from molecular details of the interplay between transmembrane

From Molecular Details of the Interplay betweenTransmembrane Helices of the Thyrotropin Receptor toGeneral Aspects of Signal Transduction in Family AG-protein-coupled Receptors (GPCRs)*

Received for publication, October 25, 2010, and in revised form, May 10, 2011 Published, JBC Papers in Press, May 17, 2011, DOI 10.1074/jbc.M110.196980

Gunnar Kleinau‡§1, Inna Hoyer‡1, Annika Kreuchwig‡1, Ann-Karin Haas‡, Claudia Rutz‡, Jens Furkert‡,Catherine L. Worth¶, Gerd Krause‡2, and Ralf Schulein‡

From the ‡Leibniz-Institut fur Molekulare Pharmakologie, 13125 Berlin, Germany, §Institute of Experimental PediatricEndocrinology, Charite Universitatsmedizin, 13353 Berlin, Germany, and ¶Institute of Physiology, Charite Universitatsmedizin,13125 Berlin, Germany

Transmembrane helices (TMHs) 5 and 6 are known to beimportant for signal transduction by G-protein-coupled recep-tors (GPCRs).Our aimwas to characterize the interface betweenTMH5 and TMH6 of the thyrotropin receptor (TSHR) to gainmolecular insights into aspects of signal transduction and regu-lation. A proline at TMH5 position 5.50 is highly conserved infamily A GPCRs and causes a twist in the helix structure. Muta-tion of the TSHR-specific alanine (Ala-5935.50) at this positionto proline resulted in a 20-fold reduction of cell surface expres-sion. This indicates that TMH5 in the TSHR might have aconformation different from most other family A GPCRs byforming a regular �-helix. Furthermore, linking our own andprevious data from directed mutagenesis with structural infor-mation led to suggestions of distinct pairs of interacting resi-dues between TMH5 and TMH6 that are responsible for stabi-lizing either the basal or the active state. Our insights suggestthat the inactive state conformation is constrained by a core setof polar interactions among TMHs 2, 3, 6, and 7 and in contrastthat the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings mightbe relevant for all family A GPCRs as supported by a statisticalanalysis of residue properties between the TMHs of a vast num-ber of GPCR sequences.

G-protein-coupled receptors (GPCRs)3 are mediators be-tween extracellular stimuli and intracellular signaling cascades.Knowledge concerning themechanisms of receptor andG-pro-tein activation has grown exponentially in the past few decades(1–6). GPCRs are anchored in the membrane, and differentparts of GPCRs are responsible for specific intra- as well as

intermolecular functions during a sequential signal transduc-tion process. Despite individual and selective properties ofparticular receptors in each of these steps, general aspects arecommon for family A GPCRs. A “global toggle-switching”mechanism is proposed based on a vast amount of experimentaland structural data (7) whereby a vertical see-sawmovement oftransmembrane helix (TMH) 6 occurs around a pivot. Conse-quently, spatial movement of particular TMHs relative to eachother characterizes receptor activation; this occurs to the great-est extent between TMHs 5, 6, and 7, respectively (8, 9). Theconformations and structural rearrangements are supported byamino acids acting as “microswitches” (10). Some of these res-idues are well known for family A GPCRs such as the highlyconservedNPXXY (TMH7) andDRY (TMH3)motifs. DifferentGPCR conformations are causally related to different signalingactivity states (3, 10, 11). Furthermore, differences betweenactivated conformations depend on particular interactingligands, and they are probably involved in determination ofG-protein subtype preferences (12–17). The previously pub-lished GPCR structures of inactive receptor conformations (forreviews, see Refs. 15, 18, and 19) compared with the recentlypublished active conformation of opsin and �2-adrenergicreceptor (20, 21) confirmed a distinct spatial movement of spe-cific transmembrane helices relative to each other, in particularof TMHs 5 and 6, during receptor activation.However, underlying details and general questions of GPCR

signal transduction still remain open. For instance, which resi-dues constrain the inactive state, and which are responsible formovement of the helices? Structural and functional specificitiescaused by different residues at conserved positions of GPCRsubtypes are also of interest for further investigation (22, 23).Therefore, our aim was to characterize the interface of aminoacids between TMH5 and TMH6 of the thyrotropin receptor(TSHR) in detail to gain deeper insight into properties that areresponsible for the inactive (or basal) and the active conforma-tions. This we studied by site-directed mutations, structuralhomologymodeling, and by statistical analysis of a vast numberof GPCR sequences.As in other GPCRs, the TSHR topology is characterized by a

serpentine domain comprising seven TMHs connected bythree intracellular loops and three extracellular loops (24). In

* This work was supported by the Deutsche Forschungsgemeinschaft GrantsKR1273/4-1 and KL2334/2-1.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed: Leibniz-Inst. fur Molekulare

Pharmakologie, Robert-Rossle-Str. 10, 13125 Berlin, Germany. Tel.: 49-30-94793228; Fax: 49-30-94793230; E-mail: [email protected].

3 The abbreviations used are: GPCR, G-protein-coupled receptor; GPHR,glycoprotein hormone receptor; TSHR, thyroid-stimulating hormonereceptor, thyrotropin receptor; TSH, thyroid-stimulating hormone; TMH,transmembrane helix; CAM, constitutively activating mutation; EndoH,endo-�-N-acetylglucosaminidase H; PNGaseF, peptide-N-glycosidase F;SCA, specific constitutive activity; IP, inositol phosphate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 29, pp. 25859 –25871, July 22, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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addition, a few amino acids that are highly conserved in otherfamilyAGPCRs such as the proline at position 5.50 inTMH5orthe essential tryptophan at position 6.48 in TMH6 are differentin the TSHR. They are replaced in the TSHR by an alanine(Ala-593) at position 5.50 and by a methionine at position 6.48(Met-637). Furthermore, by analyzing available functional datafor TSHR residues in TMH5 and TMH6 in a glycoprotein hor-mone receptor (GPHR) information resource (25), it becomesapparent that several amino acids that form the interfacebetween the activation-important TMHs 5 and 6 have not yetbeen investigated. Therefore, we analyzed those amino acidsand performed site-directed mutagenesis followed by func-tional characterization of themutant receptors. In combinationwith previous findings, our data provide (i) hints for details ofinteracting partners at transmembrane helices 5 and 6, (ii)insight into general aspects and specific details of signalingmechanisms during TSHR activation, and (iii) structural andfunctional consequences of variations at conserved positions infamily A GPCRs. Finally, this study focused on the causal rela-tionship among amino acid sequence, the three-dimensionalstructural properties, and the particular functions proceedingat the interfaces along the transmembrane helices of both theTSHR and other family A GPCRs.

EXPERIMENTAL PROCEDURES

Molecular Homology Models of Different TSHR Confor-mations—Crystal structures in the inactive conformation serv-ing for GPCR homologymodeling have been published for sev-eral family A GPCRs (26–34). These structures were silencedby keeping the receptors in a rigid conformation (thermostabi-lized) by one of the followingmethods: (a) inverse agonistswereused as ligands, (b) receptors were modified by silencing muta-tions, or (c) theywere fusedwith lysozyme (for reviews, seeRefs.18, 19, 35, and 36). In contrast, the new x-ray structure of opsinlacks the inverse agonist ligand retinal and has structural fea-tures predicted for an active receptor conformation (20). Forstructural comparisons between an inactive TSHR and an acti-vated TSHR conformation, we used homology models for bothan inactive TSHR model based on rhodopsin (Protein DataBank code 2I35) and an activated model using opsin (ProteinData Bank code 3CAP) as a template.For TMH5, two different conformational models were gen-

erated. Model 1 has the proline bulge at position 5.50 from thetemplate structures, and model 2 has a regular �-helical con-formation around position 5.50 due to the lack of a prolinecompared with opsin/rhodopsin (Fig. 1). Gaps of missing resi-dues in the loops of the template structure were closed by the“LoopSearch” tool in Sybyl 8.1 (Tripos Inc., St. Louis,MO). Sidechains and loops of homology models were subjected to conju-gate gradient minimizations (until converging at a terminationgradient of 0.05 kcal/mol � Å) and molecular dynamics simu-lation (4 ns) by fixing the backbone of the TMHs. Finally, themodels were minimized without constraints using the AMBER7.0 force field. To facilitate comparison with other GPCRs, weused both the amino acid numbering scheme of the entireTSHR with its signal peptide and the Ballesteros-Weinsteinnomenclature for family A GPCRs (37).

Structure images were produced using PyMOL software(version 0.99; Schrodinger, LLC). Fig. 7 was produced using theUCSF Chimera package from the Resource for Biocomputing,Visualization, and Informatics at the University of California,San Francisco.Simulation (Morph) of Conformational Transition between

Basal and Activated Conformations—The transition betweenthe basal and activated conformations of the TSHR homologymodels was simulated using the YaleMorph Server. This servertries to find a possible pathway between different protein con-formations using an adiabatic mapping approach (38). At eachintermediate step (frame), the structure is linearly interpolated,and the potential energy of the system is minimized. Themotions in the morph are consistent with experimental knowl-edge and provide a useful tool for visualizing and analyzing the

FIGURE 1. Transmembrane helix 5 exerts different conformationsdependent on sequence specificities at position 5.50. Two superimposedTSHR homology models (TMH3, TMH4, TMH5, and TMH6 are displayed) dis-playing possible different conformations of the second half of TMH5 towardthe extracellular side. TSHR model 1 (cyan TMH5) is based on the rhodopsintemplate. In most of the family A GPCRs, a proline (Pro5935.50; cyan stick) islocated at TMH5 position 5.50. As a consequence, a proline-supported bulgecaused by a missing H-bond to the backbone can be observed; it causes atwist of the preceding part of TMH5 (cyan) in comparison with a regular �-he-lix. TSHR model 2 (orange TMH5) shows a regular �-helix for TMH5 (without abulge) because in the TSHR an alanine (Ala5935.50) can be found at this posi-tion instead of a proline. This lack of twisted conformation affects the constit-uents of the interfaces between TMH3 and TMH5, TMH4, or TMH6, respec-tively. As a result, the spatial orientations of side chains (sticks) are remarkablydifferent in the models. Side chain Leu-5875.44 (circled in red) is pointingtoward the membrane in model 1, but in model 2, it points to TMH6. L587Vmutation data of constitutive activation (Ref. 46 and Fig. 5) are consistent withthe �-helix conformation in the second model.

Signal Transduction and Regulation at TSHR

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structural changes that might occur during activation of theTSHR. In our web application (25), we offer an applet to studythe conformational transition between the basal and activatedconformation of the TSHR. Themorph is displayed using Jmol.Analysis of Conservation Patterns of Polar and Non-polar

Amino Acids in Transmembrane Helices of Family A GPCRs—The amino acid sequences of the transmembrane helices of5025 family A GPCRs were taken from the GPCR-SequenceStructure Feature Extractor (SSFE). To remove redundancyfrom this data set, cd-hit (39)was used to filter theGPCRs basedon the criterion of having 70% sequence similarity or less. Theresulting data set (comprising 574 sequences) was then used tocalculate the frequency of polar and non-polar amino acids ateach position in the transmembrane helices. Polar residueswere defined as Arg, Asp, Glu, His, Lys, Ser, Thr, and Tyr withthe remaining residues being defined as non-polar. The fre-quency of gaps was also calculated. Using the “define feature”tool of Chimera, the frequencies of polar and non-polar resi-dues were plotted onto the three-dimensional structure ofbovine rhodopsin (Protein Data Bank code 1U19). Thisrequired that the total frequency of polar and non-polar aminoacids in each position be scaled to 100%. Therefore, the numberof gaps at a particular position was subtracted from the totalnumber of GPCRs, and the frequencies of polar and non-polarresidues were recalculated based on the number of GPCRs nothaving a gap at that position. Hence, a sliding scale of conser-vation of polar andnon-polar residues could be plotted onto thethree-dimensional structure with positions (i) conserved forpolar residues being 0, (ii) non-conserved being 50%, and (iii)conserved for non-polar residues being 100%.Construction of Vectors and Site-directed Mutagenesis of

TSHR—The restriction site KpnI was introduced into con-struct hTSHR-pSVL by the QuikChange site-directed mu-tagenesis kit (Stratagene). The resulting plasmid was digestedwithKpnI andBamHI (NewEnglandBiolabs), and the fragmentencoding the wild type (WT) TSHR sequence was inserted intothe pcDNA3 vector (Invitrogen). Mutations were generated bythe QuikChange site-directed mutagenesis kit (Stratagene)using the human TSHR-pcDNA3 as template.For generation of TSHR-A593P�GFP, A593P-TSHR-pcDNA3

was digested with ScaI and BstEII (New England Biolabs). Thefragment encoding the A593P mutation was inserted intoGFP-N1 vector. GFP was fused C-terminally to the TSHR,thereby deleting the stop codon. All constructs were verified bydideoxy sequencing.Cell Culture and Transfection—HEK 293 cells (Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ)) were grown in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal calf serum (Biochrom) at 37 °C ina humidified 5% CO2 incubator. For determination of intracel-lular cAMP accumulation, cells were seeded in 24-well plates(7.5 � 104 cells/well) and transiently transfected after 24 husing 0.6 �g of DNA/well and Lipofectamine 2000 (Invitrogen)according to the supplier’s recommendations. For determina-tion of receptor cell surface expression, cells were seeded in6-well plates (3 � 105 cells/well) and transiently transfectedwith 2.4 �g of DNA/well. For the analysis of the cells by confo-cal laser scanning microscopy, cells were seeded in 35-mm-

diameter dishes (1.5 � 105 cells/dish) containing a poly-L-ly-sine-coated coverslip and transiently transfected using 0.8�g ofDNA/dish. For immunoprecipitation experiments and glycosy-lation state analyses, cells were seeded in 100-mm-diameterdishes (4 � 106 cells/dish) and transiently transfected with 18�g of DNA/dish.Determination of Cell Surface Expression by Flow Cytometry

Measurements—TheTSH receptor cell surface expression levelwas quantified by flow cytometry (FACSCalibur, BD Biosci-ences). All steps were performed at 4 °C or on ice. Approxi-mately 48 h after transfection, cells were detached from thedishes using 1 mM EDTA and 1 mM EGTA in PBS. Suspendedcells were spun at 300 � g for 3 min, and the supernatant wasdiscarded. Cells were incubated for 10min in FACS buffer (PBScontaining 0.5% BSA) and centrifuged again. Cells were incu-bated for 30 min with a 1:200 dilution of a mouse anti-humanTSHR antibody directed against an extracellular domain (2C11(1 mg/ml), Serotec) in FACS buffer. Cells were washed twicewith FACS buffer and incubated for 30 min with a 1:400 dilu-tion of DyLight�488-conjugated goat anti-mouse IgG (1mg/ml; Serotec). Thereafter, cells werewashed twice and resus-pended in FACS buffer. The fluorescence signals of at least 1 �104 cells/tube were measured, and receptor expression at thecell surface was expressed as percentage of the wild type con-trol. Damaged cells were excluded from the analysis by detect-ing them with 7-aminoactinomycin D (BD Biosciences).Determination of Intracellular Cyclic AMP Accumulation by

RIA—After transfection, cells were cultured for 48 h andstimulated for 1 h at 37 °C with stimulation buffer (DMEMsupplemented with 10mMHEPES, 0.5% BSA, 0.25mM 3-isobu-tyl-1-methylxanthine) alone or stimulation buffer containingincreasing concentrations of bovine TSH (1 �IU/ml to 0.1IU/ml; SigmaAldrich). Stimulationmediumwas aspirated, andreactions were stopped by lysis of the cells with 0.1% trifluoro-acetic acid, 0.005% Triton X-100 for 30 min at 4 °C. Superna-tants were collected, heated at 95 °C for 10 min, dried in arotation vacuum concentrator (Alpha-RCV, Martin ChristGefriertrocknungsanlagenGmbH), and finally stored at�20 °Cuntil use. After reconstitution and acetylation of the samples(40), the cAMP content was determined using cAMP-125I-ty-rosyl methyl ester (10,000 cpm; IBL International) and poly-clonal rabbit anti-cAMP antibody (final dilution, 1:160,000).Sampleswere incubated overnight at 4 °C. The antibody-boundfraction was precipitated using 50 �l of Saccel (IBL Interna-tional). The radioactivity of the precipitate was determined in a�-counter.Determination of [3H]Inositol Phosphate—After transfection,

cells were cultivated for 24 h. The DMEM was supplementedwith 74 kBq/ml myo-[2-3H]inositol (Amersham Biosciences),and cells were incubated for an additional 24 h. Cells werewashed with stimulation buffer (culture medium with 10 mM

HEPES, 0.5% BSA, and 10 mM LiCl) and then stimulated for 60min at 37 °C with bovine TSH in stimulation buffer. Stimula-tion medium was removed, and cells were lysed with 0.1 M

NaOH. By subsequent addition of 0.2 M formic acid and dilu-tion buffer (5 mM sodium tetraborate, 0.5 mM EDTA), theappropriate conditions (pH and ion strength) were adjusted.The lysates were spun, and supernatants were subjected to




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anion exchange chromatography on Sep-Pak AccellTM PlusQMAcartridges (Waters). [3H]Inositol 1,4,5-trisphosphatewaseluted with 0.1 M formic acid and 0.4 M ammonium formate.Radioactivity was determined in a liquid scintillation counter.For normalization, the individual counting results were relatedto the total amount ofmyo-[2-3H]inositol incorporated into theHEK 293 cells.Confocal Laser Scanning Microscopy—After transfection,

cells were incubated overnight, washed once with PBS, andtransferred immediately into a self-made chamber (details areavailable on request). For the co-localization of the TSH recep-tor with the plasma membrane marker trypan blue, live cellswere covered with 1 ml of PBS, and trypan blue was added to afinal concentration of 0.05%. After 1 min of incubation, GFPand trypan blue signals were visualized at room temperatureusing a Zeiss LSM510 invert confocal laser scanning micro-scope (objective lens, 100�/1.3 oil; optical section, �0.9 �m;multitrack mode; GFP: � excitation, 488 nm, argon laser; bandpass filter, 500–530 nm; trypan blue: � excitation, 543 nm, heli-um-neon laser; long pass filter, 560 nm). The overlay of bothsignals was computed using the Zeiss LSM510 software (release3.2; Carl Zeiss AG, Jena, Germany). Images were imported intoPhotoshop software (release 6.0; Adobe Systems Inc., San Jose,CA), and contrast was adjusted to approximate the originalimage.Immunoprecipitation, EndoH and PNGaseF Treatment,

SDS-PAGE, and Immunoblotting—After transfection, cellswere cultivated for 24 h, washed twice with PBS-CM (PBS, 1mMMgCl2, 0.1 mMCaCl2, pH 7.4) and lysed for 1 h with 1ml oflysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.1%(w/v) SDS, 1% (v/v) Triton X-100, pH 8.0 supplemented with0.5 mM PMSF, 0.5 mM benzamidine, 1.4 �g/ml aprotinin, and3.2 �g/ml trypsin inhibitor). Insoluble debris were removed bycentrifugation (30 min at 12,000 � g). The supernatant wassupplemented with a 1:200 dilution of monoclonal mouse anti-TSHR antibody (4C1 (1 mg/ml), Serotec) coupled to proteinA-Sepharose CL-4B beads (3.5 mg/1 ml of PBS), and the sam-ples were incubated overnight. Receptors were precipitated (2min at 700 � g) and washed once with 1 ml of washing buffer 1(50 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 0.1% (w/v) SDS,0.5% (v/v) TritonX-100, pH 8.0) and twice with 1ml of washingbuffer 2 (50 mM Tris-HCl, 1 mM EDTA, 0.1% (w/v) SDS, 1%(v/v) TritonX-100, pH 7.4). Precipitated receptors were treatedwith EndoH or PNGaseF according to the supplier’s recom-mendations or left untreated. Samples were supplementedwithRotiLoad sample buffer (Carl Roth GmbH and Co. KG), incu-

bated for 5 min at 95 °C, and analyzed by SDS-PAGE (8% SDS)and immunoblotting. Immunoblots were carried out asdescribed (84) using a 1:200 dilution of a monoclonal mouseanti-human TSHR antibody directed against an extracellulardomain (2C11 (1 mg/ml), Serotec) and a 1:10,000 dilution ofDyLight680-conjugated goat anti-mouse IgG (1mg/ml; Pierce)as secondary antibody. The quantification was done using aninfrared imager (Odyssey 2.1, LI-COR Biosciences).Determination of Specific Constitutive Activity (SCA)—Be-

cause of the linear relationship between basal cAMP accumu-lation and receptor cell surface expression (41), basal cAMPwas normalized to cell surface expression for each of the con-structs. To this end, specific basal (constitutive) activity wasdefined as described recently by Mueller et al. (42). The valueswere normalized to the basal activity of the WT TSHR, whichwas set to 1.

RESULTS

Side Chain Variations of Alanine 5935.50 to Proline, Valine,and Glycine Cause Fold Defects or Constitutive Activation ofSignaling—Inmost of the family AGPCRs, a proline is found atposition 5.50 in TMH5 (for a review, see Ref. 7). Like all otherGPHRs, the TSHR possess an alanine in TMH5 instead of aproline. Therefore, we exchanged Ala-5935.50 to proline butalso replaced this alanine by larger and smaller side chains toreveal insights into the functionality of this position.Cell Surface Expression—Cell surface expression of the

mutants A593P, A593V, and A593G (Table 1) was decreased incomparison with the wild type; in particular, mutant A593Preached only 6% of the wild type level. The fact that cell surfaceexpression of mutant A593P was strongly reduced could beexplained by a folding defect of the receptor caused by themutation. In this case, the mutant receptor should be recog-nized and retained by the quality control system of the earlysecretory pathway and finally subjected to proteolysis by theendoplasmic reticulum-associated degradation pathway. Toaddress the question of whether the mutant is retained intra-cellularly, the wild type and mutant receptors were C-termi-nally tagged with GFP. HEK 293 cells were transiently trans-fected with the constructs, and co-localization of the GFPfluorescence signals of the receptor with those of the plasmamembrane marker trypan blue was analyzed by confocal laserscanning microscopy (Fig. 2A).In the case of the wild type TSHR�GFP, both plasma mem-

brane and intracellular signals were detectable, demonstratingthat the receptor reaches the cell surface. The intracellular sig-

TABLE 1Functional characterization of TSHR mutations at Ala-593 in TMH5 (position 5.50)HEK 293 cells were transiently transfected with the WT TSHR or mutated TSHRs. The TSHR cell surface expression levels were quantified by flow cytometry measure-ments and expressed as the percentage of the wild type control. Data represent mean values of two independent experiments each carried out in duplicates �S.E.Accumulation of cAMP following TSH stimulation of intact cells was measured by a cAMP RIA. Data represent mean values of two independent experiments, each carriedout in duplicates �S.E. EC50 values are shown as the geometric mean (95% confidence limits). The assignment of the SCA was analyzed as described byMueller et al. (42).

Cell surface expression,FACS percentageof TSHRWT

cAMP accumulation

Basal n-fold WT EC50 n-fold WTTSH-stimulated

percentage of TSHR SCA

WT 100 1 1 100 1A593G 76.1 � 9.4 2.13 � 0.12 0.72 (0.63–0.82) 99 � 7 2.80A593V 41.3 � 5.4 0.33 � 0.10 1.54 (1.32–1.80) 103 � 14 0.80A593P 6.6 � 0.2 0.19 � 0.06 1.87 (0.56–2.43) 24 � 11 3.17




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nals may represent transport intermediates en route to the cellsurface and/or a misfolded receptor population present evenin the case of the wild type receptor. In contrast, only intracel-lular signals were detectable in the case of mutant TSHR-A593P�GFP. In agreement with these results, only the wild typeuntagged TSHR could be detected at the cell surface in flowcytometry measurements using an antibody directed againstthe TSHR N-terminal tail (Fig. 2B). If the mutant receptor isindeed retained in the early secretory pathway, only immatureglycosylated forms of the receptors should be present. Toaddress this question, the untagged receptors were precipitatedfrom transiently transfected HEK 293 cells and detected bySDS-PAGE/immunoblotting (Fig. 2C). For the wild type recep-tor, two immunoreactive protein bands were detected. Theupper band represents mature complex glycosylated receptors,and the lower band represents immature high mannose-glyco-sylated receptors (identity of the protein bands was verified by

analysis of the glycosylation state using EndoH and PNGaseFtreatments; see Fig. 2D). In the case ofmutant A593P, only highmannose forms were found, demonstrating that the receptor isunable to leave the early secretory pathway. These data indicatethat the A593P mutation indeed causes a folding defect that isrecognized by the quality control system of the early secretorypathway.cAMP Accumulation Determined by RIA Measurements—

We determined basal and TSH-induced cAMP accumulationto characterize signaling properties of the mutants. The maxi-mum cAMP accumulation of the mutants A593V and A593Gafter stimulation with bovine TSHwas comparable with that ofwild type (Table 1).For the mutants A593V, A593G, and A593P, we also mea-

sured concentration-response curves (10 points; Fig. 3). Themutant EC50 values were comparable with wild type. For themutant A593P, decreased basal and maximum values wereobtained; these might be caused by the lower cell surface ex-pression levels (Table 1).Mutation A593G induced constitutive TSHR activation,

which was confirmed by SCA determination (Table 1). MutantA593P also showed a 3-fold increase in SCA with a cell surfaceexpression of around 7% compared with wild type.Regular �-Helical Conformation of TMH5 in TSHR—Taking

these functional data into consideration, we conclude thatTSHR alanine 5935.50 likely causes a different helix confor-mation in TMH5 compared with other family A GPCRs thathave the conserved proline at this position.Moreover, an initial TSHR model (model 1) based on the

available GPCR crystal structures resulted in a twisted helix 5caused by a proline-induced bulge, which is not consistent withparticular TSHR mutation data. In model 1, the side chain ofLeu-5875.44 points toward the membrane (Fig. 1), but even theslight side-chain alteration L587V led to constitutive activation.However, a regular �-helical conformation of TMH5 in a mod-ified TSHRmodel (model 2) showedmore consistency with themutation data. In this second model, the spatially shifted side-chain orientation of Leu-5875.44 points toward TMH6 andinteracts with Leu-6456.56, mutation of which also led to con-

FIGURE 2. Receptor mutant A593P5. 50 is unable to leave early secretorypathway in transiently transfected HEK 293 cells due to misfold. A, sub-cellular localization of the constructs TSHR�GFP and TSHR-A593P�GFP by con-focal laser scanning microscopy. The GFP fluorescence signals of the recep-tors (green; left panel) were recorded and computer-overlaid with trypan blueplasma membrane signals (red; center panel). Co-localization is indicated byyellow color (right panel). The horizontal (xy) scans show representative cells.Scale bar, 10 �m. Similar data were obtained in three independent experi-ments. B, quantification of the untagged TSHR and mutant A593P at the cellsurface by flow cytometry measurements. Plasma membrane receptors of104 cells were quantified using a monoclonal mouse anti-TSHR antibodydirected against an extracellular domain and DyLight488-conjugated goatanti-mouse IgG. Columns represent mean values of three independent exper-iments (�S.E.). C, glycosylation state analysis of the untagged TSHR andmutant A593P TSHR. Receptors were immunoprecipitated from cell lysatesusing a monoclonal mouse anti-TSHR antibody and detected by SDS-PAGE/immunoblotting using a monoclonal mouse anti-TSHR antibody andDyLight680-conjugated goat anti-mouse IgG. The immunoblot is represen-tative of three independent experiments. D, verification of the identity of theprotein bands for the untagged TSHR. The receptors were precipitated anddetected as described above. Samples were treated with EndoH or PNGaseFprior to SDS-PAGE or left untreated (�). High mannose-glycosylated recep-tors are EndoH- and PNGaseF-sensitive (§); complex glycosylated receptorsare EndoH-resistant and PNGaseF-sensitive (#). Non-glycosylated receptorsare indicated ($). The immunoblot is representative of three independentexperiments.

FIGURE 3. Concentration-response curves for wild type TSHR andmutants A593G, A593V, and A593P. HEK 293 cells were transiently trans-fected with wild type TSHR or mutant receptors and exposed to increasingconcentrations of bovine TSH in the presence of 1 mM isobutylmethylxan-thine. Data points represent mean values of a single experiment performed induplicate. The concentration-response curve is a representative of two inde-pendent experiments for wild type TSHR and mutant receptors.




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stitutive receptor activation (Fig. 1). Consequently, in theTSHRlacking a proline, the amino acids are likely shifted one position,and the interfaces between TMH5 and neighboring helices like3, 4, and 6 are remarkably different compared with a TMH5with a helical twist. Thus, for TSHR and likely also for otherGPHRs, a regular �-helix conformation of TMH5 should beconsidered in structural models.Functional Characterization of TSHRMutations at Potential

Interface between TMH5 and TMH6—Analyzing the aminoacids suggested by the TSHR homologymodel to constitute thepotential interface between TMH5 and TMH6, we selected 10positions where the properties of the residues would bechanged by performing site-directed mutagenesis (Fig. 4).Using the freely accessible Internet tool that provides interac-tive functional information on GPHRs (25, 43), we found thatthese positions had not yet been characterized. To investigatethe functional importance of side chains, mutations were cho-sen so that only slight alterations in length and bulkiness weremade and that the hydrophobicity was conserved. The mutantreceptors were characterized regarding their cell surfaceexpression and cAMPaccumulation in the basal state andTSH-induced activity state (Table 2).Quantification of Cell Surface Expression of Receptors by Flow

Cytometry Measurements—The cell surface expression of nineof the generated mutants from the interface between TMHs 5and 6 were comparable with the wild type TSHR in a rangebetween 79 and 120% (Table 2). Only mutant F642I showed adecreased expression level (57%). Therefore, we do not discussfurther the signaling data for mutation F642I as it might beinfluenced by a decreased number of receptors at the cellsurface.cAMP Accumulation Determined by RIA Measurements—

The maximum cAMP accumulation of the mutants after stim-ulation with bovine TSH was comparable with the wild type,ranging between 87 and 125% of the wild type level. Threemutants (A638V, F642I, and A644V) decreased TSH-inducedsignaling activity, which is indicated by higher EC50 levels,

FIGURE 4. Amino acids constituting potential interface between heli-ces 5 and 6 of TSHR. The homology model of the TSHR serpentine domain(white backbone) with amino acid side chains (sticks) potentially constitut-ing the interface between TMH5 and TMH6 (both helices are coloredbrown) shows the spatial localization and orientation of side chains. Resi-dues that were not investigated previously but studied here by side-chainsubstitution are depicted in green, and their labels are bold and enlarged.ICL, intracellular loop; ECL, extracellular loop; Ntt, N-terminal tail; Ctt, C-ter-minal tail.

TABLE 2Functional characterization of TSHR mutations in TMH5 and TMH6HEK 293 cells were transiently transfected with the wild type or mutant TSHRs. The cell surface expression levels of the receptors were quantified by flow cytometrymeasurements and expressed as the percentage of the wild type control. Accumulation of cAMP following TSH stimulation was determined by a cAMP RIA.Determination of IP accumulation was done by �3H�inositol incorporation studies, and results for cAMP accumulation were measured by RIA. Data are given asmean � S.E. of two independent experiments, each carried out in duplicate. EC50 values are shown as the geometric mean (95% confidence limits). ND, notdetermined.

Transfectedconstruct

Ballesteros-Weinsteinnumbering

Cell surface expression,FACS percentageof TSHR WT

cAMP accumulation IP accumulation

Basal n-fold wt EC50 n-fold wt

TSH-stimulatedpercentage of

TSHR SCA Basal n-fold WT

TSH-stimulatedpercentage of

TSHR

WT TSHR 100 1 1 100 1 1 100TMH5L580V 5.37 113 � 4 0.95 � 0.17 1.14 (1.08–1.21) 102 � 8 0.84 ND NDI583V 5.40 100 � 6 1.32 � 0.19 0.77 (0.75–0.80) 101 � 22 1.32 ND NDV584A 5.41 93 � 8 1.35 � 0.26 1.60 (1.46–1.76) 125 � 28 1.45 ND NDI591V 5.48 93 � 7 0.98 � 0.17 1.88 (1.87–1.89) 94 � 8 1.05 ND NDF594I 5.51 85 � 8 0.13 � 0.02a 4.48 (4.47–4.49)b 92 � 20 0.15 0.89 � 0.07 41 � 0

TMH6F634I 6.45 79 � 9 0.13 � 0.02a 6.34 (5.42–7.41)b 89 � 9 0.16 0.89 � 0.04 15 � 1A638V 6.49 104 � 6 0.38 � 0.05a 7.85 (6.33–9.73)b 95 � 10 0.37 0.78 � 0.17 22 � 6F642I 6.53 57 � 6 0.25 � 0.05a 8.21 (7.46–9.04)b 62 � 3 0.44 0.90 � 0.10 8 � 1A644V 6.55 79 � 5 1.36 � 0.13 8.88 (7.10–11.12)b 87 � 11 1.72 0.73 � 0.07 19 � 0I648V 6.59 120 � 13 0.56 � 0.05a 1.51 (1.10–2.09) 93 � 14 0.47 ND ND

aAmino acids with decreased basal signaling activity by mutation.b Mutations with an increased or slightly increased EC50 compared with wild type.




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whereas mutants F594I and F634I showed slightly increasedEC50 values. Four mutants (F594I in TMH5 and F634I, A638V,and I648V in TMH6) showed decreased basal signaling activityof TSHR (Table 2), whereas A644V was characterized by aslightly increased constitutive activity of 1.7-fold overwild type.IP Determination—Single side-chain substitutions F634I,

A638V, F642I, and A644V in TMH6 and F594I in TMH5 werefound to decrease the capability of the TSHR for basal cAMPaccumulation and to partially decrease the capability of theTSHR for TSH-stimulated cAMP accumulation. Therefore, forthese mutations, we also measured the Gq-mediated IP accu-mulation, which is known to be of physiological relevance tothyroid growth (44). All of these mutations led to a strongdecrease in the maximum IP accumulation induced by bovineTSH in a range between 8 and 41% compared with wild type.Sequence Conservation and Spatial Distribution of Polar and

Non-polar Residues Analyzed in Family A GPCR Sequences—According to the model of the active TSHR conformation, thefollowing direct side-chain interactions occur: Leu-5805.37-Ile-6486.59, Ile-5835.40-Ile-6486.59, Leu-5875.44-Leu-6456.56, Phe-5945.51-Phe-6346.45(and Met-6376.48), Cys-5985.55-Ile-6306.41,and Tyr-6015.58-Met-6266.37 (the first residue of each pair islocated in TMH5, and the second is located in TMH6). Thedynamic process of helical movement in the transition betweenboth conformations (inactive/active) can be studied in detailusing structural morphings to visualize the helical shifts and

side-chain reorientations that potentially occur during TSHRactivation.However, the majority of mutations at these positions led to

(partial) receptor inactivation (Table 2 and Figs. 5 and 6), sup-porting their significance to the active conformation. It can beconcluded that interactions between helices 5 and 6 are essen-tial for stabilization of the active state, and it is of note that thesewild type residues are exclusively hydrophobic. Therefore, weanalyzed the spatial distribution of the conserved polar andnon-polar residues in family A GPCRs. The frequencies werecalculated from a set of 574 family A GPCRs with a sequencesimilarity of 70% or less in the transmembrane helix regions. Aset of polar residues in TMHs 1, 2, 3, and 7 is conservedthroughout family A and most likely helps to stabilize the inac-tive state (Fig. 7). Non-polar residues are conserved in TMHs 3,5, and 6 and form a non-polar interface.

DISCUSSION

One of the important questions of signal transduction byGPCRs relates to the details of the structural-functional inter-play between TMH5 and TMH6. Here we ask the questionwhich of the residues that are forming the interface of TMH5and TMH6 are responsible for stabilization of the basal stateandwhich residues are responsible formovement of the helicesto support the active conformation. Inspection of the researchresource for glycoprotein receptors (43) revealed for the TSHR

FIGURE 5. Functional results of site-directed mutagenesis are mapped onto structural model of TMH5 and TMH6 interface. A, the labels of residuesstudied in this project are marked with *. The side chains are colored according to modified signaling parameters revealed by functional studies of mutants: red,constitutively activating mutations; yellow, inverse agonistic mutations (decreased basal activity) or inactivating mutations for TSH-induced activation; blue,both constitutively activating and inactivating mutations are known; brown, similar to wild type. Distinct interacting pairs of opposing residues (ovals) alsoshow concurrent functional data by mutations. Pairs of constitutive activating mutations (red oval) indicate sites for stabilization of the basal activity, whereasthe residues showing pairs of inactivating mutations (yellow and blue ovals) are sites that are likely to be involved in forming an active conformation. B, the tableis synchronized with the TSHR homology model and presents residues at the interface between TMH5 and TMH6 (A), but in addition, each side-chainsubstitution is referenced (75– 83). The color code is similar to that in A. ICL, intracellular loop; ECL, extracellular loop; Ntt, N-terminal tail; Ctt, C-terminal tail;hTSHR, human TSHR.




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that 13 of the potential 24 interface residues have previouslybeen characterized by mutations. Receptor mutations areknown to generate active and inactive receptor conformations.Thus, we hypothesized that mutational analysis of the remain-ing amino acids would then complete the data for the interfacebetweenTMH5 andTMH6 andwould provide insights into theintramolecular mechanisms underlying stabilization and acti-vation. Here w combined the structural data of inactive andactive GPCR conformations and the statistical occurrence ofamino acid properties at helix interfaces in a large set of GPCRsequences with functional data from the TSHR to pinpointdeterminants that regulate different receptor conformations.InterplayingTransmembraneHelices 5 and 6Regulate Recep-

tor Conformations and Activity States—In agreement with oth-ers (45), we assume that constitutively activating mutations(CAMs) lead to a disturbance of the GPCR ground state bysupporting the active conformation. In contrast, inactivatingmutations that do not impair receptor cell surface expression,binding properties, or direct interactions with the G-proteinmight prevent stabilization of the activated state, which is nor-mally conducted by the wild type amino acid. In other words,inactivating mutations are indicators of wild type amino acidsacting as stabilizers or conductors of activated conformation(s).We mapped the functional data from this study together withpreviously published mutagenesis studies onto the TSHRhomology models (Fig. 5) to reveal deeper insights into the roleand interplay between TMH5 and TMH6 (see Fig. 5B for refer-ences of mutants at particular positions).We found that partic-ular interacting pairs of residues also show concurrent func-tional data by mutations. Our conclusions from the combinedfunctional-structural insights for pairwise residue interactionsbetween TMH5 and TMH6 follow.The side-chain interaction between leucine 5875.44 (TMH5)

and leucine 6456.56 (TMH6) close to the extracellular site issupported by constitutive TSHR activation induced by muta-tions at both positions (46). This interaction was disrupted byside-chain alterations of either Leu-5875.44 or Leu-6456.56 andled finally to an activated receptor conformation. Mutation ofthe two alanines, Ala-6386.49 and Ala-6446.55, in TMH6 tovaline, an enlargement of the side chain, resulted in partial inac-tivation of TSHR whereby A644V mediated a slight increase inbasal cAMP accumulation. These modifications cause stericrepulsion to surrounding amino acids and likely cause sterichindrance of the spatial rearrangement of TMH6. In contrast,side-chain substitutions of the two phenylalanines 5945.51

FIGURE 6. Comparing active and inactive conformation of TSHR: structural rearrangements and details of related microswitches in transmembranedomain. A, TSHR amino acids at positions known to be important for family A GPCR activation are highlighted on superimposed models (side view) of theinactive (white backbone) and the active (green) TSHR conformations. Differences in the backbone orientation are observed between the cytoplasmic ends ofthe fifth and sixth helices (colored arrows beside helices 5 and 6). B, top view from the extracellular side into the active TSHR conformation with clippedextracellular loops showing that several important signaling-sensitive residues are clustered and interacting with each other: cluster 1 (red circle), residues ofTMHs 3, 5, and 7 (Arg3.50, Tyr5.58, and Tyr7.53); cluster 2 (blue circle), between TMH5 and TMH7 (Phe5.51, Met6.48, and Phe6.45). Cluster 1 is close to the intracellularside, and according to the crystal structure of the opsin-transducin peptide complex, they interact with each other (dotted lines for H-bonds) or directly with aG-protein fragment (21). Cluster 2 is located in the middle section of the TSHR and involves aromatic-hydrophobic residue interactions. C, strong differences inorientations and interactions between the side chains can be observed depending on the structural conformation (white, inactive; dark green, active). Duringactivation, specific residues have to undergo larger movements inward or outward (indicated by half-round arrows). It is known that conserved hydrophilicamino acids (dark gray sticks) in the transmembrane region, Asp-633 (6.44, TMH6), Asn-674 (7.49, TMH7), and Asp-460 (2.50, TMH2), interact with each other byhydrophilic interactions (68, 70). A release of these constraints leads to a movement of TMH6, which undergoes a defined conformational shift. In consequence,the TSHR can be subdivided in a simplified way into two main regions. 1) Between TMHs 2, 3, 6, and 7, the inactive state is constrained (pink half). 2) BetweenTMH5 and TMH6, the active state is stabilized (green half). TMH6 seems to be involved in both processes and mediates the activity switch by their conforma-tional rearrangement. ICL, intracellular loop; ECL, extracellular loop; Ntt, N-terminal tail; Ctt, C-terminal tail.

FIGURE 7. Spatial distribution of conservation degree of polar and non-polar residues in 574 family A GPCRs. The frequency of occurrence for polarand non-polar (hydrophobic) residues (shown in red and green, respectively)are mapped onto the three-dimensional structure of inactive bovine rhodop-sin (Protein Data Bank code 1U19). The frequencies were calculated from a setof 574 family A GPCRs with a sequence similarity of 70% or less in the trans-membrane helix regions. A, a set of polar residues in TMHs 1, 2, 3, and 7 isconserved throughout family A, and these residues most likely help to stabi-lize the inactive state. Non-polar residues are conserved in TMHs 3, 5, and 6and form a non-polar interface that is probably important for stabilizing theactive state. B, the conservation of these central polar core residues and thenon-polar interfaces throughout family A suggests a fundamental role forthem in activity regulation.




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(TMH5) and 6346.45 (TMH6) led to a partially decreased TSHRsignaling capability (Table 2), suggesting the involvement ofthese opposing positions (Fig. 5) in themaintenance of an activeTSHR conformation by aromatic-hydrophobic residue interac-tions. Both residues might be essential for a “microswitch”mechanism together with TMH6 residues (see above). Further-more, the residues Tyr-6015.58 (TMH5) and Met-6266.37(TMH6) are located spatially close to each other two turnsalong toward the intracellular side. For both residues, CAMsand inactivating mutations are reported, indicating that theseresidues are likely to be involved in stabilizing the activated aswell as basal conformations (dual functionality; Fig. 5). It isparticularly interesting that these residues change their relativespatial orientation during the activation-related helical move-ment at the intracellular end. In the active conformation, theamino acid Tyr-6015.58 points toward TMH3 and forms aninteraction network with Arg-5193.50 (TMH3) and Tyr-6787.73(TMH7) (Fig. 6). For Tyr-6055.62 (TMH5) and Ile-6226.33(TMH6), inactivating mutations have been reported (see Fig.5B references).With respect to their location close to the intra-cellular side, they are potential points for direct G-proteininteraction (13). Close to the intracellular end of TMH6, CAMsare known at Asp-6196.30, a residue thought to participate ina helix-capping mechanism (47) or to establish a salt bridgewith Arg-5193.50 (TMH3) (for a review, see Ref. 7) in theinactive conformation. The potential interaction partnerin the inactive state, Arg-5193.50 (TMH3), forms a hydrogenbond with Tyr-6015.58 (TMH5) in the active conformation(Fig. 6C). It can be summarized that the arginine at position3.50 (TMH3) is a key player for stabilization of both theinactive and active conformations and also directly interactswith the �-subunit of G-proteins (21). With respect to familyA GPCRs without a positively charged amino acid residue atthis position, alternative mechanisms must be assumed (forreviews, see Refs. 48 and 49).For one functional observation, a detailed explanation can-

not be fully provided by a monomeric TSHR model. The sidechain of Phe-6426.53 points toward the membrane, and muta-tion to isoleucine led to partial TSHR inactivation. Only apotential dimer arrangement of the TSHR where the contactinterface would be defined at or close to TMH6 could provide adirect (intermolecular) interaction partner. TSHR dimer con-formations have been shown to be obligate (50), and it has beendemonstrated that intermolecular contacts in the transmem-brane region are the main determinants of such TSHR andGPHR dimer arrangements (51–53). Known phenomena pub-lished for the TSHR support the hypothesis of TMH6 contactsbetween TSHR monomers like the occurrence of pathogenicCAMs at TMH6 residues, which also point toward the mem-brane (F631V (54) or I635V (55)). For such mutations, mecha-nisms of activationmight be determined by themodification ofthe potential monomer-monomer interface related to activitystates of the TSHR (56). Our mutation at Phe-6426.53 supportsthis hypothesis.Position 5.50 and TMH5 Are Significant for Receptor Signal

Transduction—TheTMH5 of TSHR lacks a proline that is con-served in most other family A GPCRs. The proline mutation ofAla-5935.50 in TMH5 led to a misfolded TSHR. Our initial

TSHRmodel based on a proline-containing structural templateshowed inconsistencies with other TMH5 mutation data aswell. Therefore, for TMH5 of the TSHR, we suggest an alterna-tive model, model 2, which has a regular �-helix for TMH5 incontrast to the available crystal structures of family A GPCRs.Interestingly, the mutants A593G and A593P showed anincreased basal activity, which indicates the importance of ori-entation and adjustment of TMH5 in relation to other helicesfor the activation mechanism. Furthermore, at TSHR positionAla-5935.50, the pathogenic CAM A593N (57) and the patho-genic inactivating mutation A593V (58) have been reported.Based on our functional characterization of the A593G andA593Vmutations, we can extract two important results relatedto these pathogenic findings. (i) The pathogenic inactivatingmutant A593V is characterized by a decreased expression anddecreased basal activity (not TSH-induced stimulation), whichmight therefore be amain reason for the observedmild congen-ital primary hypothyroidism. (ii) This impeded basal activity islikely caused by altered interaction with the counterpart aminoacid Val-5093.40 in TMH3 (59). From our models, we assumethat contact between the side chains Ala-5935.50 and Val-5093.40 is involved with regulation of the basally active confor-mation. Introduction of a valine at position 5.50 inTMH5 led toa more retained interaction between the two valine side chainsby interlocking and as a consequence of restriction of theTMH5-TMH3 interplay at this particular site. Thus, the capac-ity for release or an adequate flexibility of this interacting resi-due pair, 5.50–3.40, might be important for receptor activityregulation in the TSHR. This is supported by our constitutiveactive mutations A593G and A593P and the pathogenic CAMsA593N (58) and V509A (60). Moreover, this principle findingcorresponds with recent insights from the crystal structure ofan activated �2-adrenergic receptor conformation where posi-tion 5.50 in TMH5 and the region around position 3.40 werefound to be directly related to receptor signaling regulation(60). This insight from the comparison of recent GPCR struc-tures is in accordance with results of studies on the histamineH1 receptor (61).Polar Side-chain Interactions between TMHs 2, 3, 6, and 7

Constrain Inactive Conformation, whereas Hydrophobic Inter-actions between TMHs 5 and 6 Constrain Active Conformation—From the above described and discussed observations, a mainmessage can be extracted: only two mutations in TMH5 arereported to cause slight constitutive TSHR activation. In con-trast, several mutations are reported for TMH5 that lead toreceptor signaling inactivation. Based on these facts as well asthe higher number of direct contacts in the activated comparedwith the inactive TSHR model, we here suggest a dominantly“stabilizing” function of TMH5 for TMH6 in the active state.How is this hypothesis in accordance with and supported byprevious findings for the TSHR?In the TSHR, a methionine is located at position 6.48 in

TMH6 instead of a tryptophan, which is found in around 80%ofthe rhodopsin-like GPCRs (22). This tryptophanmoves slightlyduring activation and stabilizes the active GPCR conformationby forming new interactions with TMH5 (for a review, see Ref.62). It is assumed that in the active state the aromatic trypto-phan interacts with position 5.47 in TMH5. The significance of




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this tryptophan in ligand-induced signal transduction wasshown for rhodopsin (63) and other GPCRs like the adenosinereceptor (64) and 5-HT2 receptor (65). Furthermore, this tryp-tophan is also known to be involved in the maintenance of thebasal state, which has been investigated for the thyrotropin-releasing hormone receptor subtypes 1 and 2 (66). Methionine6.48 in the TSHR (Met-6376.48) might undergo comparablemovement toward TMH5 during activation as assumed for thetryptophan in other GPCRs and interact in the active state witharomatic residues in helices 5 and 6 (Phe-5945.51 and Phe-6346.45). Interestingly, introduction of an aromatic and bulkytryptophan to TSHR at position 6.48 instead of the methioninecannot be tolerated spatially and leads to repulsion by sur-rounding amino acids and hence causes constitutive receptoractivation (46).It is known that conserved hydrophilic polar amino acids in

the TSHR transmembrane region like Asp-6336.44 (TMH6),Asn-6747.49 (TMH7), Asp-4602.50 (TMH2), and Arg-5193.50(TMH3) are mandatory for the signal transduction process(67–70). Arginine 519 at position 3.50 is a key player in manyfamily A GPCRs. Aspartate 4602.50, Asn-6747.49, and Asp-6336.44 interact (68, 70) by hydrophilic interactions and stabi-lize the inactive conformation (Fig. 6C). For these amino acids,several CAMshave been identified bymutagenesis studies or bynaturally occurring mutations.From Details of Microswitches in TSHR to General Intramo-

lecular GPCR Activation Mechanism—Taking this detailedinformation from functional studies and structural insightsinto account, we suggest the following scenario for activa-tion of the TSHR in the transmembrane region. Polar con-tacts between TMHs 2, 3, 6, and 7 preserve the inactivereceptor state. Water molecules might participate in thisinteraction network as suggested for rhodopsin (71), �2-ad-renergic receptor (72), and the MC4R (73). Signal induction(by ligands or mutation) must release such constraints formovement of TMH6 to take place (Figs. 5 and 6). Induced bythis release, the intracellular end of TMH6moves outward asobserved in the active opsin crystal structure. WhereasTMH6 is likely to be involved in stabilization of both theinactive and the active state, TMH5 functions as an “activestate stabilizer” in interplay with TMH6 (Fig. 6C). In contrastto the outward movement of TMH6, the TMH5 undergoes aslight inward movement (Fig. 6A). This bidirectional rear-rangement is locked by specific and mostly non-polar resi-due interactions with the TMH5-TMH6 interface (Fig. 6C),which might also prevent a movement of TMH6 back intothe inactive conformation. In consequence, the TSHR can besubdivided into two main regions: 1) The inactive state isconstrained by a polar core set between TMHs 2, 3, and 7(Fig. 6C, pink half), 2) stabilization of the active state is sup-ported by hydrophobic interfaces between TMH5 andTMH6 (Fig. 6C, green half). Particular amino acids contrib-ute to this process of helical rearrangement, which mightwork sequentially as revealed by insights from studies onrhodopsin (74).To assess whether our data can be generally applied to

family A GPCRs, we statistically analyzed the occurrence ofpolar and non-polar residues in all TMHs in a large set of

family A GPCRs. The amino acid sequences of the trans-membrane helices of 574 family A GPCRs were taken, andthe frequency of polar and non-polar amino acids at eachposition of the transmembrane helices was calculated (Fig.7). Indeed, a set of polar residues in TMHs 1, 2, 3, and 7 areconserved throughout family A and most likely help to sta-bilize the inactive state of the family A GPCRs. Non-polarresidues are conserved in TMHs 3, 5, and 6 and form a non-polar interface that seems to be important for stabilizing theactive state of family A GPCRs. Conservation of these centralpolar core residues and the non-polar interfaces throughoutfamily A suggests their fundamental role in regulating familyA GPCR activity.Taken together, we have described mechanisms of TSHR

activity regulation at the molecular level. Our results togetherwith previous data enabled us to assign particular residues ofthe TMH5-TMH6 interface as interacting pairs with functionalrelevance. Interacting residues showing constitutive activationby mutation indicate determinants for stabilization of the basal(non-active) conformation. Residue pairs showing inactivatingmutations indicate their involvement in forming the active con-formation. We suggest that the inactive TSHR conformation(and that of other family A GPCRs) is constrained by interact-ing polar residues between TMHs 2, 3, and 6, whereas interact-ing hydrophobic residues betweenTMH5andTMH6constrainthe active state. We also suggest that the TMH5 in TSHR doesnot have a proline-induced helical kink and bulge and has adifferent conformation compared with other GPCRs. Adetailed understanding of intramolecular mechanisms forGPCR activity regulation is of vital importance because itshould also reveal molecular knowledge for better understand-ing dysfunctions of GPCRs.

REFERENCES1. Kristiansen, K. (2004) Pharmacol. Ther. 103, 21–802. Wess, J., Han, S. J., Kim, S. K., Jacobson, K. A., and Li, J. H. (2008) Trends

Pharmacol. Sci. 29, 616–6253. Kobilka, B. K., and Deupi, X. (2007) Trends Pharmacol. Sci. 28, 397–4064. Hofmann, K. P., Scheerer, P., Hildebrand, P. W., Choe, H. W., Park, J. H.,

Heck, M., and Ernst, O. P. (2009) Trends Biochem. Sci. 34, 540–5525. Van Eps, N., Oldham, W. M., Hamm, H. E., and Hubbell, W. L. (2006)

Proc. Natl. Acad. Sci. U.S.A. 103, 16194–161996. Oldham,W.M., andHamm,H. E. (2008)Nat. Rev.Mol. Cell Biol. 9, 60–717. Schwartz, T.W., Frimurer, T. M., Holst, B., Rosenkilde, M.M., and Elling,

C. E. (2006) Annu. Rev. Pharmacol. Toxicol. 46, 481–5198. Scheerer, P., Heck, M., Goede, A., Park, J. H., Choe, H. W., Ernst, O. P.,

Hofmann, K. P., and Hildebrand, P.W. (2009) Proc. Natl. Acad. Sci. U.S.A.106, 10660–10665

9. Schertler, G. F. (2008) Nature 453, 292–29310. Ahuja, S., and Smith, S. O. (2009) Trends Pharmacol. Sci. 30, 494–50211. Seifert, R., and Wenzel-Seifert, K. (2002) Naunyn Schmiedebergs Arch.

Pharmacol. 366, 381–41612. Wong, S. K. (2003) Neurosignals 12, 1–1213. Kleinau, G., Jaeschke, H., Worth, C. L., Mueller, S., Gonzalez, J., Paschke,

R., and Krause, G. (2010) PLoS One 5, e974514. Raymond, J. R. (1995) Am. J. Physiol. Renal Physiol. 269, F141–F15815. Rosenbaum, D. M., Rasmussen, S. G., and Kobilka, B. K. (2009) Nature

459, 356–36316. Wenzel-Seifert, K., and Seifert, R. (2000)Mol. Pharmacol. 58, 954–96617. Zurn, A., Zabel, U., Vilardaga, J. P., Schindelin, H., Lohse, M. J., and Hoff-

mann, C. (2009)Mol. Pharmacol. 75, 534–54118. Hanson, M. A., and Stevens, R. C. (2009) Structure 17, 8–14




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

19. Kobilka, B., and Schertler, G. F. (2008) Trends Pharmacol. Sci. 29,79–83

20. Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W., and Ernst, O. P.(2008) Nature 454, 183–187

21. Scheerer, P., Park, J. H., Hildebrand, P. W., Kim, Y. J., Krauss, N., Choe,H. W., Hofmann, K. P., and Ernst, O. P. (2008) Nature 455, 497–502

22. Sum, C. S., Tikhonova, I. G., Costanzi, S., and Gershengorn, M. C. (2009)J. Biol. Chem. 284, 3529–3536

23. Worth, C. L., Kleinau, G., and Krause, G. (2009) PLoS One 4, e701124. Rapoport, B., Chazenbalk, G. D., Jaume, J. C., andMcLachlan, S.M. (1998)

Endocr. Rev. 19, 673–71625. Kleinau, G., Kreuchwig, A., Worth, C. L., and Krause, G. (2010) Hum.

Mutat. 31, E1519-E152526. Cherezov, V., Rosenbaum, D.M., Hanson,M. A., Rasmussen, S. G., Thian,

F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., andStevens, R. C. (2007) Science 318, 1258–1265

27. Jaakola, V. P., Griffith, M. T., Hanson, M. A., Cherezov, V., Chien, E. Y.,Lane, J. R., Ijzerman, A. P., and Stevens, R. C. (2008) Science 322,1211–1217

28. Murakami, M., and Kouyama, T. (2008) Nature 453, 363–36729. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A.,Motoshima, H., Fox,

B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto,M., and Miyano, M. (2000) Science 289, 739–745

30. Rasmussen, S. G., Choi, H. J., Rosenbaum, D. M., Kobilka, T. S., Thian,F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R., Sanishvili, R., Fisch-etti, R. F., Schertler, G. F., Weis, W. I., and Kobilka, B. K. (2007) Nature450, 383–387

31. Salom, D., Lodowski, D. T., Stenkamp, R. E., Le Trong, I., Golczak, M.,Jastrzebska, B., Harris, T., Ballesteros, J. A., and Palczewski, K. (2006) Proc.Natl. Acad. Sci. U.S.A. 103, 16123–16128

32. Shimamura, T., Hiraki, K., Takahashi, N., Hori, T., Ago, H., Masuda, K.,Takio, K., Ishiguro, M., and Miyano, M. (2008) J. Biol. Chem. 283,17753–17756

33. Standfuss, J., Xie, G., Edwards, P. C., Burghammer, M., Oprian, D. D., andSchertler, G. F. (2007) J. Mol. Biol. 372, 1179–1188

34. Warne, T., Serrano-Vega, M. J., Baker, J. G., Moukhametzianov, R., Ed-wards, P. C., Henderson, R., Leslie, A. G., Tate, C. G., and Schertler, G. F.(2008) Nature 454, 486–491

35. Lodowski, D. T., Angel, T. E., and Palczewski, K. (2009) Photochem. Pho-tobiol. 85, 425–430

36. Blois, T. M., and Bowie, J. U. (2009) Protein Sci. 18, 1335–134237. Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25,

366–42838. Krebs, W. G., and Gerstein, M. (2000) Nucleic Acids Res. 28, 1665–167539. Li, W., and Godzik, A. (2006) Bioinformatics 22, 1658–165940. Smith, B. J., Wales, M. R., and Perry, M. J. (1993) Appl. Biochem. Biotech-

nol. 41, 189–21841. Vlaeminck-Guillem, V., Ho, S. C., Rodien, P., Vassart, G., and Costagliola,

S. (2002)Mol. Endocrinol. 16, 736–74642. Mueller, S., Jaeschke, H., and Paschke, R. (2010) Methods Enzymol. 485,

421–43643. Kleinau, G., Brehm, M., Wiedemann, U., Labudde, D., Leser, U., and

Krause, G. (2007)Mol. Endocrinol. 21, 574–58044. Kero, J., Ahmed, K., Wettschureck, N., Tunaru, S., Wintermantel, T.,

Greiner, E., Schutz, G., and Offermanns, S. (2007) J. Clin. Investig. 117,2399–2407

45. Parnot, C.,Miserey-Lenkei, S., Bardin, S., Corvol, P., andClauser, E. (2002)Trends Endocrinol. Metab. 13, 336–343

46. Kleinau, G., Haas, A. K., Neumann, S., Worth, C. L., Hoyer, I., Furkert, J.,Rutz, C., Gershengorn,M. C., Schulein, R., and Krause, G. (2010) FASEB J.24, 2347–2354

47. Schulz, A., Bruns, K., Henklein, P., Krause, G., Schubert, M., Gudermann,T., Wray, V., Schultz, G., and Schoneberg, T. (2000) J. Biol. Chem. 275,37860–37869

48. Fanelli, F., and De Benedetti, P. G. (2005) Chem. Rev. 105, 3297–335149. Schoneberg, T., Schulz, A., and Gudermann, T. (2002) Rev. Physiol.

Biochem. Pharmacol. 144, 143–22750. Persani, L., Calebiro, D., and Bonomi, M. (2007)Nat. Clin. Pract. Endocri-

nol. Metab. 3, 180–19051. Urizar, E., Montanelli, L., Loy, T., Bonomi, M., Swillens, S., Gales, C.,

Bouvier, M., Smits, G., Vassart, G., and Costagliola, S. (2005) EMBO J. 24,1954–1964

52. Guan, R., Feng, X., Wu, X., Zhang, M., Zhang, X., Hebert, T. E., andSegaloff, D. L. (2009) J. Biol. Chem. 284, 7483–7494

53. Guan, R., Wu, X., Feng, X., Zhang, M., Hebert, T. E., and Segaloff, D. L.(2010) Cell. Signal. 22, 247–256

54. Gozu,H., Avsar,M., Bircan, R., Claus,M., Sahin, S., Sezgin,O., Deyneli, O.,Paschke, R., Cirakoglu, B., and Akalin, S. (2005) Thyroid 15, 389–397

55. Castro, I., Lima, L., Seoane, R., and Lado-Abeal, J. (2009) Thyroid 19,645–649

56. Biebermann, H., Winkler, F., and Kleinau, G. (2010) Front. Biosci. 15,913–933

57. Sykiotis, G. P., Neumann, S., Georgopoulos, N. A., Sgourou, A., Papachat-zopoulou, A., Markou, K. B., Kyriazopoulou, V., Paschke, R., Vagenakis,A. G., and Papavassiliou, A. G. (2003) Biochem. Biophys. Res. Commun.301, 1051–1056

58. Fricke-Otto, S., Pfarr, N.,Muhlenberg, R., and Pohlenz, J. (2005) Exp. Clin.Endocrinol. Diabetes 113, 582–585

59. Karges, B., Krause, G., Homoki, J., Debatin, K.M., de Roux, N., andKarges,W. (2005) J. Endocrinol. 186, 377–385

60. Rasmussen, S.G., Choi,H. J., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S.,Devree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A.,Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J.,Weis,W. I., and Kobilka, B. K. (2011) Nature 469, 175–180

61. Sansuk, K., Deupi, X., Torrecillas, I. R., Jongejan, A., Nijmeijer, S., Bakker,R. A., Pardo, L., and Leurs, R. (2011)Mol. Pharmacol. 79, 262–269

62. Smit, M. J., Vischer, H. F., Bakker, R. A., Jongejan, A., Timmerman, H.,Pardo, L., and Leurs, R. (2007) Annu. Rev. Pharmacol. Toxicol. 47, 53–87

63. Tikhonova, I. G., Best, R. B., Engel, S., Gershengorn, M. C., Hummer, G.,and Costanzi, S. (2008) J. Am. Chem. Soc. 130, 10141–10149

64. Hallmen, C., and Wiese, M. (2006) J. Comput. Aided Mol. Des. 20,673–684

65. Rashid, M., Manivet, P., Nishio, H., Pratuangdejkul, J., Rajab, M., Ishiguro,M., Launay, J. M., and Nagatomo, T. (2003) Life Sci. 73, 193–207

66. Deflorian, F., Engel, S., Colson, A. O., Raaka, B. M., Gershengorn, M. C.,and Costanzi, S. (2008) Proteins 71, 783–794

67. Tsunekawa, K., Onigata, K., Morimura, T., Kasahara, T., Nishiyama, S.,Kamoda, T., Mori, M., Morikawa, A., and Murakami, M. (2006) Thyroid16, 471–479

68. Neumann, S., Krause, G., Chey, S., and Paschke, R. (2001)Mol. Endocrinol.15, 1294–1305

69. Claeysen, S., Govaerts, C., Lefort, A., Van Sande, J., Costagliola, S., Pardo,L., and Vassart, G. (2002) FEBS Lett. 517, 195–200

70. Urizar, E., Claeysen, S., Deupí, X., Govaerts, C., Costagliola, S., Vassart, G.,and Pardo, L. (2005) J. Biol. Chem. 280, 17135–17141

71. Angel, T. E., Gupta, S., Jastrzebska, B., Palczewski, K., and Chance, M. R.(2009) Proc. Natl. Acad. Sci. U.S.A. 106, 14367–14372

72. Nygaard, R., Valentin-Hansen, L., Mokrosinski, J., Frimurer, T. M., andSchwartz, T. W. (2010) J. Biol. Chem. 285, 19625–19636

73. Tarnow, P., Rediger, A., Brumm, H., Ambrugger, P., Rettenbacher, E.,Widhalm, K., Hinney, A., Kleinau, G., Schaefer,M., Hebebrand, J., Krause,G., Gruters, A., and Biebermann, H. (2008) Obes. Facts 1, 155–162

74. Ye, S., Zaitseva, E., Caltabiano,G., Schertler, G. F., Sakmar, T. P., Deupi, X.,and Vogel, R. (2010) Nature 464, 1386–1389

75. Neumann, S., Huang, W., Titus, S., Krause, G., Kleinau, G., Alberobello,A. T., Zheng, W., Southall, N. T., Inglese, J., Austin, C. P., Celi, F. S.,Gavrilova, O., Thomas, C. J., Raaka, B. M., and Gershengorn, M. C. (2009)Proc. Natl. Acad. Sci. U.S.A. 106, 12471–12476

76. Kosugi, S., and Mori, T. (1996) Biochem. Biophys. Res. Commun. 222,713–717

77. Biebermann, H., Schoneberg, T., Schulz, A., Krause, G., Gruters, A.,Schultz, G., and Gudermann, T. (1998) FASEB J. 12, 1461–1471

78. Claus, M., Neumann, S., Kleinau, G., Krause, G., and Paschke, R. (2006) J.Mol. Med. 84, 943–954

79. Cordella, A., DeMarco, A., Calebiro, D., de Filippis, T., Radetti, G.,Weber,G., Vigone,M.C., Cappa,M., Sartorio, A., Busnelli,M., Bonomi,M., Chini,




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

B., Beck-Peccoz, P., and Persani, L. (2007) Endocr. Abstr. 14, OC1–180. Parma, J., Duprez, L., Van Sande, J., Cochaux, P., Gervy, C., Mockel, J.,

Dumont, J., and Vassart, G. (1993) Nature 365, 649–65181. Ringkananont, U., Van Durme, J., Montanelli, L., Ugrasbul, F., Yu, Y.M.,Weiss,

R. E., Refetoff, S., andGrasberger,H. (2006)Mol. Endocrinol.20, 893–90382. Gozu, H. I., Bircan, R., Krohn, K., Muller, S., Vural, S., Gezen, C., Sargin,

H., Yavuzer, D., Sargin, M., Cirakoglu, B., and Paschke, R. (2006) Eur. J.Endocrinol. 155, 535–545

83. Tonacchera,M., Chiovato, L., Pinchera, A., Agretti, P., Fiore, E., Cetani, F.,Rocchi, R., Viacava, P., Miccoli, P., and Vitti, P. (1998) J. Clin. Endocrinol.Metab. 83, 492–498

84. Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203–209




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Furkert, Catherine L. Worth, Gerd Krause and Ralf SchüleinGunnar Kleinau, Inna Hoyer, Annika Kreuchwig, Ann-Karin Haas, Claudia Rutz, Jens

G-protein-coupled Receptors (GPCRs)Thyrotropin Receptor to General Aspects of Signal Transduction in Family A

From Molecular Details of the Interplay between Transmembrane Helices of the

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