full functional efficiency, but not for the structural stability of the receptor. The double mutants F85L/Y89S and Y89S/L104F show a level of expression reduced by ~50%, a functional response strongly affected for F85L/Y89S and almost completely abolished for Y89S/L104F, underlining the increase in structural perturbation caused by the additional mutations.
Combining the experimental results with our modeling approach leads to the following picture; the function of the CCR5 receptor is strongly dependent on the interface between the extracellular ends of TM2 and TM3, which is determined by a series of interhelical interactions. The structural and dynamical properties of this interface are maintained by, at least, two polar interactions: Tyr-89-Thr-99 and Phe-85-Leu-104. These interactions are in equilibrium with each other, providing balanced structural constraints. Perturbing this interface by mutating one of these residues would modify this equilibrium and affect the function of the receptor.
This model implies that, in the case of the CCR2 receptor, the TM2-TM3 interface is organized differently, and that the substitutions tested here are counterbalanced by other changes in the sequence. In particular, one can spot the T993.23A change between the two receptors (see Fig. 1), but other changes (like A922.66N or Q932.67E) could also be important. Interestingly, a chimeric construct involving ICL1, TM2, ECL1, and TM3 of CCR2 in a CCR5 background appeared to be well expressed and functional (35), suggesting that interactions between TM2 and TM3 are sufficient to obtain structural stability and functionality (as the other helices were strictly from CCR5).
The Extracellular Ends of TM2 and TM3 Are Involved in the Activation Mechanism-- By mutating the Thr-2.56-X-Pro-2.58 motif, we previously have shown that the structural integrity of TM2 is crucial for CCR5 function (13). According to the nature of the substitution (various residues in place of Thr-822.56, or Ala instead of Pro-842.58), we could modulate the extent of the structural perturbation, which was translated into a functional defect. However, the different agonists of CCR5 were affected differently by these mutations, suggesting that this part of the receptor is involved in the activation process in a ligand-specific manner.
We proposed that this TXP motif governs the structural and dynamical properties of the extracellular end of TM2. By looking at the specific role of Phe-852.59 and Tyr-892.63, we now probe the elements involved in the ligand-induced activation in this particular part of the TM bundle. Interestingly, we find the same trend as that observed after mutating the TXP motif; the ligand sensitivity to the mutations is variable, RANTES being the least affected agonist, whereas MCP-2 is the most sensitive.
Structural Modeling of Mutants Involving the Middle of TM3-- Phe-109 and Phe-112, variable between CCR5 and CCR2, are not facing TM2 in the CCR5 model, but are oriented toward TM5 and TM6. We therefore undertook full bundle MD simulation to model (see "Experimental Procedures") the possible effects of the F1093.33H, F1123.36Y, and F109H/F112Y mutations, and the putative interaction between these residues and TM5 and/or TM6.
Although the F109H change does not modify the pattern of helix-helix
interactions as compared with the wt receptor (data not shown),
changing Phe-112 for Tyr modifies the interactions between TM3 and TM6
(Fig. 7, panels E and F). In our
simulation, the hydroxyl group added by the mutation forms a H-bond
with Asn-2526.52 in TM6. In the wt receptor,
this Asn H-bonds back to the backbone carbonyl of
Trp-2486.48, and also interacts through its
H1 atom with the aromatic ring of Phe-112 (Fig.
7E). Pointing inside the bundle, residue Asn-2526.52 is highly conserved among
CC-chemokine receptors, and position 6.52 is known to be
functionally important in several rhodopsin-like GPCRs (6). In
particular, this position is involved in ligand binding, ligand
selectivity, or receptor activation in a significant number of peptide
(48-52) and neurotransmitter (16, 17, 53) receptors, suggesting a
possible functional role of the corresponding residue in chemokine
receptors as well. In addition, the H-bond proposed here to form with
the backbone could also be important. The conserved Pro, present in TM6
of most rhodopsin-like receptors (Pro-6.50) is known to be
crucial for their function, most probably through the structural action
of the proline. It has been proposed that, in the context of an
-helix, polar residues H-bonding the backbone in the vicinity of a
Pro could significantly modulate the structure of the proline-induced
deformation, and therefore be functionally relevant (13, 54).
Interestingly, in the F112Y mutant, the polar interaction between
Tyr-1123.36 and
Asn-2522.52 changes the conformation of the
asparagine away from the backbone, hence preventing it from H-bonding
with the backbone carbonyl of Trp-2486.48.
Therefore, the polar interaction between Tyr-112 and Asn-252 could strongly modify the functional properties of Asn-252 by interfering with its putative interaction with the ligand and/or its structural role. It is important to note that, in contrast to the mutations located in the TM2-TM3 interface, the F112Y mutation affects all four agonists in a similar manner. The activation profile is qualitatively similar to that of the wt receptor (the potency order is preserved), but the potency and efficacy of agonists are reduced ~3-fold.
As detailed under "Results," the double F109H/F112Y mutant exhibits
a behavior similar to that of the wt receptor, hence showing that
adding the F109H mutation restores the function lost in the F112Y
mutant. Although such functional recovery may suggest a direct
interaction between the two residues, this is very unlikely in the
context of an -helix, in which a His and a Tyr cannot interact
together when separated by three positions. In our model, the F109H
substitution modifies indirectly the effect of the F112Y mutation (Fig. 7G). Although Tyr-112 now interacts with the
backbone carbonyl of Gly-2025.46,
His-109 interacts with Asn-252, leading to a reorientation of the side
chain that mimics the wt situation, including a recovery of the H-bond
between Asn-2526.52 and the carbonyl of
Trp-2486.48. The effect of the single F112Y
mutation is therefore counterbalanced by the addition of the histidine,
which allows the reorientation of Asn-2526.52,
leading to an almost normal behavior of this putative functional motif.
Conclusions--
The wealth of structural, biophysical, and
biochemical data accumulated during the last years provides valuable
insights into the activation mechanism of GPCRs. It is well established
that the activation process requires helix motions; in particular, movements of TM3, TM6, and TM7 have been evidenced (5, 6). The
importance of the TM2-TM3 interface in the activation of CCR5 support
the concept that other parts of the TM bundle are also involved in this
mechanism. As suggested previously (13), we hypothesize that the active
role of the TM2-TM3 interface in the activation process is somehow
specific to chemokine receptors, as suggested by the conservation of
the TXP motif in TM2, and of the aromatic cluster in the
extracellular ends of TM2 and TM3. Although our work suggests that TM2
(and possibly TM3) would actually move upon chemokine-induced
activation, biophysical approaches would be necessary to demonstrate
the motion of these helices during activation.
![]() |
ACKNOWLEDGEMENTS |
---|
Expert technical assistance was provided by M. J. Simons. We thank Mathias Mack for kindly providing monoclonal antibodies and Juan Ballesteros for inspiring 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 Centre de Recherche Inter-universitaire en Vaccinologie, the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming, the 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 SAF2002-01509, a grant from Fundació La Marató TV3, and Improving Human Potential of the European Community Grant HPRI-CT-1999-00071). Computer facilities were provided by the Centre 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.
§ Current address: Dept. of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143.
¶ These two authors contributed equally to this work.
§§ To whom correspondence should be addressed: IRIBHN, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium. Tel.: 32-2-555-41-71; Fax: 32-2-555-46-55; E-mail: mparment@ulb.ac.be.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M205685200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HIV, human
immunodeficiency virus;
GPCR, G protein-coupled receptor;
TM, transmembrane helix;
ECL, extracellular loop;
ICL, intracellular loop;
MD, molecular dynamics;
MIP, macrophage inflammatory protein;
MCP, monocyte chemotactic protein;
wt, wild-type;
RANTES, regulated on
activation normal T cell expressed and secreted;
PDB, Protein Data
Bank;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
CHO, Chinese hamster ovary;
MAP, mitogen-activated protein;
DMEM, Dulbecco's modified Eagle's medium;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Baggiolini, M. (1998) Nature 392, 565-568[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gerard, C., and Rollins, B. J. (2001) Nat. Immunol. 2, 108-115[CrossRef][Medline] [Order article via Infotrieve] |
3. | Berger, E. A., Murphy, P. M., and Farber, J. M. (1999) Annu. Rev. Immunol. 17, 657-700[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. | Meng, E. C., and Bourne, H. R. (2001) Trends Pharmacol. Sci. 22, 587-593[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Gether, U.
(2000)
Endocr. Rev.
21,
90-113 |
7. |
Farrens, D. L.,
Altenbach, C.,
Yang, K.,
Hubbell, W. L.,
and Khorana, H. G.
(1996)
Science
274,
768-770 |
8. |
Dunham, T. D.,
and Farrens, D. L.
(1999)
J. Biol. Chem.
274,
1683-1690 |
9. |
Gether, U.,
Lin, S.,
Ghanouni, P.,
Ballesteros, J. A.,
Weinstein, H.,
and Kobilka, B. K.
(1997)
EMBO J.
16,
6737-6747 |
10. |
Marjamaki, A.,
Frang, H.,
Pihlavisto, M.,
Hoffren, A. M.,
Salminen, T.,
Johnson, M. S.,
Kallio, J.,
Javitch, J. A.,
and Scheinin, M.
(1999)
J. Biol. Chem.
274,
21867-21872 |
11. |
Abdulaev, N. G.,
and Ridge, K. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12854-12859 |
12. |
Ji, T. H.,
Grossmann, M.,
and Ji, I.
(1998)
J. Biol. Chem.
273,
17299-17302 |
13. |
Govaerts, C.,
Blanpain, C.,
Deupi, X.,
Ballet, S.,
Ballesteros, J. A.,
Wodak, S. J.,
Vassart, G.,
Pardo, L.,
and Parmentier, M.
(2001)
J. Biol. Chem.
276,
13217-13225 |
14. | Wess, J., Gdula, D., and Brann, M. R. (1991) EMBO J. 10, 3729-3734[Abstract] |
15. | Fong, T. M., Cascieri, M. A., Yu, H., Bansal, A., Swain, C., and Strader, C. D. (1993) Nature 362, 350-353[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Kim, J.,
Wess, J.,
van Rhee, A. M.,
Schoneberg, T.,
and Jacobson, K. A.
(1995)
J. Biol. Chem.
270,
13987-13997 |
17. | Cho, W., Taylor, L. P., Mansour, A., and Akil, H. (1995) J. Neurochem. 65, 2105-2115[Medline] [Order article via Infotrieve] |
18. | Lee, S. Y., Zhu, S. Z., and el Fakahany, E. E. (1996) Recept. Signal. Transduct. 6, 43-52[Medline] [Order article via Infotrieve] |
19. |
Roth, B. L.,
Shoham, M.,
Choudhary, M. S.,
and Khan, N.
(1997)
Mol. Pharmacol.
52,
259-266 |
20. | Javitch, J. A., Ballesteros, J. A., Weinstein, H., and Chen, J. (1998) Biochemistry 37, 998-1006[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Colson, A. O.,
Perlman, J. H.,
Jinsi-Parimoo, A.,
Nussenzveig, D. R.,
Osman, R.,
and Gershengorn, M. C.
(1998)
Mol. Pharmacol.
54,
968-978 |
22. | Rhee, M. H., Nevo, I., Bayewitch, M. L., Zagoory, O., and Vogel, Z. (2000) J. Neurochem. 75, 2485-2491[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Simpson, M. M.,
Ballesteros, J. A.,
Chiappa, V.,
Chen, J.,
Suehiro, M.,
Hartman, D. S.,
Godel, T.,
Snyder, L. A.,
Sakmar, T. P.,
and Javitch, J. A.
(1999)
Mol. Pharmacol.
56,
1116-1126 |
24. | Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25, 366-428 |
25. |
Lopez-Rodriguez, M. L.,
Vicente, B.,
Deupi, X.,
Barrondo, S.,
Olivella, M.,
Morcillo, M. J.,
Behamu, B.,
Ballesteros, J. A.,
Salles, J.,
and Pardo, L.
(2002)
Mol. Pharmacol.
62,
15-21 |
26. |
Ballesteros, J.,
Deupi, X.,
Olivella, M.,
Haaksma, E.,
and Pardo, L.
(2000)
Biophys. J.
79,
2754-2760 |
27. |
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 |
28. | Kelley, L. A., Gardner, S. P., and Sutcliffe, M. J. (1996) Protein Eng. 9, 1063-1065[Medline] [Order article via Infotrieve] |
29. | Bower, M. J., Cohen, F. E., and Dunbrack, R. L. (1997) J. Mol. Biol. 267, 1268-1282[CrossRef][Medline] [Order article via Infotrieve] |
30. | Tsuzuki, S., Honda, K., Uchimaru, T., Mikami, M., and Tanabe, K. (2000) J. Am. Chem. Soc 122, 3746-3753[CrossRef] |
31. |
Olivella, M.,
Deupi, X.,
Govaerts, C.,
and Pardo, L.
(2002)
Biophys. J.
82,
3207-3213 |
32. | Darden, T., York, D., and Pedersen, L. (1993) J. Chem. Phys. 98, 10089-10092[CrossRef] |
33. | 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 |
34. | 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 |
35. |
Samson, M.,
LaRosa, G.,
Libert, F.,
Paindavoine, P.,
Detheux, M.,
Vassart, G.,
and Parmentier, M.
(1997)
J. Biol. Chem.
272,
24934-24941 |
36. |
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 |
37. | 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] |
38. |
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 |
39. |
Blanpain, C.,
Wittamer, V.,
Vanderwinden, J. M.,
Boom, A.,
Renneboog, B.,
Lee, B., Le,
Poul, E., El,
Asmar, L.,
Govaerts, C.,
Vassart, G.,
Doms, R. W.,
and Parmentier, M.
(2001)
J. Biol. Chem.
276,
23795-23804 |
40. |
Blanpain, C.,
Vanderwinden, J. M.,
Cihak, J.,
Wittamer, V., Le,
Poul, E.,
Issafras, H.,
Stangassinger, M.,
Vassart, G.,
Marullo, S.,
Schlndorff, D.,
Parmentier, M.,
and Mack, M.
(2002)
Mol. Biol. Cell
13,
723-737 |
41. | Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379[Medline] [Order article via Infotrieve] |
42. |
Lee, B.,
Sharron, M.,
Blanpain, C.,
Doranz, B. J.,
Vakili, J.,
Setoh, P.,
Berg, E.,
Liu, G.,
Guy, H. R.,
Durell, S. R.,
Parmentier, M.,
Chang, C. N.,
Price, K.,
Tsang, M.,
and Doms, R. W.
(1999)
J. Biol. Chem.
274,
9617-9626 |
43. | Steiner, T., and Koellner, G. (2001) J. Mol. Biol. 305, 535-557[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Dairaghi, D. J.,
Franz-Bacon, K.,
Callas, E.,
Cupp, J.,
Schall, T. J.,
Tamraz, S. A.,
Boehme, S. A.,
Taylor, N.,
and Bacon, K. B.
(1998)
Blood
91,
2905-2913 |
45. |
Misse, D.,
Esteve, P. O.,
Renneboog, B.,
Vidal, M.,
Cerutti, M., St,
Pierre, Y.,
Yssel, H.,
Parmentier, M.,
and Veas, F.
(2001)
Blood
98,
541-547 |
46. | Burley, S. K., and Petsko, G. A. (1985) Science 229, 23-28[Medline] [Order article via Infotrieve] |
47. | Suzuki, S., Green, P. G., Bumgarner, R. E., Dasgupta, S., Goddard, W. A., III, and Blake, G. A. (1992) Science 257, 942-945 |
48. | Zoffmann, S., Gether, U., and Schwartz, T. W. (1993) FEBS Lett. 336, 506-510[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Fong, T. M., Yu, H.,
Cascieri, M. A.,
Underwood, D.,
Swain, C. J.,
and Strader, C. D.
(1994)
J. Biol. Chem.
269,
2728-2732 |
50. | Kask, K., Berthold, M., Kahl, U., Nordvall, G., and Bartfai, T. (1996) EMBO J. 15, 236-244[Abstract] |
51. | Turner, C. A., Cooper, S., and Pulakat, L. (1999) Biochem. Biophys. Res. Commun. 257, 704-707[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Huang, X. P.,
Nagy, P. I.,
Williams, F. E.,
Peseckis, S. M.,
and Messer, W. S., Jr.
(1999)
Br. J. Pharmacol.
126,
735-745 |
53. | Granas, C., Nordvall, G., and Larhammar, D. (1998) J. Recept. Signal. Transduct. Res. 18, 225-241[Medline] [Order article via Infotrieve] |
54. |
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 |