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Signal transmission through the CXC chemokinereceptor 4 (CXCR4)
transmembrane helicesMelanie P. Wescotta, Irina Kufarevab, Cheryl
Paesa, Jason R. Goodmana, Yana Thakera, Bridget A. Puffera, Eli
Berdougoa,Joseph B. Ruckera, Tracy M. Handelb, and Benjamin J.
Doranza,1
aIntegral Molecular, Philadelphia, PA 19104; and bSkaggs School
of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego, La Jolla,CA 92093
Edited by K. Christopher Garcia, Stanford University, Stanford,
CA, and approved July 6, 2016 (received for review January 23,
2016)
The atomic-level mechanisms by which G protein-coupled
receptors(GPCRs) transmit extracellular ligand binding events
through theirtransmembrane helices to activate intracellular G
proteins remainunclear. Using a comprehensive library of mutations
covering all352 residues of the GPCR CXC chemokine receptor 4
(CXCR4), weidentified 41 amino acids that are required for
signaling induced bythe chemokine ligand CXCL12 (stromal
cell-derived factor 1). CXCR4variants with each of these mutations
do not signal properly butremain folded, based on receptor surface
trafficking, reactivity toconformationally sensitive monoclonal
antibodies, and ligand bind-ing. When visualized on the structure
of CXCR4, the majority ofthese residues form a continuous
intramolecular signaling chainthrough the transmembrane helices;
this chain connects chemokinebinding residues on the extracellular
side of CXCR4 to G protein-coupling residues on its intracellular
side. Integrated into a cohesivemodel of signal transmission, these
CXCR4 residues cluster into fivefunctional groups that mediate (i)
chemokine engagement, (ii) signalinitiation, (iii) signal
propagation, (iv) microswitch activation, and(v) G protein
coupling. Propagation of the signal passes through a“hydrophobic
bridge” on helix VI that coordinates with nearly everyknown GPCR
signaling motif. Our results agree with known con-served mechanisms
of GPCR activation and significantly expand onunderstanding the
structural principles of CXCR4 signaling.
GPCR activation | G protein | chemokine receptor | hydrophobic
bridge |shotgun mutagenesis
The CXC chemokine receptor 4 (CXCR4) belongs to the
Gprotein-coupled receptor (GPCR) superfamily of proteins,the
largest class of integral membrane proteins encoded in thehuman
genome, comprising greater than 30% of current drugtargets.
Deregulation of CXCR4 expression in multiple humancancers, its role
in hematopoietic stem cell migration, and theutilization of CXCR4
by HIV-1 for T-cell entry, make this re-ceptor an increasingly
important therapeutic target (1). OneFDA-approved drug against
CXCR4 is currently on the market(Mozobil, for hematopoietic stem
cell mobilization), and multi-ple additional drugs against this
target are in development foroncology and other indications (2).The
crystal structures of class A GPCR superfamily members
in their active and inactive conformations (reviewed in refs.
3and 4) provide unprecedented insight into the structural basis
ofligand binding, G protein coupling, and activation of GPCRs
viarearrangements of transmembrane (TM) helices. GPCR helicesV and
VI in particular, and in some cases III and VII, are knownto
undergo significant conformational changes upon activation(5–7).
However, static images alone have not been able to ex-plain the
residue-level mechanisms underlying the dynamic he-lical shifts
that mediate GPCR signal transduction. Additionally,only inactive
state structures have been solved for CXCR4 andmost other GPCRs (8,
9). Over the last two decades, extensivemutagenesis studies of
GPCRs in general [collectively describing>8,000 mutations
(gpcrdb.org)] and of CXCR4 in particular(covering 81 primarily
extracellular residues of 352 total) (10) haveidentified individual
residues that are critical for receptor signaling.
Whereas many of the individual critical residues and motifs
havebeen described, the complete intramolecular signal
transmissionchain remains unclear.Here we report a cohesive model
for the mechanism by which
CXCR4 transmits the signal induced by its extracellular
chemo-kine ligand CXCL12 [also known as stromal cell-derived factor
1(SDF-1)] to the intracellular G protein. Using a
comprehensivelibrary of 728 mutants covering all 352 residues of
CXCR4, weexperimentally identified 41 amino acids that are required
forsignal transmission. Our results complement structural studies
ofGPCRs and expand on previous mutagenesis studies from
diverselaboratories to form a comprehensive functional model that
ex-plains how CXCR4 transmits an extracellular ligand binding
eventthrough its TM domains to dynamically affect helical shifts
andintracellular G protein coupling.
ResultsComprehensive Mutagenesis Identifies Critical Residues
for CXCR4Signaling. To identify critical residues required for
CXCL12-dependent CXCR4 signal transmission, a comprehensive
“shotgunmutagenesis” library of receptor variants was created with
at leastone mutation at each of the 352 residues of CXCR4 (11). The
li-brary contains a total of 728 mutant clones, representing an
averageof 2.7 substitutions at each amino acid position. The entire
CXCR4mutation library was transfected into mammalian cells in a
384-wellarray format (one clone per well) and evaluated in parallel
forCXCL12-dependent activation as measured by a calcium flux
assay(Fig. 1A). The addition of 20 nM CXCL12 to cells expressing
WTCXCR4, but not mock-transfected cells, resulted in robust
receptoractivation, measured as an increase in cellular
fluorescence. A highconcentration of CXCL12 (20 nM, approximately
three times theKD) was selected for stimulation to identify
mutations that are themost important for (i.e., resulted in the
most severe impairment of)CXCR4 signaling.
Significance
Our study helps answer the question of how G
protein-coupledreceptors bind an extracellular ligand and relay
this signalthrough its transmembrane helices into an intracellular
signalingevent. This has been the central thesis that G
protein-coupledreceptor structural studies have sought to address
through staticsnapshots of the crystallized proteins. Our
functional approachusing CXC chemokine receptor 4 as a model
complements thesestructures by identifying the dynamic atomic
pathway fromchemokine engagement to G protein coupling.
Author contributions: J.B.R. and B.J.D. designed research;
M.P.W., J.R.G., Y.T., and B.A.P.performed research; M.P.W., I.K.,
C.P., J.R.G., Y.T., B.A.P., E.B., J.B.R., T.M.H., and
B.J.D.analyzed data; and M.P.W., I.K., E.B., T.M.H., and B.J.D.
wrote the paper.
Conflict of interest statement: B.J.D. and J.B.R. are
shareholders of Integral Molecular.
This article is a PNAS Direct Submission.1To whom correspondence
should be addressed. Email: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1601278113/-/DCSupplemental.
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To differentiate nonsignaling mutant proteins from
poorlytrafficking or misfolded proteins, each CXCR4 variant was
alsoindependently tested for surface expression using an
N-terminalFLAG epitope tag and for global folding using the
conformation-dependent anti-CXCR4 monoclonal antibody 12G5 (Fig.
1B). Weidentified a total of 41 positions in CXCR4 where
mutationsresulted in significantly reduced CXCR4 activation (less
than twoSDs below wild-type, 80% 12G5 reactivity) or surface
trafficking (>80% FLAG re-activity) (Fig. 1C and SI Appendix,
Table S1). Each mutant wasfurther tested for reactivity with three
additional conformationallysensitive anti-CXCR4 MAbs, which
confirmed that the selectedproteins were correctly folded in nearly
every case (SI Appendix,Fig. S1). Mutants with the greatest
impairment of signaling (whosecalcium flux value plus one SD
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(iv) microswitch activation, and (v) G protein coupling. The
fol-lowing sections describe the composition of these functional
cat-egories and their role in signal transmission.CXCR4 residues
involved in chemokine engagement. The interaction ofCXCL12 with
CXCR4 is known to be mediated by two distinctepitopes (1, 8, 12,
16–22). In the first [chemokine recognition site 1(CRS1)], the
unstructured N terminus of CXCR4 interacts with theglobular core of
the chemokine, specifically with the N loop and the40s loop of
CXCL12. In the second site [chemokine recognition site2 (CRS2)],
the distal flexible N terminus of CXCL12 reaches intothe TM binding
pocket of CXCR4 to trigger signaling. Most of thecritical residues
that we identified on the extracellular side of thereceptor cluster
in the CRS2 binding pocket (blue and green layers
in Fig. 3), consistent with the fact that CRS2 interactions are
theprimary driver for both affinity and signaling in CXCR4 (9).Our
study identified seven solvent-exposed critical residues
on the extracellular face of the receptor that are positioned
tomediate chemokine engagement (Fig. 3B, blue). These en-gagement
residues include D972.63 at the top of helix II andD187ECL2 in
ECL2, both of which have previously been impli-cated in binding
CXCL12 (9, 17, 22, 23). On the other side of thepocket, D2626.58
toward the top of helix VI (consistent withprevious studies, ref.
23) as well as H2817.32 at the top of helix VIIwere identified,
both in direct proximity of the chemokine in thestructure (9) and
the models. Residues F189ECL2, N192ECL2, andL267ECL3 on the
extracellular face of CXCR4 are also consistentwith a potential
role in chemokine engagement.Transmembrane residues that initiate
signal transmission. Four of the 41critical residues are solvent
accessible but are located at the verybottom of the binding pocket
and directly contact buried criticalresidues involved in signaling
(Fig. 3C, green). This unique position
inactiveactive
inactiveactive
inactiveactive
inactiveactive
inactiveactive
chemokine engagementchemokine engagement
microswitch activationmicroswitch activationG protein couplingG
protein coupling
signal initiationsignal initiation
signal propagationsignal propagation
R134
L226
R134
L226
I
II
VIV
IV
III
VII
III
VIV
IV
III
VII
I
II
VI
V
IV
III
VII
I
II
VI
V
IVIII
VII
I
II
VI
V
IV III
VII
S131
V242
Y302
Y219
Y219
Y302
W252F248
I245
F292
W94Y116
H203
D187F189
N192
D97H281D262
L267
A B
C
D
F E
toCXCR4N-term
W94
H203
F189
Y45Y45E288E288
D97H281D262
A291A291
CXCL12CXCL12
toCXCR4C-term
toCXCR4C-term
Fig. 3. Functional classes of critical residues in CXCR4. (A)
Critical residues ofCXCR4 are divided into functional groups based
on the CXCR4:CXCL12model and CXCR4:vMIP-II structure. Critical
residues that contact each other,CXCL12, or G protein are shown in
color; other critical residues identified areshown in gray. The
active state of the CXCR4:CXCL12 complex is shown.CXCR4 residues
are involved in (B) chemokine engagement at the mouth ofthe
orthosteric ligand binding pocket (blue), (C) signal initiation at
the baseof the ligand binding pocket (green), (D) signal
propagation through theTM domain helices that include the
hydrophobic bridge (yellow), (E) mi-croswitch activation that
transmits hydrophobic bridge conformationalchanges (red), and (F) G
protein coupling (purple). Subpanels show top-down views of
interactions in each group. In the subpanels, dark and lightcolors
represent the active and inactive states of the CXCR4:CXCL12
complexmodel, respectively.
Fig. 4. CXCR4 signal transmission through the TM helices.
Critical CXCR4residues identified are involved in chemokine
engagement (blue), signal initia-tion (green), signal propagation
(yellow), microswitch activation (red), and Gprotein coupling
(purple). The dashed horizontal line highlights the N-terminalamine
of CXCL12 (black). Oxygen atoms are shown in red, nitrogen atoms
inblue. Solid connecting lines indicate interatomic distances that
are
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suggests their role as signal initiators responsive to
chemokinebinding. One of these CXCR4 residues, W942.60, is highly
conservedamong chemokine receptors, has been implicated in binding
thesmall molecule antagonist IT1t and vMIP-II (8, 9), and
theequivalent residue in the chemokine receptor CCR5 (W862.60)
hasalso been shown to play a role in ligand binding (24).
Additionalsignal initiator residues identified in our screen
include Y451.39,Y1163.32, and E2887.39, all previously reported as
binding and/or signaling determinants in CXCR4 (13, 19, 22, 25). In
theCXCR4:CXCL12 complex model, each of these four residuesdirectly
contacts or is in close proximity to the distal N terminus ofCXCL12
(residues K1 and P2), which is widely recognized as thecritical
domain of the chemokine that initiates signaling (16,
26).Transmembrane residues involved in signal propagation. Eight
criticalresidues from our dataset are located in the core of the
receptorand form a continuous intramolecular chain between the
resi-dues involved in signal initiation on the extracellular side
and the
activation microswitches on the intracellular side,
suggestingtheir role in signal propagation (Fig. 3D, yellow). Three
of thesecritical residues are buried directly below the CXCR4
signalinitiator residues discussed above. These residues are
F2927.43, whichhas been previously shown to be important for CXCR4
activity (17,19), A2917.42, which has a known role in introducing
constitu-tive activity in CCR5 (27), and W2526.48, which is part of
thewell-characterized CWxP rotamer motif (28, 29).
Homologouspositions in the A2AR structure (H2787.43 and S2777.42)
havebeen shown to move closer to helix III upon agonist
binding(30), supporting an important role for these residues in
GPCRsignal transmission.Directly below W2526.48, a string of five
residues on helix VI
(F2486.44, L2466.42, I2456.41, L2446.40, and V2426.38) were
iden-tified as critical. Interestingly, these five hydrophobic
residuesappear to form a “bridge” linking nearly every key
signalingmotif in GPCRs, including the CWxP rotamer, NPxxY, DRY,and
Y(x)5KL microswitch motifs (Fig. 5A). Moreover, thesemotifs move
closer to this hydrophobic bridge in the active statemodel of CXCR4
(Fig. 5 B and C), suggesting that the hydro-phobic bridge enables
helix and side chain repacking during theconformational transition.
Previous mutation analyses at the 6.44and 6.40 positions in
different receptors support a role for theseresidues in mediating
the transition between inactive and activeGPCR states (3, 31–33).
Whereas the identity of these residues isnot absolutely conserved,
the hydrophobic nature and helicalcompatibility of these residues
is strictly conserved among allchemokine receptors (Fig. 5D) and
other GPCRs (10). Two ofthese residues were deemed critical based
on substitution toproline (L2446.40P and L2466.42P). Introducing a
proline in thisregion of CXCR4 can eliminate signaling without
altering ex-tracellular structure or ligand binding (8), suggesting
that thehelical conformation of the intracellular half of TM6 is
criticallyimportant for signal transmission.Activation
microswitches that control G protein coupling. Highly con-served
microswitches within GPCRs are known to control the Gprotein
interface during the inactive-to-active state transition. Ourscreen
identified three amino acids that are critical microswitchresidues
in CXCR4, S1313.47, Y2195.58, and Y3027.53 (Fig. 3E, red).Residues
Y2195.58 and Y3027.53 are predicted to significantly changeposition
in the active state to form the structural support for the Gprotein
interface (Fig. 3E, dark vs. light red). Residue Y2195.58 isthe
first position of the Y(x)5KL microswitch motif, whereas
residueY3027.53 is the last position of the conserved NPxxY motif,
bothrecognized as critical determinants of GPCR activation (29).
Theimportance of S1313.47 and Y2195.58 is also supported by studies
ofrhodopsin where the direct interaction of homologous residues
hasbeen reported to stabilize the activated state (34). Notably,
hydro-phobic bridge residue V2426.38 structurally resides directly
in thecenter of the microswitch functional group, suggesting that
it mayserve as the trigger residue that activates the
microswitches.CXCR4 residues that directly couple to G protein.
Finally, our screenalso identified two highly conserved critical
residues on the in-tracellular side of the receptor, R1343.50 and
L2265.65 (Fig. 3F,purple residues). R1343.50 is part of the
well-known “DRY” boxmotif and L2265.65 is the last residue of the
Y(x)5KL motif, con-sistent with both residues contributing directly
to G protein cou-pling. The homologous positions are directly
involved in bindingthe C terminus of G protein in the crystal
structures of the activestate ternary complex of the β2 adrenergic
receptor (β2AR) (30)and bRho (35, 36). By homology with β2AR and
bRho, 15 residueson the intracellular face of CXCR4 could be
involved in G proteincoupling, with 9 of them either identical or
similar to the corre-sponding residues in β2AR (SI Appendix, Fig.
S3). Our screentested mutations at all 15 CXCR4 positions (SI
Appendix, TableS3), and identified two of them as critical, likely
representinghotspots within an otherwise large distributed
interface. Muta-tions at the remaining 13 positions expressed well
and were
A D
CWxP
NPxxYNPxxY
DRYDRY
Y(x)5KLY(x)5KL
CWxP
hydrophobicbridge
242
243
244
245
246
247
248
249
250
251
252
254
253
IIMVIAFLICWLPGIIMGTFTLCWLP
bRhoβ2AR
VILILAFFACWLPCXCR4
FVIMIIFFLFWTPFTIMIVYFLFWTPFVIMAVFFIFWTPFAVVVLFLGFWTPFTIMIVYFLFWAPIAVVLVFLACQIPIAVVVVFIVFQLPLIVVIASLLFWVPITVLTVFVLSQFPVALVAAFVVLQLPFAVVLIFLLCWLPFAVVLIFLLCWLPVVVVVAFALCWTPILVTSIFFLCWSPFLVMAVFLLTQMPLLVVIVFFLFWTPFAIVVAYFLSWGPMNILWAWFIFWWPAALVVAFFVLWFPFSYVVVFLVCWLPLTVVIVFIVTQLPFAIMVVFLLMWAPVTIIITFFLCWCPIAVVLVFIIFWLP
CCR1CCR2CCR3CCR4CCR5CCR6CCR7CCR8CCR9CCR10CXCR1CXCR2CXCR3CXCR5CXCR6CX3CR1XCR1ACKR1ACKR2ACKR3ACKR4CCRL2CML1US28ORF74
VAVVLLFFVFCFP
CXCL12
aliphaticaromaticpolar
cysteineproline
DRY
VII
V
III
DRY
inactive
NPxxYNPxxY
VI
Y(x)5KLY(x)5KLB
DRY
VII
V
III
NPxxYNPxxY
DRY
Y(x)5KLY(x)5KLactive
VI
CWxPCWxP
CCWxP
Fig. 5. Interactions of the hydrophobic bridge with GPCR
signaling motifs.(A) An active-state model of the CXCR4:CXCL12
complex shows contacts ofhydrophobic bridge residues (yellow) with
motifs known to be important inGPCR activation. These include the
CWxP motif (yellow) that is part of thesignal propagation
functional group, the Y(x)5KL and NPxxY motifs (red)that are part
of the microswitch functional group, and the DRY box motif(purple)
that is part of the G protein-coupling functional group. The
lowerhalf of helix VI is shown as a cylinder. (B and C) The
hydrophobic bridge andknown GPCR motifs are shown (bottom-up view)
in the inactive and activestates of CXCR4, respectively. The motifs
move closer to the hydrophobicbridge in the active state. Oxygen
atoms are shown in red, nitrogen atoms inblue. (D) Sequence
alignments of homologous residues corresponding to theCXCR4
hydrophobic bridge V2426.38-ILILA-F2486.44 (highlighted at Top
aswide horizontal bar) in bRho, β2AR, and the other chemokine
receptors,showing that residues are not strictly conserved but need
to be hydrophobic.
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conformationally folded, but did not meet our threshold
criteriafor impaired signaling. However, the exact set of CXCR4
residuesinvolved in G protein coupling remains unknown, and there
arelikely to be significant differences in how different GPCRs bind
Gproteins (37).
DiscussionA Functional Model for CXCR4 Signal Transmission.
Helical move-ments are well known to be essential in the GPCR
signaltransduction process, but static structural information alone
hasnot been able to fully distinguish the functionally critical
aminoacids within these helices that mediate this dynamic process.
Theidentification of 41 critical CXCR4 residues forming a
continuousintramolecular signaling chain through the TM helices
enables usto propose a comprehensive, hypothesis-based functional
modelfor signal transmission. Although many previous
mutagenesisstudies have been conducted on CXCR4 and other GPCRs,
ourresults provide unbiased, full-coverage characterization of all
352residues in the receptor, with the results derived from a
singlelaboratory using an internally consistent set of assays and
protocolsthat account for expression and folding of receptor
variants.Overall, the critical CXCR4 activation residues identified
in ourstudy agree with common activation mechanisms proposed
forclass A GPCRs, such as the prominent role of helix VI and the
roleof various conserved motifs and microswitches.Critical residues
of CXCR4 identified here also overlap with
the water-mediated polar residue network that facilitates
theinactive-to-active transition in the μ-opioid receptor (35, 38).
All17 homologous residue positions were tested in our screen,13
expressed and folded well, and 8 were identified as
criticalsignaling residues in our assays (Y1163.32, L1273.43,
Y2195.58,L2446.40, W2526.48, E2887.39, A2917.42, and Y3027.53).
These find-ings suggest that CXCR4 also features a polar residue
networkthat may play a role in its activation.Six crystal
structures of CXCR4 have been reported to date, all
featuring a common parallel homodimer interface (8, 9). Our
datasuggest that dimerization involving this interface may not be
im-portant for CXCL12-induced Ca+2 mobilization in our assay
sys-tem; among 11 homodimer interface residues that were mutated
inour screen, only N192ECL2 and L267ECL3 had any significant
effect.All mutants of the homodimer interface were properly folded
andtrafficked to the cell surface (SI Appendix, Table S4), also
sug-gesting that this interface does not play a role in folding or
traf-ficking. However, we cannot exclude the role of this
dimerizationinterface in other processes, such as internalization
or other types ofCXCR4 signaling (39) or of alternative
oligomerization interfaces.
Unbiased High-Throughput Screening for Function. The
functionalmodel described here integrates our comprehensive
mutationaldataset with crystal structures and models of CXCR4
complexesinto a cohesive model of CXCL12-mediated CXCR4
activation.Although our final model of how each residue functions
is hy-pothesis-driven, critical residues were identified using
stringentcriteria: critical mutants had to express, fold, and
traffic similar toWT CXCR4 yet impair signaling in response to a
saturating con-centration of ligand (∼3× KD). It is likely that
mutations thatproduce more modest effects on signaling escaped
detection underthese criteria. Our analysis does not distinguish
effects of CXCR4mutations on CXCL12 potency vs. efficacy, but it is
unlikely thatfurther increases in CXCL12 concentrations would
identify addi-tional residues more important to CXCR4 signal
transmission.Amino acids within all known GPCR signaling motifs
were
identified in our studies as critical signal transmission
residues.Other positions within these motifs are also likely
involved, but thetested mutations at these positions did not
express or fold wellenough, or did not significantly affect
signaling (SI Appendix,Table S5). It is also possible that CXCR4
signal transmissionpathways could be different when coupled to
different G proteins,
when signaling through different pathways, or for
constitutiveactivation.The use of random and unbiased mutagenesis
across all 352
residues of CXCR4 led to the identification of a number of
well-expressing but signaling-deficient mutants, such as
W94R2.60,D97G2.63, and E288G7.39, that would not have been possible
with al-anine mutations. For example, W94A2.60, D97A2.63, and
E288A7.39
have been previously shown to reduce receptor expression (12,
13,22). Substitutions to proline can impact expression and folding,
butmany were well tolerated. For example, L244P6.40 and
L246P6.42
support the critical helical nature of TM6. However, random
mu-tagenesis also has limits; some of the positions tested had
ratherextreme substitutions that affected receptor folding,
whereasothers had only conservative substitutions that failed to
impactsignaling. To compensate for the random changes, we tested
2.7different amino acid substitutions per position on average,
buttesting more substitutions would likely have revealed
additionalcritical residues.
ConclusionsAn interconnected chain of residues responsible for
transmittingextracellular ligand signals to intracellular G
proteins is likely aconserved mechanism across all GPCRs. Thus, we
expect theresults of our study to be largely applicable to other
GPCRs,especially for the propagation, microswitch, and G
protein-cou-pling groups. The chemokine engagement and signal
initiationgroups are likely to be more ligand specific, so we
expect them tobe most applicable to other chemokine
receptors.Understanding the activation dynamics of GPCRs has
implica-
tions for studying the effects of mutations and naturally
occurringvariants, for structure determination efforts, and for the
devel-opment of novel therapeutic targeting strategies using
allostericligands. Modulation of CXCR4–CXCL12 signaling, in
particular,has implications for controlling cancer metastasis,
preventing HIVinfection, and promoting immune and stem cell
trafficking. Thepresent study provides a missing link in
understanding the dynamicmultistep activation mechanisms of the
GPCR CXCR4.
Experimental MethodsAll experiments in this project were
approved by the Institutional BiosafetyCommittee and management of
Integral Molecular. No human subjectmaterials were used.
Preparation of CXCR4 Shotgun Mutagenesis Mutation Library. A
shotgunmutagenesis mutation library was created as previously
described (40).Briefly, a parental plasmid expressing full-length
human CXCR4 cDNA wasconstructed with an N-terminal FLAG epitope tag
and a C-terminal V5 epi-tope tag. Using the parental cDNA construct
as a template, a library ofrandom mutations was created using
PCR-based mutagenesis (Diversify PCRRandom Mutagenesis Kit,
Clontech). Each mutant clone was sequence veri-fied. A complete
mutation library was assembled by selection of 2.7 mutantclones per
residue, spanning the entire protein, and preferably repre-senting
a conserved and nonconserved substitution at each position. A
totalof 551 CXCR4 variants contained single mutations and the
remaining 172clones contained mutations at two or more
positions.
Calcium Flux Assay. The FLIPR Calcium 4 Assay (Molecular
Devices) was per-formed according to the manufacturer’s protocol,
with minor modifications.The CXCR4 mutation library and controls
[WT (+) and vector alone (−)] weretransfected and expressed in
canine Cf2Th cells (selected because they lackendogenous CXCR4) in
384-well microplate format. Twenty-four hours post-transfection,
cells were washed twice in HBSS/Hepes supplemented with 10
μMindomethacin, then incubated with 1× loading dye for 1.5 h at 37
°C. The plateswere transferred to a FlexStation II-384 plate
reader, and wells were injected (att = 20 s) with 20 nM (final)
human recombinant CXCL12 (PeproTech), andfluorescence was measured
for 70 s, reading every 3 s (Ex485/Em525).
Immunodetection Assays. The CXCR4mutation librarywas expressed
in HEK-293Tcells. Twenty-four hours posttransfection, cells were
washed and fixed in4% (vol/vol) paraformaldehyde, incubated with
anti-FLAG M2 monoclonal
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antibody (Stratagene, no. 200472), or anti-CXCR4 monoclonal
antibodies 12G5(a gift of James Hoxie, University of Pennsylvania,
Philadelphia), 44708, 44712,or 44716 (R&D Systems), followed by
goat anti-mouse Cy3-conjugated sec-ondary antibodies (Jackson
ImmunoResearch Laboratories). Microplates weremeasured on a NovaRay
imager (Alpha Innotech).
Ligand Binding FRET Assay. Selected FLAG-tagged CXCR4 clones and
controlswere arrayed in duplicate in 96-well microplates and
expressed in HEK-293Tcells. Twenty-four hours posttransfection,
cells were washed twice in HBSSsupplemented with 10 mM Hepes, then
incubated for 1 h at room temper-ature with the donor fluorophore,
an anti-FLAG (M2)-Terbium-labeled an-tibody (Cisbio). Following
five washes in PBS and one wash in 1× ligandbinding buffer, cells
were incubated for 3 h at room temperature with theacceptor
fluorophore solution, 60 nM Tag-lite CXCR4 receptor red
agonist(L0012RED). Fluorescent-positive agonist-bound cells were
detected at 665-nmemission using a Perkin-Elmer Envision 2100.
Data Analyses. The maximum calcium flux value for each clone was
calculatedfrom the peak flux value at t = 30 s minus the trace
baseline value, thenbackground subtracted using negative control
values on the same plate andnormalized to express each mutant
activity as a percentage of wild-type. Theaverage (n = 5) calcium
flux value for each clone was compared with a 62%cut-off threshold
[100 − (2 × [SD of wild-type controls])] to identify clonesthat
signal significantly below wild-type levels. Similarly, the
immunofluo-rescence values for each clone were calculated from raw
plate data, back-ground subtracted, and normalized to express each
mutant as a percentage
of wild-type. The average FLAG (n = 5) or 12G5 (n = 3)
immunofluorescencevalue for each clone was compared with an 80%
cut-off threshold to identifyclones that react with each MAb at
near wild-type levels.
Structural Modeling. Amodel of the complex between wild-type
CXCR4 in theinactive state and CXCL12 was previously published (9).
A model of the activestate for this complex was obtained using
gradient minimization with re-straints in Molsoft ICM molecular
modeling package. For that, consensusintramolecular distance
changes were first obtained by comparing the activeand inactive
state structures for each of β2AR, AA2AR, and bRho (7, 35, 36,41).
The calculated distance changes were converted into target
distances byadding them to the homologous intramolecular distances
measured withinthe inactive CXCR4 structure and then imposed onto
the model as harmonicdistance restraints. An additional set of
restraints was derived from the in-active state intramolecular
hydrogen bonds and used to maintain the re-ceptor secondary
structure during energy minimization. The 105 steps ofgradient
minimization were performed using a fully flexible representationof
the receptor. The final model was visually inspected for the
absence ofsteric conflicts and for consistency of the microswitch
residue rotamerswith the signature of an active state as described
for crystallized activestate GPCRs.
ACKNOWLEDGMENTS. This work was supported by NIH Grants
R44-GM076779 (to B.J.D.), R01-GM071872 (to I.K.), and R01-AI118985,
R01-GM117424, R21-AI121918, and R21-AI122211 (to I.K. and
T.M.H.).
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