thirdextracellularloop(ec3)-nterminusinteractionis ...nism is described as “molecular switches”...

Third Extracellular Loop (EC3)-N Terminus Interaction IsImportant for Seven-transmembrane Domain ReceptorFunctionIMPLICATIONS FOR AN ACTIVATION MICROSWITCH REGION*

Received for publication, March 31, 2010, and in revised form, July 25, 2010 Published, JBC Papers in Press, July 27, 2010, DOI 10.1074/jbc.M110.129213

Soumendra Rana and Thomas J. Baranski1

From the Division of Metabolism, Endocrinology, and Lipid Research, Department of Medicine, Washington University School ofMedicine, St. Louis, Missouri 63110

The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined bythree intra- and three extracellular loops, whose local confor-mations are important in receptor signaling. Many 7TM recep-tors contain a cysteine residue in the third extracellular loop(EC3) and a complementary cysteine residue on theN terminus.The functional role of such EC3-N terminus conserved cysteinepairs remains unclear. This study explores the role of the EC3-Nterminus cysteine pairs on receptor conformation andGproteinactivation by disrupting them in the chemokine receptorCXCR4,while engineering anovel EC3-N terminus cysteinepairinto the complement factor 5a receptor (C5aR), a chemoattract-ant receptor that lacks it. Mutated CXCR4 and C5aRs wereexpressed in engineered yeast. Mutation of the cysteine pairwith the serine pair (C28S/C274S) in constitutively activemutant CXCR4 abrogated the receptor activation, whereasmutation with the aromatic pair (C28F–C274F) or the saltbridge pair (C28R/C274E), respectively, rescued or retained thereceptor activation in response to CXCL12. In this context, thecysteine pair (Cys30 and Cys272) engineered into the EC3-N ter-minus (Ser30 and Ser272) of a novel constitutively active mutantof C5aR restrained the constitutive signaling without affectingthe C5a-induced activation. Further mutational studies demon-strated a previously unappreciated role for Ser272 on EC3 of C5aRand its interaction with the N terminus, thus defining a newmicroswitch regionwithin theC5aR. Similar resultswere obtainedwith mutated CXCR4 and C5aRs expressed in COS-7 cells. Thesestudies demonstrate a novel role of the EC3-N terminus cysteinepairs in G protein-coupled receptor activation and signaling.

G protein-coupled receptors (GPCRs),2 often referred to asseven-transmembrane domain receptors, are one of the mostbiologically important superfamilies of receptors (1). Phyloge-netic analysis classifies the superfamily of receptors into gluta-mate, rhodopsin, adhesive, frizzled, and secretin families (2).

The rhodopsin family alone includes�750GPCRs (3).�60%ofGPCRs respond to sensory and olfactory neurons; �30%respond to protein and peptide hormones, amino acids, bio-genic amines, and lipids, whereas the remaining GPCRsrespond to nutrients and metabolites. In response to ligands,the receptors recruit either G proteins or other intracellulareffector proteins (4), which transduce the extracellular chemi-cal signals and further stimulate intracellular signaling cascades(5). This extraordinary ability to sense such a diverse array ofchemical signals with selective precision classifies GPCRs as a“druggable proteome” (6) that generates �10% of the globalpharma revenue (7).Given their cellular and pharmacological importance, under-

standing the receptor function at the molecular level is a pre-requisite, and indeed the recent advancements in the highresolution crystals of rhodopsin-like GPCRs have been signifi-cantly informative (8–12). Nevertheless, a central questionremains how the canonical receptor topology interconvertsbetween several possible conformations in response to diversechemical stimuli. Traditionally, the GPCR activation mecha-nism is described as “molecular switches” identified in con-served “microdomains” (13) and recently reviewed elsewhere(14, 15). Briefly, ligand/agonist binding or an activating pointmutation (CAM) triggers a series of molecular macroswitches,such as the global rotamer toggling (16, 17) on TM6 or thedisruption of an ionic lock (18, 19), between TM3 and TM6, tounlock the G protein-binding site in the intracellular face of thereceptors leading to G protein activation. However, �80% ofthe rhodopsin family receptors do not have the putative resi-dues to support the described “rotamer toggle” switch, the“ionic lock,” or both (20). Although a universal activationmechanism is also suggested by the ability of humanreceptors toactivate evolutionarily distant yeastGproteins, how this is accom-plished isdifficult to reconcilewithina frameworkof lowsequencehomology (�20%) between receptors (21), the variable length ofthe peptide loops, and the diversity of N termini in receptors.Indeed, the significant variation observed in pharmacologicalproperties of receptors (22, 23) within the same family (24) doesnot favor a single, unified activationmechanismmodel.Within the rhodopsin family of GPCRs, the EC3 loop varies

in length between 4 and 27 residues and plays a key role in theactivation of the neuropeptide class of receptors (25). Minoralterations affecting its folding or structure have been shown toimpair both ligand binding and signaling (26–32). This study

* This work was supported, in whole or in part, by National Institutes of HealthGrants GM071634 and GM071634-03S109 (to T. J. B.).

1 To whom correspondence should be addressed: Dept. of Medicine, Divisionof Metabolism, Endocrinology, and Lipid Research, Washington UniversitySchool of Medicine, Campus Box 8127, 660 S. Euclid Ave., St. Louis, MO63110. Tel.: 314-747-3997; Fax: 314-362-7641; E-mail: [email protected].

2 The abbreviations used are: GPCR, G protein-coupled receptor; TM, trans-membrane; CAM, constitutive active mutant; IP3, inositol triphosphate; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 41, pp. 31472–31483, October 8, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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focuses specifically on conserved cysteine residues in EC3 withrespect to the NPXXYmotif in TM7. Using the Ballesteros andWeinstein numbering system that designates the most con-served residue in the TM as X.50 (for example, in TM7, theproline position in the NPXXY is 7.50), C7.25 is the position atwhich cysteine residues are conserved in EC3.TheEC3 cysteine(C7.25) in several receptors has been proposed to participate ina disulfide linkage with another conserved cysteine in the Nterminus. These paired cysteine residues are highly conservedamong chemokine-binding receptors and have been demon-strated to be important for both EC3 structure and perhapshigh affinity ligand binding (30, 33–36). However, their exactrole in structure-function relationship is still unclear.This study focuses on the role of such cysteine pairs in ligand

binding, activation, and maintaining the overall inactive/activeconformation of the receptors. Two representative rhodopsinfamily receptors that signal through G�i subunits have beenchosen for this study as follows: CXCR4 (37), activated by che-mokine CXCL12 (SDF-1), and C5aR, activated by anaphyla-toxinC5a (38).We chose to express the receptors in engineeredyeast (39) because this system offers several advantages for thisanalysis as follows: there are no other endogenous GPCRs thatmight interact with the mutant receptors, and there is only oneG protein. We find that the EC3-N terminus cysteine pairsserve different functions in these receptors. The cysteine pairadds functionality similar to a weak agonist by increasing thereceptor activation in CXCR4, although it conformationallyconstrains an activated receptor similar to an inverse agonistwhen engineered into theC5aR.Moreover, ourmutational datasuggest a more involved and coordinated interaction betweenEC3 and the N terminus that is essential in these receptors tomaintain the active sites for the ligands and the overall confor-mation of the receptors for signaling.

EXPERIMENTAL PROCEDURES

Yeast Strains—The Saccharomyces cerevisiae strain BY1173(39) with the following genotype [MATa His3 Leu2 Trp1 Ura3Can1 gpa1�::ade2�::3XHA far1�::ura3� Fus1::PFus1-His3LEU2::Leu2 PFus1LacZ SST2�::ura3� ste2�::G418R Trp1::GPA1/G�i(1/2)] has been used in this study unless otherwisespecificallymentioned. This specific strain expresses a chimericyeast-human G� subunit (GPA1-G�i(1/2)/(5)) in which theC-terminal five amino acids of human G�i(1/2) replaced theC-terminal five amino acids of the yeast G� subunit, GPA1.This strain also carries a deletion allele of SST2, which is knownto down-regulate the pheromone-response pathway by accel-erating the GTPase activity of GPA1. The engineered MAPKmating pathway (1) in BY1173 is coupled to the FUS1-LacZreporter enzyme,which stimulates basal production of�-galac-tosidase upon ligand-dependent or -independent activation ofthe receptors coupling to the chimeric G protein.Yeast Transformation—All the receptor and ligand plasmids

were transformed to the yeast following the standard lithium-acetate protocol. All plasmids used for transformation are2-�m-based and contained an REP3 element for autonomousreplication in S. cerevisiae and ampicillin-R for selection inEscherichia coli. All the receptor plasmids possess an ADE2-selectable marker, which expresses the gene of interest under

the control of the constitutive phosphoglycerate kinase (PGK1)promoter. All the ligand plasmids possess a URA3-selectablemarker, which expresses the gene of interest under the controlof the constitutive alcohol dehydrogenase (ADH) promoter.Control transformations were made using empty ADE2 andempty URA3 plasmids to control for plasmid copy number inconditions that lack receptor, ligand, or both. Positive transfor-mants were selected by allowing the yeast to grow at 30 °C onadenine-uracil dropout solid media.

�-Galactosidase Assays—Three positive transformants wereselected from each plate and subjected to liquid�-galactosidaseassays. The yeast cells were grown overnight in appropriatesynthetic dropout medium until confluent. The confluent cul-ture was diluted to �0.15 A600 0.5 ml�1 and further grown at30 °C in a humidified environment for 4–5 h. For dose-re-sponse studies, an appropriate concentration of the peptideagonist was added to the cells expressing the respective recep-tor mutants. The expression of the �-galactosidase was moni-tored by lysing the cells with 50�l of buffer consisting of a 50:50mixture of 5% (w/v) Triton X-100 (Bio-Rad) in 250 mM PIPES,pH 6.8, and 4.86 mg ml�1 chlorophenol red galactosidase(Roche Applied Science) in 25 mM PIPES, pH 6.8, and incubat-ing in the dark at 37 °C. �-Galactosidase activity was deter-mined by terminating the reaction after �1–1.5 h by adding50 �l of 1 M Na2CO3 and measuring A570 on a Bio-Rad model680 microplate reader. Color development for yeast cellsexpressing the wild type CXCR4 required overnight incuba-tion with lysis buffer. Data were plotted and analyzed usingPrism 5 (GraphPad software).Construction of Mutant Receptor and Ligands—Point muta-

tions were introduced into the receptor and ligand plasmids bysite-directed mutagenesis using Pfu Turbo polymerase (Strat-agene). The presence of the only desired point mutation wasconfirmed by sequencing both strands of the entire gene at theProtein and Nucleic Acid Chemistry Laboratory, WashingtonUniversity School of Medicine, St. Louis, MO.Western Blots—The expression levels of some single/double

point mutants of C5aR were assessed by Western blot. Yeastcells carrying empty ADE2 plasmid or plasmids encoding wildtype/mutants were grown overnight in adenine dropout liquidmedia. The overnight cultures were adjusted to �1 A600 ml�1.Yeast cells were lysed in a 50:50 mixture of 4� LDS NuPAGEbuffer (Invitrogen) and 62.5 mM Tris, pH �7.5, supplementedwith 1 �g/ml leupeptin, 1 �g/ml aprotinin, and 500 �M phen-ylmethylsulfonyl fluoride.�200mgof 0.5-mmglass beads (Bio-spec Products, Bartlesville, OK) was added to the mixture, fol-lowed by vortexing at high speed for 5 min. The emulsion washeated for 10 min at 50 °C, cooled on ice, and centrifuged for 5min. 25 �l of each supernatant was resolved on a NuPAGE�Novex 4–12% BisTris gel, transferred to nitrocellulose mem-brane, and immunoblotted with a rabbit polyclonal anti-C5aRantibody raised against full-length endogenous C5aR (NovusBiologicals, Littleton, CO). The blot was then washed andprobedwith horseradish peroxidase conjugated anti-rabbit IgG(Santa Cruz Biotechnology) at room temperature. Proteinbands were detected using ECL methods.Sequence Analysis of the GPCRs—All sequences were down-

loaded from the GPCR data base and aligned with ClustalX-

EC3-N Terminus Interaction in 7TM Receptor Function

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2.0.11 program. The sequence alignment presented in Fig. 1 hasbeen further manually annotated.Mammalian Cell Culture and Transfections—Mutant C5aR

receptor plasmids were constructed by site-directed mutagen-esis using Pfu Turbo polymerase (Stratagene) in IRES vector(Clontech) encoding G�16.Wild type andmutant CXCR4 plas-mids were constructed by two-step restriction endonucleasedigestion followed by two-step ligation to introduce receptorandG�16 genes into the IRES vector. COS-7 cellsmaintained inDulbecco’s modified Eagle’s medium (DMEM) with 10% fetalbovine serum, 100 �g/ml streptomycin sulfate, 100 units/mlpenicillin G were transiently transfected with wild type ormutant receptors using Lipofectamine LTX with plus reagents(Invitrogen) according to the manufacturer’s protocol.Inositol Phosphate Accumulation Assays—COS-7 cells ex-

pressing a promiscuous G�16 subunit with either wild type ormutant receptorswere supplementedwith 1�Ci of [3H]inositol(PerkinElmer Life Sciences) for 24 h followed by stimulation inthe absence or presence of 10–100 nM ligands at 37 °C for 1 h.Accumulated IP3 fractions were separated as described else-where (40) and counted in Scintiverse scintillation fluid (Fisher)until 5% significance.Membrane Preparation and Saturation Binding Assays—

Membranes of COS-7 cells expressing wild type or mutantreceptors were prepared as described elsewhere (41). Themembranes (1–10 �g of total protein) were thawed on ice andincubated for 1 h with 0.1 nM 125I-labeled C5a in binding buffer(Hanks’ balanced salt solution, supplemented with 25 mM

HEPES, pH 7.4, and 0.1% BSA). Binding reactions were per-formed on ice in the presence of varying concentrations ofrecombinant nonradioactive C5a (Sigma) and terminated byvacuum filtration through GF/C filters (Whatman) presoakedin 0.1% poly(ethyleneimine) (Sigma) with a Millipore(Temecula, CA) harvester. Filters were rapidlywashedwith ice-cold binding buffer and then added to scintillation tubes with 3ml of the scintillation fluid. Same protocol was followed using0.1 nM 125I-labeled CXCL12 binding to CXCR4 membranes inthe presence of varying concentrations of recombinant nonra-dioactive CXCL12 (Abcam Inc.). Data were analyzed withGraphPad Prism 5.0.Molecular Modeling—Approximately 20% overall sequence

identity was shared between C5aR and rhodopsin. Besides thelow level of sequence identity, the 2.8-Å resolution x-ray struc-ture of bovine rhodopsin (Protein Data Bank code 1F88) waschosen as the starting three-dimensional template for generat-ing homology-based model coordinates for C5aR, recruitingthe program MODELLER (42). The conserved disulfide be-tween TM3 and EC2 (Cys109–Cys188) of C5aR could be mod-eled successfully with MODELLER. To realize the putativeengineered disulfide bond between theN terminus and the EC3loop, small peptides corresponding to the EC3 and N terminuswere modeled manually, and the hypothetical �, � angles weregrafted onto the MODELLER defined coordinates. The modi-fied C5aR coordinate was then submitted to careful energyminimization in a cubic box with periodic boundary involvinggromos-96 43a1 force field (43) as implemented in GROMACS3.3.1. Placed in center of a periodic box, large enough to accom-modate�10.5 nm solvent layer on each side, the receptor coor-

dinate was energy minimized to 100 kJ mol�1 nm�1 tolerancewith steepest descent, first in vacuum and then in the presenceof simple point charge (SPC) watermodel, as provided inGRO-MACS.Numerical integrationswere performed in step size of 2fs. Bonds were constrained with SHAKE to the tolerance0.0001. Nonbonded pair list cut-off was 1.4 nm with shift func-tion. Solvent densitywas set to the value corresponding to 1 atmat 300 K. Receptor and solvent were coupled independently toBerendsen bath at 300 K, to the coupling time constant 0.1 ps.Amide bond geometry, including the �, � angles of individualresidues were analyzed to assess the general quality of themodel. For comparison purposes, the crystal structure of wildtype and engineered rhodopsin were also energy-minimized.The figures were made using the program PyMOL.

RESULTS

SequenceAlignment of Rhodopsin Family Receptors—HumanGPCRswere aligned in search of conserved cysteine residues onEC3 within 25 amino acids relative to the highly conservedP7.50 of NPXXY (TM7) motif and on the N terminus within20–25 amino acids relative to the highly conserved N1.50(TM1). The alignment identified 48 receptors within 15 sub-families of rhodopsin family of GPCRs (summarized in Table1), which have such EC3-N terminus cysteine pairs. Of the 48receptors, 24 receptorswere identified as chemokine or chemo-kine-type receptors. However, many other receptors (Table 1)such as angiotensin, purigenic, and cysteinyl leukotriene recep-tors were also found to possess such EC3-N terminus cysteinepairs. Receptors with multiple cysteine residues either in EC3or in theN terminuswere excluded from this analysis; examplesincluded the muscarinic acetylcholine, dopamine, MC4R, nic-otinic, and serotonin type-7 receptors. Interestingly, most ofthe receptors possessed the cysteine pairs at a highly conservedposition on EC3 (C7.25), and on N terminus (C1.22), althoughsome variability was noted for EC3 and N-terminal cysteinepositions in some receptors (Table 1). The minor positionaldifference could be due to the differences of both the N termi-nus and loop lengths in receptors. The CXCR4 receptor waschosen as a representative of chemokine family receptor forfurther study. To better understand the functional role of such

TABLE 1Summary of receptors with conserved cysteine residues on EC3 andN terminus

Receptor subfamilies Totalno.a

Cysteine positions onEC3 and N erminus

GPR182 2 (C7.25, C1.22)Angiotensin type 1 1 (C7.25, C1.22)Angiotensin type 2 1 (C7.25, C1.22)B1 bradykinin 1 (C7.25, C1.22)B2 bradykinin 1 (C7.25, C1.22)Chemokine 16 (C7.25, C1.22)Chemokine-type receptors 8 (C7.25, C1.22)EBV-induced 2 (C7.25, C1.22)Leukotriene type 1 1 (C7.25, C1.23)Leukotriene type 2 1 (C7.27, C1.25)Lysosphingolipid andlysophosphatidic acid

2 (C7.25, C1.24)

Lysophosphatidic acid 4 1 (C7.25, C1.24)Prostacyclin or prostaglandin 3 (C7.24, C1.24)Purigenic type 5 (C7.26, C1.23)Uncharacterized 3 (C7.25, C1.22)

a Data are based on GPCR database.




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cysteine pairs, a novel EC3-N terminus cysteine pair (Cys30 andCys272) was engineered into C5aR, a chemoattractant receptorthat lacks such a cysteine pair.Sequence Alignment of CXCR4 and C5aR with Rhodopsin—

Sequence alignment to rhodopsin, presented in Fig. 1, demon-strates that CXCR4 has an extra pair of conserved cysteine res-idues in EC3 (C7.25) and the N terminus (C1.22), which inprinciple can support a putative disulfide linkage, whereas bothrhodopsin and C5aR lacks this cysteine pair. Considering rho-dopsin crystal structure as a template, the predicted EC3 loop isof 15 residues in CXCR4, and of 10 residues in C5aR comparedwith 6 residues in rhodopsin. Moreover, the alignment high-lights the known activation microswitches such as W6.48 ofXWXP (TM6), R3.50 of (D/E)RY (TM3), Y5.58 of XY (TM5),and Y7.53 of NPXXY (TM7) motifs that are conserved inCXCR4, C5aR, and rhodopsin. The W6.48 (TM6) representsthe global “rotamer toggle” microswitch in GPCRs. R3.50(TM3), D3.49 (TM3), and E6.30 (TM6) represent the ionic lockmicroswitch in GPCRs. In contrast to rhodopsin, CXCR4 andC5aR do not conserve the ionic lock because they lack a nega-tively charged residue at the appropriate position in TM6(K6.30, and S6.30, respectively). Y5.58 (TM5) is recently knownto latch R3.50 (TM3) in an active state of opsin (11). Y7.53 ofTM7 is an allosteric activationmicroswitch participating in for-mation of complex hydrogen bond networks with conservedwater molecules (44).Signaling Profile of Mutant Human CXCR4 in Yeast—Muta-

tions C28A, C274A, or C28A/C274A of the N-terminal (Cys28)and EC3 (Cys274) cysteine residues has been shown to signifi-cantly alter CXCR4 function as a co-receptor in HIV-1 Env-mediated cell fusion in a strain-specific manner (35). In tissueculture studies, mutation of C28A or C274A has also beenshown to decrease the specific binding of CXCL12 by �50%amounting to an �6-fold increase in the Kd value for mutant

receptors compared with wild typeCXCR4 (30). However, the role ofthese cysteine residues in CXCL12-dependent or -independent CXCR4signaling is essentially not known.We mutated C28S, C274S or C28S/C274S both in wild type and CAMof CXCR4 and expressed the recep-tors in yeast strain BY1173, whichfeatures a chimera of amino acids1–467 of GPA1 followed by thelast five amino acids of humanG�i2 (39). In the isolated environ-ment of the yeast cell, a singlehumanGPCR can be studied in theabsence of other receptors com-peting for G protein binding, andthe readout of receptor activationis clear because only a single Gprotein chimera is expressed. Theyeast strain used in this study wasengineered so that receptor signal-ing leads to activation of the yeast-mating pathway resulting in ex-

pression of a PFUS1-�-galactosidase reporter gene (39).Wild type CXCR4 displayed weak signaling in response to

theCXCL12 (45) thatwas co-expressed in the yeast (Fig. 2, rightpanel, lane A). Upon overnight incubation with substrates,yeast co-expressingWT-CXCR4andCXCL12displayed ligand-dependent signaling (1.34 � 0.27-fold; n � 2, data not shown);however, the signal was near the basal activity of the systemmaking it difficult to assess the effects of cysteine mutations inthe context of wild type CXCR4. Thus, we chose to study theeffect of cysteine mutation on CXCR4 signaling in the TM3mutant N119S (N/S3.35) (Fig. 2, left panel), which displaysrobust constitutive signaling and responds to agonist both inyeast and mammalian cells (46, 47). As noted in Fig. 2, rightpanel, the N119S mutant CXCR4 displayed near-maximal sig-naling that increased in response to CXCL12 co-expression(1.35� 0.18-fold; n� 5). Individual C28S andC274Smutationsdisplayed �50% reduced constitutive active signaling, whichupon CXCL12 stimulation displayed an apparently similar foldincrease in signaling (Fig. 2, right panel). In contrast, the doublemutation C28S/C274S completely impaired the constitutiveactivity ofCXCR4 and abrogated theCXCL12-mediated signal-ing. These data clearly suggest that the double cysteine muta-tions have an additive effect on the receptor activity relative toindividual cysteine mutations. If the sole function of the cys-teine pair were to form a disulfide bridge, onewould expect thatthe individual mutations would give similar results when com-pared with the double mutation.Rescuing the Constitutive Signaling of CXCR4 in Yeast—The

complete loss of constitutive signaling in the C28S/C274Smutant CXCR4 and its failure to respond to CXCL12 (Fig. 2,lane E) could be attributed to folding defects leading toimproper receptor trafficking or to the loss of overall confor-mational integrity due to the absence of the EC3-N terminuscysteine pair. Mutation of conserved cysteine pairs with serine

FIGURE 1. Sequence alignment of CXCR4 and C5aR with rhodopsin highlighting the most conservedresidues and the known activation switches in GPCRs. Ballesteros and Weinstein nomenclature assignsX.50, to the “fingerprint residues” shown as white letters with black background in each transmembranedomain. Important transmembrane activation domains are shaded in gray. Conserved cysteine residues par-ticipating in disulfide bond formation are shown with gray boxes. Residues shown with black boxes indicate lackof complementary charged residues at 6.30 to support the ionic lock activation switch. Point mutation knownto confer constitutive activity in CXCR4 is shown with white box. Residues shown as underlined have beensubjected to mutagenesis for engineering C5aR. Residues marked with stars have been mutated to cysteine inother studies for engineering an extra disulfide bond between EC3 and N terminus of rhodopsin.




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has been shown to completely impair the antimicrobial effectsof Mesentericin Y105 (48), a known bacteriocin. In this setting,noncovalent interactions such as hydrophobic or �-stackingcould functionally replace the conserved disulfide linkage intype IIa bacteriocin antimicrobial peptides (49, 50). To examinesuch possibilities in CXCR4, we looked for mutant pairs thatintroduce aromatic-aromatic (C28F/C274F) or salt bridgeinteractions (C28R/C274E) between EC3 and N terminus,which can potentially rescue the function by acting as a surro-gate to disulfide linkage.As illustrated in Fig. 2, right panel, the double mutant C28F/

C274F did not rescue the constitutive signaling of CXCR4, butit did display potent CXCL12-stimulated signaling (Fig. 2, rightpanel, compare lane E with lane F). On the other hand, thedouble mutant C28R/C274E rescued both the constitutive sig-naling as well as CXCL12-dependent signaling similar to theCXCR4-CAM (Fig. 2, right panel, compare lane Bwith lane G).Energetically, single exposed salt bridge interactions (0.8/1kcal/mol) could be weaker than the hydrophobic �-stackinginteractions (�1.3/�2 kcal/mol), which can providemore con-formational stability to the receptor. The energetic componentof the engineered interactionsmay account for the difference inconstitutive signaling displayed by C28F/C274F and C28R/C274Emutant receptors. These data demonstrate that an inter-action between EC3 and the N terminus of CXCR4 could beessential for receptor activation and that this interaction doesnot need to be mediated by cysteine pairs.

Engineering C5aR into a Chemo-kine-type Receptor—Chemokine-binding GPCRs have evolved withan extra cysteine pair in the N ter-minus and EC3 loop, which sepa-rates them from other GPCRs inthe rhodopsin family. Except forCXCR6, the chemokine bindingreceptors possess a cysteine residueat a highly conserved position onEC3 (C7.25), corresponding toP7.50 of NPXXY (TM7) motif,which is complemented by anothercysteine residue at a highly con-served position (C1.22), corre-sponding to N1.50 on the N termi-nus with the exception of CXCR5(C1.19). We wanted to know theeffects of introducing the conservedcysteine pair in a GPCR that lacks it.For this analysis, we chose to engi-neer the cysteine pair into the C5aR.As highlighted in Fig. 1, Ser272(S7.25) of C5aR aligned with theconserved cysteine of CXCR4 onEC3 and was therefore mutated tocysteine. This position also corre-sponds to Asp282 in bovine rhodop-sin, which has been successfullyengineered to form a disulfide bondbetween Asp282 of EC3 and Asn2 of

N terminus, as demonstrated in a crystal structure (51). Tosample the flexibility of the N terminus and its probability toform the putative disulfide linkage with S272C of EC3, two res-idues, Asp27 (D1.22) and Ser30 (S1.25), respectively, on the Nterminus of C5aRwere chosen for cysteinemutation (Fig. 3, leftpanel).Signaling Profile of Engineered C5aR in Yeast—Mutation

S30C (Fig. 3,middle panel) or D27C (Fig. 3, right panel) on theN terminus did not display any appreciable loss in signalingcompared with wild type C5aR in response to C5a, althoughS30C mutants displayed more C5a-independent activity com-pared with both D27C and wild type C5aR. This is in accord-ance with the earlier observations that the N terminus toleratesmany point mutations (52). On the other hand, S272C muta-tion on EC3 (Fig. 3) failed to demonstrate C5a-mediated signal-ing. The loss of function due to the S272C mutation could berescued by the additional mutation of S30C or D27C mutationin theN terminus of C5aR (Fig. 3,middle or right panel, respec-tively). As noted in Fig. 4, introduction of S272A or S272Tmutation on EC3 of C5aR also severely impaired C5a-mediatedsignaling, suggesting a strict requirement for Ser272 in EC3 ofC5aR.Surprisingly, the S30C mutation on the N terminus rescued

S272A and S272T nonfunctional mutant C5aRs (Fig. 4). Thisresult demonstrates that the rescue effect observed in theD27C/S272C and S30C/S272Cmay not be uniquely dependenton introduction of a disulfide bridge but could be due to local

FIGURE 2. Signaling profile of mutant CXCR4 receptors co-expressed with CXCL12 in yeast. Left panel,canonical receptor topology for CXCR4. The N119S mutation on TM3 is known to produce constitutive activesignaling in CXCR4. Cys28 and Cys274, presumed to form a disulfide linkage between N terminus and EC3 loop,is highlighted in white with black background. Right panel illustrates the contrasting functional effect of C28S,C274S, C28S/C274S, C28F/C274F, and C28R/C274E on constitutively active mutant CXCR4 signaling. Lane Aillustrates the weak signaling induced by CXCL12 when co-expressed with WT-CXCR4 in yeast. Vector repre-sents the �-galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each barrepresents the means � S.D. of signaling activity for three independent transformants, and data are represent-ative of at least two independent experiments. Unpaired t test suggests statistical significance with ***, p �0.0005, and *, p � 0.05 versus no CXCL12.




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hydrophobic or conformationalinteractions between the mem-brane-proximal portion of the Nterminus of C5aR and EC3. Onepossible mechanism by which theD27C or S30Cmight rescue activityof the mutated Ser272 C5aRs couldbe by binding the unpaired cysteineon the C5a ligand. In our previouswork (53), saturation mutagenesisidentified positions in the mem-brane-proximal portion of the Nterminus into which cysteine muta-tions occurred that could appar-ently interact with the unpaired cys-teines present in the C5a ligand. Toexclude this possibility in this study,we co-expressed the “cysteine-less”C5a ligand that contains a C27Rmutationwith the S30C-Ser272-mu-tated C5aRs. Illustrated in Fig. 4, the

cysteine-less C5a ligand efficiently activated the S30C-S272Tand S30C-S272AC5aR, as well as thewild typeC5aR.Wild typeC5aR displayed maximal signaling in response to human C5a,which contains an unpaired cysteine (Cys27), and a mutatedC5a in which the unpaired cysteine was substituted for a con-served residue in the rat C5a sequence (C27R). This result isconsistentwith previous findings fromour group (53) and dem-onstrates that the Cys27 on C5a is not required for C5aR signal-ing. Moreover, this result demonstrates that the signalingobserved for the single cysteine-mutated C5aR is unlikely to bedue to the formation of a disulfide bond between the C5a ligandand the receptor.Western blot analyses ofwhole yeast lysates ofthese single and double mutants of C5aR (presented in Fig. 5)demonstrate that these mutants were expressed well in yeast,although at different levels. Of note, the expression level did notcorrelate with the strength of signaling. For example, the weaksignaling mutant S272A C5aR was expressed more highly thanthe wild type C5aR or the strong signaling mutant D27C/S272C-C5aR (Fig. 5, compare lane E with lanes B and D). Thislack of correlation between receptor expression and activitymight reflect that the receptors are expressed in excess of the Gproteins and are therefore not limiting.Previous work by several groups implicated EC3 residues for

interactions with the arginine residue at the C terminus (Arg74)of the C5a ligand. Mutational studies support that Asp282located at the EC3/TM7 junction in the C5a receptor forms asalt bridge interaction with Arg74. In support of this model, byremoving Arg74 fromC5a (C5a-des-Arg74, a natural metaboliteof C5a generated by proteolysis), theC5a-des-Arg74 ligand acti-vated theD282A-C5aRwith similar potency comparedwith thefull-length C5a (54). To test if the S272A mutation affects thepotential interaction of Arg74 with EC3, we generated a C5a-des-Arg74 ligand that could be expressed in yeast. In our studiesin yeast, C5a stimulated weak signaling in D282A-C5aR,whereas C5a-des-Arg74 induced potent signaling in D282A-C5aR (Fig. 6), consistent with the earlier studies in mammaliancells (54, 55). However, both C5a and C5a-des-Arg74 failed to

FIGURE 3. Engineering C5aR to a chemokine-type receptor; signaling profile of mutant C5aR receptors,co-expressed with C5a in yeast. Left panel, canonical receptor topology for C5aR. The N119S mutation onTM3 is a novel constitutively active mutant identified for C5aR in this study. Asp27, Ser30, and Ser272, highlightedin white with black background, have been mutated to cysteine for engineering a possible extra disulfidelinkage between the N terminus and EC3 loop of C5aR. Middle and right panel, respectively, illustrate the effectof S30C, S272C, S30C/S272C, and D27C, S272C, D27C/S272C on C5a-stimulated C5aR signaling. Vector repre-sents the �-galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each barrepresents means � S.D. of signaling activity for three independent transformants, and data are representativeof at least two independent experiments.

FIGURE 4. Signaling profile of wild type and mutant C5aR receptors, co-expressed with wild type and C27R C5a in yeast. The nonfunctionalmutants, such as S272A and S272T, have not been tested with C27R C5a.Vector represents the �-galactosidase activity of the engineered yeast in theabsence of both receptor and ligand. Each bar represents means � S.D. ofsignaling activity for three independent transformants, and data are repre-sentative of at least two independent experiments.

FIGURE 5. Effect of point mutations on expression profile of C5aR in yeastas assessed by Western blot. 25 �l of whole yeast lysates expressing themutant or the wild type receptors were resolved on a 4 –12% BisTris gel anddetected as described under “Experimental Procedures.” The single and dou-ble asterisks, respectively, indicate the full-length and proteolytic fragmentsof the receptors. n/a, not applicable.




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stimulate signaling in S272A-C5aR (Fig. 6), suggesting that theS272A mutation in EC3 induces a conformational change thatmight compromise the active site and the affinity of the C5a forC5aR. To test this possibility, we assessed the ability of the10-mer peptide agonist (YSFKPMPLaR) (54, 56) to activatewild type and mutant C5aRs. As noted in Fig. 7, left panel, �10�M of the peptide was able to stimulate strong signaling both inwild type andmutantC5aRs including S272A-C5aR. In concen-trations ranging from 100 pM to 10 �M (Fig. 7, right panel), thepeptide displayed EC50 of 4.6 � 0.14 nM for wild type C5aR,which increased�120-fold (488� 14 nM) for S272A-C5aR. Onthe other hand, both the double mutants S30C/S272A andS30C/S272C displayed moderately increased EC50 values of38 � 1.4 nM (�9-fold) and 108 � 1.4 nM (�23-fold), respec-

tively, for the peptide. These data demonstrate that themutatedC5a receptors were competent to transduce signals, and thelack of C5a-induced signaling in S272A/S272T/S272C mutantC5aRs could be due to decreased C5a affinity toward themutants. These data further support that the rescue effectobserved in doublemutants S30C/S272Aor S30C/S272C is dueto the conformational interactions between the membrane-proximal portion of the N terminus of C5aR and EC3. The�2.5-fold difference observed in EC50 values between S30C/S272A and S30C/S272C affirms that these mutant receptorslikely have similar conformations. This supports the hypothesisthat GPCRs can exist in a multitude of conformational ensem-bles in equilibrium and can differentially modulate G proteinbinding with respect to their ligands (57, 58).Identification of CAM of Human C5aR—Alignment of

CXCR4 and C5aR (Fig. 1) revealed that like CXCR4 C5aR alsohas an asparagine in TM3 (N3.35). Sequence alignment of 736human GPCRs of the rhodopsin family demonstrated a polar(Cys/Ser/Thr/Asn), nonpolar (Gly/Ala/Val/Ile/Leu/Met) or anaromatic (Phe/Tyr) residue in 47, 34, and 18% of the receptorsat this position, respectively. We found that N3.35 (TM3) is aconserved residue among 15% of the receptors. Themost abun-dant amino acids at N3.35 are Ser/Cys/Thr in �32% of thereceptors, followed byGly/Ala in�14% of the receptors.Muta-tion of N3.35 to Gly/Ala/Ser has been previously shown to con-fer robust constitutive active signaling inCXCR4 (47) andmod-erate constitutive signaling in angiotensin II AT1 receptor (59,60). Based on this, we constructed the TM3mutant, Asn/S3.35of C5aR. As illustrated in Fig. 8, left panel, the N119S mutantC5aR displayed strong constitutive activity in yeast, with amodest increase in signaling upon stimulation with C5a to alevel comparable with that observed with maximal wild typeC5aR signaling, thus identifying a robust novel CAM of C5aR.Effect of Engineered Cysteine Pair on Constitutive Signaling of

C5aR in Yeast—As noted in Fig. 8, neither the D27C nor theS30C mutation in the N terminusaffected the constitutive signalingor C5a-stimulated signaling, con-gruent with results observed whenthese mutations were introducedinto the wild type C5aR (Fig. 3). Theloss of function in the wild typeC5aR imparted by the S272Cmutant could be rescuedwith eitheran S30C or a D27C mutation (Fig.3), suggesting that this region of theN terminus of C5aR might directlyinteract with EC3. Thus, the impor-tance of Ser272 on EC3 conforma-tion and its ability to form the puta-tive disulfide linkage with S30Con N terminus was systematicallyprobed by substituting Ser272, re-spectively, to S272T, S272A, andS272C in the CAM of C5aR.In contrast to wild type EC3

mutants (Figs. 3 and 4), the intro-duction of S272A/S272C/S272T

FIGURE 6. Comparison of signaling profile of wild type, D282A, andS272A mutant C5aR receptors, co-expressed with C5a and metaboliteC5a-des-Arg74 in yeast. C5a-des-Arg74 stimulates only �30% of the signal-ing in wild type C5aR compared with the signaling stimulated in response toC5a. D282A mutant displays reduced signaling in response to C5a consistentwith other studies but displays near-maximal signaling in response to metab-olite C5a-des-Arg74. The nonfunctional mutant S272A neither responds toC5a nor to the metabolite C5a-des-Arg74. Vector represents the �-galactosid-ase activity of the engineered yeast in the absence of both receptor andligand. Each bar represents means � S.D. of signaling activity for three inde-pendent transformants, and data are representative of at least two indepen-dent experiments.

FIGURE 7. Quantifying the efficacy of the 10-mer peptide agonist toward wild type and mutant C5aRs.Left panel, maximal signaling stimulated in wild type, S272A, S30C/S272A, and S30C/S272C mutant C5aRs inresponse to �10 �M peptide agonist. Vector represents the �-galactosidase activity of the engineered yeast inthe absence of both receptor and ligand. Each bar represents means � S.D. of signaling activity for threeindependent transformants, and data are representative of at least two independent experiments. Right panel,dose-dependent signaling stimulated by the 10-mer peptide agonist in wild type, S272A, S30/S272A, andS30C/S272C mutant C5aRs. y axis represents �-galactosidase activity normalized to the maximum signaling,and each point represents means � S.D. of signaling activity for three independent transformants, and data arerepresentative of at least two independent experiments.




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into the CAM did not abolish receptor activity; the mutatedreceptors retained some constitutive signaling and exhibitedmaximal C5a-stimulated signaling (Figs. 8 and 9). Of note, theS30C and D27C mutations in the N terminus decreased themaximal constitutive signaling displayed by N119S-S272Cmutant without affecting the C5a-induced signaling (Fig. 8,compare lanes CwithD in both panels). However, the presenceof the S30C mutation on the N terminus did not result in asubstantial change in the constitutive signaling of S272A orS272Tmutants, and like the single point mutants, these doublemutants also displayed maximal C5a-stimulated signaling (Fig.9, compare lanes B with D and C with E, respectively). Theapparent loss in constitutive signaling in S30C/S272C C5aRCAM (Fig. 8, compare lanes A with D in both panels) may beattributed to the conformational stability induced by the cys-

teine pair in C5aR CAM. This is an interesting observation asEC3-N terminus cysteine pair displays contrasting function inCXCR4 and C5aR.Signal Transduction and Membrane Binding Assays of

Mutated Receptors Expressed in Mammalian Cells—Althoughthe yeast system allows for convenient testing of large numbersof mutated receptors, the yeast cell wall is not permeable tolarge agonists like C5a and CXCL12. Therefore, to determinebinding affinities and to confirm receptor phenotypes, weselected several C5aR and CXCR4 mutant receptors to expressin COS-7 cells. As noted in Fig. 10, left panel, wild type and theselected mutant C5aRs displayed robust inositol phosphatesignaling in response to 10 nM C5a (a concentration that is�10-fold higher than the reported EC50 for C5a (61)). TheS272C-mutated C5aR demonstrated slightly diminished C5a-mediated signaling relative to the wild type C5aR (6.68- versus4.02-fold over basal, respectively). As shown in Table 2, thisdifference might be due to decreased binding affinity (�5-foldless relative to wild type C5aR) because there is little differencein expression levels (Bmax 7.88 versus 8.43 pmol/mg, respec-tively). Introduction of the second cysteine in the N terminuspartially rescued receptor activation and C5a binding affinity.Relative to S272C-C5aR, the S30C/S272C double mutant dem-onstrated increased IP3 production and increased bindingaffinity (6.52- versus 4.02-fold over basal andKd 1.23 versus 3.61nM, respectively). The rescue effect of S30C for S272C seen inCOS-7 cells is consistent with the effects observed in yeast. Ofnote, the S272A/S272T/S272C mutant C5aRs displayed signif-

FIGURE 8. Positional effect of N-terminal and EC3 cysteines on constitu-tive signaling profile of C5aR and their respective response to C5a, co-expressed in yeast. Left and right panels, respectively, illustrate the effect ofS30C, S272C, S30C/S272C, and D27C, S272C, D27C/S272C on ligand-indepen-dent and -dependent signaling of constitutively active C5aR. The contrastingeffect of S30C/S272C, D27C/S272C, and S272C on constitutive signaling ofC5aR is noted in both panels. Vector represents the �-galactosidase activity ofthe engineered yeast in the absence of both receptor and ligand. Each barrepresents means � S.D. of signaling activity for three independent transfor-mants, and data are representative of at least two independent experiments.

FIGURE 9. Probing the side chain effect at Ser272 of EC3 on constitutivesignaling of C5aR in yeast. The constitutive signaling profile of S272T,S272A, and S272C mutants in the presence and absence of S30C at the Nterminus of C5aR. Maximal signaling is noted for all the single and doublemutants in response to C5a. Vector represents the �-galactosidase activity ofthe engineered yeast in the absence of both receptor and ligand. Each barrepresents means � S.D. of signaling activity for three independent transfor-mants, and data are representative of at least two independent experiments.

FIGURE 10. Signaling assays in COS-7 cells expressing G�16 with wild typeor mutant receptors. Left and right panels, respectively, represent C5aR andCXCR4. The cells were stimulated with 10 nM C5a (C5aR) or 100 nM CXCL12(CXCR4). IP3 accumulation values are normalized to the mock transfections,where each bar represents means � S.D. of three independent experiments.

TABLE 2Analysis of receptor binding affinities in COS-7 cell membranesThe apparent Kd and Bmax values are derived from binding assays by fitting the rawdata into one site homologous bindingmodels. Values representmeans� S.D. fromthree independent experiments.

Receptors Kd Bmax

nM pmol/mgC5aR 0.65 � 0.07 8.43 � 0.85S272C-C5aR 3.61 � 0.38 7.88 � 0.87S30C/S272C-C5aR 1.23 � 0.04 5.08 � 0.15N119S-C5aR 0.22 � 0.07 4.02 � 0.52S30C/N119S/S272C-C5aR 1.11 � 0.03 7.03 � 0.42CXCR4 3.32 � 0.24 9.41 � 0.72N119S-CXCR4 1.63 � 0.23 20.86 � 2.55C28S/N119S/C274S-CXCR4 13.33 � 1.15 16.42 � 1.70C28R/N119S/C274E-CXCR4 7.59 � 0.20 16.64 � 0.46




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icantly blunted signaling phenotypes relative to that observedfor S272C-C5aR expressed in COS-7 cells (Figs. 3 and 4 com-pared with Fig. 10). The basis for this is not clear but likelyreflects an increased sensitivity to mutations in human recep-tors expressed in yeast. The N119S mutant C5aR displayedmodest constitutive activity in COS-7 cells (Fig. 10, left panel)that was similar to that observed in yeast and was also furtherstimulated in response to C5a. With respect to wild type C5aR,the N119S mutant displayed slightly increased binding affinityfor C5a (Table 2; Kd 0.22 versus 0.65 nM). The combined cys-teine mutations S30C and S272C reduced the constitutiveactivity of N119S mutant (Fig. 10, left panel) and decreased thebinding affinity to near wild type levels (Kd 1.11 versus 0.65 nM)with no significant reduction in C5a-mediated signaling.Wild type CXCR4 expressed in COS-7 cells displayed basal

signaling in IP3 assays, which further increased upon stimula-tion with 100 nM CXCL12 (Fig. 10, right panel, 3.63-fold stim-ulation over basal). Consistentwith our studies in yeast, N119S-CXCR4 also displayed modest constitutive activity as assessedin IP3 assays. Some of the increased basal activity could be dueto an increase in receptor expression relative to wild typeCXCR4 (Table 2; Bmax 20.86 versus 9.41 pmol/mg, respec-tively). In favor of more active conformation, N119S-CXCR4demonstrated a 2-fold increase in binding affinity for CXCL12(Kd 1.63 versus 3.32 nM). Removal of the cysteine pair decreasedthe apparent constitutive activity in C28S/N119S/C274SCXCR4 (Fig. 10, lane D, right panel) and decreased the bindingaffinity for CXCL12 with no effect on receptor expression(Table 2) or its ability to be activated by 100 nM CXCL12 (Fig.10, right panel, lane D). Similar to the studies in yeast, a poten-tial salt bridge (C28R/C274E) substituted for the cysteine pairappreciably restored the constitutive activity in N119S mutantCXCR4 expressed in COS-7 cells. Although the putative saltbridge improved the binding affinity for CXCL12 in respect toC28S/N119S/C274S mutant (Table 2), no further stimulationwas observed in presence of 100 nM CXCL12 in mammaliancells (Fig. 10, right panel). These results further support animportant interaction between EC3 and the N terminus ofCXCR4.Hypothetical Molecular Model of C5aR—Functional data of

C5aR presented in Figs. 3, 4, and 7–9 suggest that Ser272plays a crucial role for maintaining EC3 structure, whichmay have a role in ligand binding and activation. It alsoappears that the S272C of EC3 perhaps favors the formationof a putative disulfide linkage with S30C/D27C by reducingthe conformational possibilities of the flexible N terminus.Furthermore, Ser30 or Asp27 could be involved in N termi-nus-EC3 packing as S30C or D27C mutants rescue the func-tion in S272A and S272T mutations. This argument is picto-rially represented via molecular models using one out of theseveral possible conformations of N terminus for the doublemutant S30C-S272C. Fig. 11A represents a conformationalsnapshot of wild type C5aR, where Ser272 of EC3 is shownwithin the permissive covalent contact with Ser30 of the Nterminus, although Fig. 11B represents the putative disulfidelinkage between Cys30 and Cys272 in engineered C5aR. For-mation of an EC3-N terminus disulfide linkage may affectthe EC3 loop structure and can potentially stabilize the

receptor conformation. Introduction of a cysteine at Asp282in EC3 of bovine rhodopsin and a corresponding cysteinesubstituted at Asn2 in the N terminus formed a disulfidebridge as evidenced in the crystal structure (51), but it didnot significantly alter the conformation of EC3 when com-pared with the wild type rhodopsin structure (Fig. 11C).However, the engineered mutant receptor displayed higherthermostability (51). In contrast, opsin bound to a G protein-like peptide displayed appreciable conformational changesin EC3 when compared with the “off” structure of dark staterhodopsin (Fig. 11D).

DISCUSSION

This study highlights the importance of EC3-N terminuscysteine pairs in receptor function. In contrast to the highlyconserved EC2-TM3 cysteine pair that plays an importantstructural role in nearly all rhodopsin family receptors, theEC3-N terminus cysteine pairs appear to add functionality toa subset of the rhodopsin family of receptors. The nature ofthis functionality varies from receptor to receptor. InCXCR4, the EC3-N terminus cysteine pair adds functionalityby stabilizing the active state of the receptor. In contrast,when engineered into the C5aR, the cysteine pair exerts theeffect of an inverse agonist in C5aR by diminishing the con-stitutive signaling of the novel C5aR CAM. These studies

FIGURE 11. Hypothetical molecular models of C5aR and illustration ofconformational changes in EC3 due to disulfide linkage and receptoractivation. Molecular models of wild type and engineered C5aR are pre-sented, respectively, in A and B. Asp27, Ser30, Asn119, and Ser272 that havebeen mutated for engineering the C5aR are highlighted in spheres.B, highlight of both the conserved and the engineered disulfide bond,respectively, between Cys109–Cys188 and Cys30–Cys272 in spheres, in con-text of other residues. Loop structures have been smoothed for clarity.C, structural alignment of EC3 of wild type (green loop with cyan helices,1F88) and engineered rhodopsin (magenta loop with sand helices, ProteinData Bank code 2J4Y). Disulfide linkage between Asp282 of EC3 with Asn2

of the N terminus increases the EC3 loop length by straightening the TM7by one helical loop. D, structural alignment of EC3 of rhodopsin (greenloop with cyan helices, Protein Data Bank code 1F88) with EC3 of appar-ently active opsin (red loop with lime helices, Protein Data Bank code3DQB). Major conformational changes noted in EC3 are associated withstraightening of both TM6 and TM7 helices.




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demonstrate a structural interaction between the EC3 andthe N termini in the C5aR and CXCR4, which suggests anovel microswitch region for C5aR and CXCR4. Moreover,the different functional phenotypes obtained for single anddouble cysteine mutants across the receptors imply a moreinvolved role of EC3-N terminus via cysteine pairs beyond asimple disulfide linkage.EC3-N terminus cysteine pairs have been studied previously

in both chemokine and non-chemokine receptors. Disruptionof these cysteine pairs in wild type CXCR4 have been shown toreduce CXCL12 binding affinity (30). In addition, the cysteinepair is not essential for CXCR4 function as a co-receptor forHIV-1 Env-mediated membrane fusion in a strain-specificmanner (35). Mutation of the cysteine pair in CCR5 completelyabolishes the detectable level of MIP-1� binding, whereas theCys-mutated receptors supported infection of R5HIV strains atslightly reduced levels (34). In CCR6, cysteine pair mutation atCys36 significantly reduced MIP-3� binding and signaling,whereas Cys288-mutated CCR6 showed only blunted signaling(33). Despite expression levels similar to wild type CCR6, nei-ther cysteine-mutated CCR6 supported chemotaxis (33). Sim-ilarly, in CXCR2, mutation of Cys286 in the EC3 completelydisrupted CXCL8 binding and CXCR2-mediated intracellularsignaling (36); however, mutation of the pair residue Cys39 inthe N terminus increased both the binding affinity slightly forCXCL8 and intracellular signaling through calcium flux. Inter-estingly, HEK cells transfected with C39S-mutated CXCR2demonstrated severely blunted chemotactic function relative tothe wild type CXCR2 (36).Earlier studies suggested cysteine pair mutations do not sig-

nificantly impair the cell surface expression levels in chemokinereceptors, although antibody staining suggested cysteine pairmutation could affect the overall receptor conformation (35).Few studies have directly examined if a disulfide bond links theEC3-N terminus cysteine pair. In CCR6, alkylation of free thiolsdemonstrated decreased mobility in SDS-PAGE for the wildtypeCCR6 indicative of cysteine thiolmodification, but no shiftwas observed in the C36S/C288S-mutated CCR6 (33). Theseresults do not rule out the possibility that the cysteines mightexist in equilibrium between free thiols and labile disulfidebridges, which could be trapped during prolonged incubationwith alkylated agents.In non-chemokine receptors like angiotensin II AT1

receptor, disruption of the EC3-N terminus cysteine pairconferred constitutive signaling without significantly affect-ing the expression levels or EC50 value for angiotensin II (62).Similarly, in AT2R mutational disruption or reduction byDTT of the EC3-N terminus cysteine pair resulted in anincreased binding affinity for both angiotensin II and recep-tor-specific antagonist PD123319 (63). The purinergicreceptors such as P2Y1 and P2Y12 are another class of non-chemokine receptors, where the EC3-N terminus cysteinepairs appear to play different roles within members of thepurinergic family. Previous studies demonstrate an essentialrole for EC3-N terminus cysteine pairs in P2Y1 ligand bind-ing (28). In contrast, disruption of the EC3-N terminus cys-teine pair in P2Y12 did not significantly affect the EC50 valuefor agonist-mediated inhibition of adenylyl cyclase activity

(64). Although the cysteine pair was not essential for P2Y12activity, the cysteines could be modified by either thiolagents or the active metabolite of the widely prescribed anti-platelet drug, clopidogrel (64).Given the different roles of cysteine pairs in both chemo-

kine and non-chemokine-type receptor function, we electedto engineer an EC3-N terminus cysteine pair into C5aR. Indoing so, we uncovered an essential role for the serinelocated at the position in EC3 at 7.25, the position at whichcysteines most likely occur in receptors containing the cys-teine pair. In our studies in yeast, mutations S272A, S272T,and S272C abrogated the C5a-stimulated signaling in thesesingle EC3 point mutants. In mammalian cells the S272Cmutant displayed appreciable signaling but at a reduced levelin reference to wild type C5aR. This is a novel finding as ourprevious studies using random saturation mutagenesis sug-gested that mutations could be tolerated at this position inEC3 (65) in the presence of other point mutations intro-duced into EC3. The loss in C5a-stimulated signaling in theS272C mutant could be rescued by introducing another cys-teine on the N terminus of C5aR (Figs. 3 and 10). In addition,the EC3-N terminus cysteine pair significantly reduced con-stitutive signaling of the C5aR CAM (Figs. 8 and 10). Theability of the N-terminal cysteine to act as second-site sup-pressors implies a direct interaction with EC3, which mayfavor a possible disulfide linkage between the EC3-N termi-nus cysteine pair in C5aR. The most direct interpretation isthat the rescue effect occurs via an intramolecular interac-tion between the N terminus and EC3. However, the possi-bility of intermolecular interactions cannot be excluded,because many GPCRs are known to oligomerize, includingthe C5aR and CXCR4. In addition, our studies in yeast dem-onstrate that a disulfide bond is not required for the rescueeffect because the S30C mutation on N terminus rescued theC5a-stimulated signaling in S272A and S272T mutant inwild type C5aR (Fig. 5). This result was particularly interest-ing as it implicates the possibility of a noncovalent, confor-mationally dynamic interaction between EC3 and N termi-nus in C5aR, a feature that was previously unappreciated.The importance of noncovalent interactions between EC3-Nterminus is further evidenced in CXCR4 (Figs. 2 and 10), asthe loss of constitutive or CXCL12-mediated signaling inC28S/C274S mutant could be restored either by introducinga salt bridge (C28R/C274E) or �-stacking interactions(C28F/C274F). Previous studies in bovine rhodopsin sug-gests that the double mutant N2C/D282C can replace thenoncovalent interactions between Asp282 (EC3) and Asn2 (Nterminus) through a disulfide bond without altering theoverall fold of the protein (51).Our data suggest that EC3 loop structure is susceptible to

mutations, and its conformational states can modulate bothligand-dependent and -independent G protein signaling. EC3forms a helical hairpin with kinked TM6 and TM7 and is posi-tioned such that any conformational transition in EC3 couldmodulate the sea-saw motion of TM6 with respect to TM7. Infact, as noted in Fig. 11D, EC3 in apparently active opsin dem-onstrates a major conformational displacement, compared




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with the inactive rhodopsin, suggesting a possible unappreci-ated role of EC3 in receptor activation.The biologic significance of the EC3-N terminus cysteine

pair remains an open and important question. The highdegree of conservation of the cysteine pair in chemokinereceptors suggests a unique role for this structural motif inchemokine biology. Our studies in CXCR4 and C5aR dem-onstrate that the interface between the N terminus and EC3might serve as a “microswitch” in many rhodopsin familymembers. The presence of thiol groups at this microswitchregion may confer on chemokine receptors the ability to reg-ulate or fine-tune the signaling properties of the receptors.This raises the interesting possibility that these cysteinepairs might be acting as redox sensors responding to perox-ides, reactive oxygen species, reducing agents, or enzymesthat modify the disulfide bridges such as protein-disulfideisomerases. Further studies will be required to understandbetter the effects of the redox environment on cellularsignaling.

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Soumendra Rana and Thomas J. BaranskiACTIVATION MICROSWITCH REGION

Seven-transmembrane Domain Receptor Function: IMPLICATIONS FOR AN Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for

doi: 10.1074/jbc.M110.129213 originally published online July 27, 20102010, 285:31472-31483.J. Biol. Chem.

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thirdextracellularloop(ec3)-nterminusinteractionis ...nism is described as “molecular switches”...

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