The role of proline residues in the structure and function ofhuman MT2 melatonin receptor

Introduction

Melatonin functions as an essential regulator of variousphysiological processes in all vertebrate species, including

entrainment of circadian and seasonal rhythms by the light-dark cycle [1], tumor suppression [2] and modulation of thefree radical levels [3]. In mammals, the two G protein-

coupled melatonin receptors (GPCR) mediate some of thehormone�s actions: MT1 [4] and MT2 [5]. Both receptorsubtypes are expressed in a variety of tissues including

specific brain structures and peripheral organs, and theiractivation modulates a wide range of intracellular messen-gers, e.g. cAMP and [Ca2+]i [1].

As with other GPCRs, melatonin receptors consist ofseven hydrophobic transmembrane (TM) domains ofa-helical structure, joined by intracellular and extracellularloops [6]. TM domains of most GPCRs contain a set of

highly conserved proline residues that presumably playimportant structural and functional roles [6]. Structurally,the proline residue can disturb an ideal geometry of TM

a-helix both by causing steric conflicts with the precedingamino acid and the lack of a backbone H-bond. The firsteffect is a consequence of the restricted torsion angle for the

N–Ca bond of proline residue that allows only a limitednumber of conformations and thus destabilizes protein

secondary structure [7]. The second effect results from thefact that the proline imino group lacks the hydrogen atom,and thus cannot form a hydrogen bond between NH and

CO groups as other amino acids [7]. Therefore, on a level ofgeneral helix geometry, the presence of proline residues can,depending on local environment, introduce highly flexible

kinks and/or twists that are considered as key dynamicelements in conformational changes linked to receptoractivation [8]. Occurrence of helix-destabilizing prolineswithin TM helices seems to be important also for proper

folding of membrane proteins by disfavoring the formationof misfolded structure [9].TM domains of the human MT2 melatonin receptor

(hMT2R) contain seven proline residues: P41 (P1.33according to the numbering by Ballesteros et al. [10]), P93(P2.57), P95 (P2.59), P158 (P4.40), P174 (P4.59), P212

(P5.50) and P266 (P6.50) (Fig. 1). Proline residue atposition 1.33 (P41 in hMT2R) is present only in the smallnumber of rhodopsin-like GPCRs (rGPCRs), including

the MT2 subtype of melatonin receptor (MT1 subtypeexpresses serine at this position), melanocortin receptor 1and several olfactory receptors. Proline P2.57 (P93 inhMT2R) is absolutely specific for all melatonin receptors,

while proline at position 2.59 (P95 in hMT2R) can be foundin many rGPCRs but not in rhodopsins. Similarly as in the

Abstract: Melatonin functions as an essential regulator of various

physiological processes in all vertebrate species. In mammals, two G protein-

coupled melatonin receptors (GPCR) mediate some melatonin�s actions:MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a

set of highly conserved proline residues that presumably play important

structural and functional roles. As TM segments of MT2 receptor display

several interesting differences in expression of specific proline residues

compared to other rhodopsin-like receptors (rGPCRs), we investigated the

role of proline residues in the structure and function of this receptor. All

prolines in TM segments of MT2 receptor were individually replaced with

alanine and/or glycine. In addition, the unusual NAxxY motif located in

TM7 was mutated to generate highly conserved NPxxY motif found in the

majority of rGPCR proteins. Following transient expression in CHO-K1

cells, binding properties of the mutant receptors and their ability to

transduce signals were analyzed using 125I-mel- and [35S]GTPcS-bindingassays, respectively. The impact of the performed mutations on the receptor

structure was assessed by molecular dynamic simulations of MT2 receptors

embedded in the fully hydrated phospholipid bilayer. Our results indicate

that residues P174, P212 and P266 are important for the ligand binding and/

or signaling of the human MT2 receptor. We also show that changes within

the unusual NAxxY sequence in the TM7 (mutations A305P and A305V)

produce defective MT2 receptors indicating an important role of this motif in

the function of melatonin receptors.

Petr Mazna1, Lenka Grycova1,Ales Balik1, Hana Zemkova1,Eliska Friedlova1, VeronikaObsilova1, Tomas Obsil1,2 andJan Teisinger1

1Institute of Physiology, Academy of Sciences

of the Czech Republic, Prague, Czech

Republic; 2Department of Physical and

Macromolecular Chemistry, Faculty of

Science, Charles University, Prague, Czech

Republic

Key words: G protein-coupled melatonin

receptors, molecular dynamics, MT2

melatonin receptor, proline

Address reprint requests to Jan Teisinger,

Institute of Physiology, Academy of Sciences

of the Czech Republic, 14220 Prague, Czech

Republic.

E-mail: teisingr@biomed.cas.cz

Received January 18, 2008;

accepted April 16, 2008.

J. Pineal Res. 2008Doi:10.1111/j.1600-079X.2008.00598.x

� 2008 The AuthorsJournal compilation � 2008 Blackwell Munksgaard

Journal of Pineal Research

1

case of P1.33, proline residue at position 4.40 (P158 in

hMT2R) is present only in the MT2 subtype and someolfactory receptors. Proline residues at positions 4.59 and5.50 (P174 and P212 in hMT2R) are conserved among 68%

and 85% of rGPCRs, respectively, whereas proline P6.50(P266 in hMT2R) is absolutely conserved. Interestingly,TM7 of all melatonin receptors lacks proline P7.50 that

forms a part of one of the most highly conserved motifs inrGPCRs, the N/DPxxY sequence (98% conservationamong all rGPCRs). This proline residue is replaced by

alanine in melatonin receptors.A large number of mutagenesis studies of rGPCRs

revealed the importance of proline residues for receptorexpression and function with particularly severe effects

obtained after mutation of conserved prolines in TM4–TM7 [11–17]. For example, in the case of the m3 muscarinicreceptor, mutation of P6.50 (TM6) impaired both receptor

expression and agonist binding while mutation of conservedP7.50 within NPxxY motif in TM7 of the m3 receptoraffected receptor activation [12]. In general, many proline

residues are highly conserved in GPCR family but thepresence of other prolines can vary significantly and mayreflect some functional and pharmacological differencesamong these receptors [18].

As TM segments of hMT2R receptor display severalinteresting differences in expression of specific prolineresidues compared to other rGPCRs, we investigated the

role of prolines in the structure and function of thisreceptor. To accomplish that, all TM proline residues inhMT2R were individually replaced with alanine and/or

glycine. In addition, the unusual NAxxY motif located in

TM7 was mutated to generate highly conserved NPxxYmotif found in the majority of rGPCR proteins. Followingtransient expression in CHO-K1 cells, binding properties of

the mutant receptors and their ability to transduce signalwere analyzed using 125I-mel- and [35S]GTPcS-bindingassays, respectively. The impact of performed mutations

on the receptor structure was assessed by moleculardynamic (MD) simulations of hMT2R embedded in thefully hydrated phospholipid bilayer. Our results indicate

that residues P174, P212 and P266 are important for theligand binding and/or signaling of hMT2R. We also showthat changes within unusual NAxxY sequence in the TM7of hMT2R (mutations A305P and A305V) produce defec-

tive receptors, indicating an important role of this motif inthe function of melatonin receptors.

Materials and methods

Construction of FLAG tagged hMT2 receptor andgeneration of receptor mutants

Subcloning of hMT2 receptor-coding cDNA (gift of DrS. M. Reppert) into pFLAG-CMV-2 plasmid (Sigma, St

Louis, MO, USA) to produce N-terminally FLAG-taggedhMT2 receptor was described previously [19]. Set of pointmutants (P41A, P93A, P95A, P158A, P174A, P174G,

P212A, P212G, P266A, P266G, A305P and A305V) wasgenerated by standard PCR-based mutagenesis (Quik-Change kit; Stratagene, La Jolla, CA, USA) using the

Fig. 1. Schematic topology of humanMT2 melatonin receptor showing relativeposition of studied residues. ECL, extra-cellular loop; ICL, intracellular loop; TM,transmembrane domain; S-S, disulphidebridge between C113 and C190. Aminoacids are given in one letter code.

Mazna et al.

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FLAG-hMT2R cDNA as a template. Oligonucleotidesintroducing specific point mutations were synthesized byVBC-Biotech (Vienna, Austria). The presence of the

mutations and the integrity of the rest of the gene wereconfirmed by automated DNA sequencing (ABI Prism,Foster City, CA, USA).

Cell culture, transfection and membrane preparation

CHO-K1 (Chinese hamster ovary) cells (ATCC) were

grown as monolayers in Ham�s F12K medium (Biochrom,Berlin, Germany) supplemented with 2 mm l-glutamine,10% (v/v) fetal bovine serum (PAA Laboratories, Pasching,

Austria), penicillin (50 U/mL) and streptomycin (50lg/mL) in 95% O2/5% CO2 at 37�C. On the day beforetransfection, the cells were trypsinized and seeded onto100-mm Petri dishes (8–10 · 106/dish). After 24 hr, the cells

were transfected with plasmid DNA (6–24 lg/dish) encod-ing the wild-type (WT) or mutant receptors using Lipofecta-mine2000 transfection reagent (Invitrogen, Carlsbad, CA,

USA) following manufacturers instructions. After addi-tional 36–48 hr of incubation, the cells were washed withPBS solution (pH 7.4) containing 10 mm EDTA, harvested

by gentle scraping, transferred to microtube and pelleted bycentrifugation (1600 · g, 6 min, 4�C). The cell pellet wasresuspended in 50 mm Tris–HCl (pH 7.4) containing 10 mm

EDTA and sonicated using an ultrasonic homogenizer(4710 Series; Cole-Parmer, Vernon Hills, IL, USA). Result-ing homogenate was centrifuged at 28,000 · g for 40 min at4�C and the pellet was either stored at )80�C or further

manipulated depending on the assay type (see below).

125I-mel-binding assays

For the analysis of 2-[125I]Iodomelatonin (125I-mel) bind-ing, the pellet obtained as described above was resuspended

in a binding buffer (50 mm Tris–HCl, pH 7.4) andincubated with 2–1250 pm

125I-mel (Amersham, Piscata-way, NJ, USA; 2000 Ci/mmol) for 1.5 hr at 37�C in a finalassay volume of 100 lL. Nonspecific binding was assessed

in the presence of 1 lm melatonin (Sigma) and typically0.5–5 lg of membrane preparation per tube was used forexperiment as determined by the method of Bradford [20].

Incubation was terminated by the addition of 3 · 3 mL ofice-cold binding buffer and bound and free ligands wereseparated by immediate vacuum filtration through What-

man GF/B filters (GE Healthcare, Buckinghamshire, UK).All samples were determined in triplicates. Radioactivitywas measured by gamma counter (Packard Cobra, GMI,

Ramsey, MN, USA). Binding data were analyzed bynonlinear regression analysis using the PRISM 4.0 program(GraphPad Software, La Jolla, CA, USA).

[35S]GTPcS-binding assays

For the analysis of [35S]GTPcS binding, the pellet obtained

as described above was resuspended in GTP-incubationmedium (50 mm Tris–HCl, 100 mm NaCl, 3 mm MgCl2,1 mm DTT, pH 7.4) and centrifuged at 28,000 · g for

40 min at 4�C. Resulting pellet was resuspended in GTP-incubation medium and diluted to give a membrane

concentration of 0.5–1 mg/mL. Triplicates of membranesamples were then assayed at 30�C in 4-mL polystyrenetubes in a final assay volume of 200 lL. The reaction was

initiated by preincubation of membranes (10–15 lg/tube)with GDP (Sigma; final concentration 10 lm) lasting 5 min,followed by the addition of agonist (melatonin or I-mel).After 35 min with agonist, [35S]GTPcS (Amersham) was

added to reaction mixture to give a final concentration of250–500 pm/tube and reaction was allowed to continue forfurther 30 min. Basal binding was measured in the absence

of agonist, and nonspecific binding was assessed usingnonradiolabeled GTPcS (Sigma; 50 lm). Incubation wasterminated by the addition of 3 · 3 mL of ice-cold GTP-

incubation medium followed by immediate vacuum filtra-tion through Whatman GF/B filters. Radioactivity in filterswas determined by liquid scintillation using Rotiszintcocktail (Roth, Karlsruhe, Germany) and Packard Tri-

Carb counter (GMI, Ramsey, MN, USA). Maximalresponses (Emax) elicited by melatonin and I-Mel (Tocris,Bristol, UK) (both at 1 lm) were expressed relative to basal

[35S]GTPcS binding in the absence of any agonist, whichwas set to be 100%. To determine melatonin and I-melEC50, both drugs were applied at concentrations from

0.1 pm to 1 lm. Data from [35S]GTPcS binding wereanalyzed with PRISM 4.0 program using a four-parameterlogistic equation.

Immunocytochemistry and confocal microscopy

For immunological studies, the cells were trypsinized 24 hr

after transfection, reseeded onto poly-l-lysine-treated cov-erslips (diameter 15 mm) and grown for additional 24 hr.Cells were rinsed three times in PBS (pH 7.4) and then fixed

for 15 min in 2% paraformaldehyde in PBS at roomtemperature (RT). To block nonspecific binding, fixed cellswere washed three times for 5 min with PBS and then

incubated for 15 min at RT in antibody diluent (3% horseserum in PBS with 0.05% Tween 20). Cells were thenincubated with anti-FLAG M2 monoclonal antibody(Sigma; 1:200) for 1 hr at 37�C. The cells were washed

three times for 5 min with PBS and incubated with horseanti-mouse fluorescein conjugated antibody (Chemicon-Millipore, Temecula, CA, USA) for 1 hr at RT. Following

three successive washes with PBS, the cells were mounted inonto glass slides using Vectashield medium (Vector Labs,Burlingame, CA, USA). Nuclei were stained with DRAQ 5

(Alexis, San Diego, CA, USA). Slides were viewed usingLeica TCS 1-B microscope (Leica, Wetzlar, Germany).

Receptor modeling and MD simulations

Homology model of hMT2R was build using the programModeller 6.0 [21] with the crystal structure of bovine

rhodopsin (PDB access code 1U19) [22] as a template. Thesequence alignment used for the homology modeling proce-dure was calculated using CLUSTALW program [23] and

contained the conserved disulfide bridge between the C113 inTM3 (C3.25) and C190 in ECL2 [22, 24, 25]. Mutations ofhMT2R were generated using DeepView/Swiss-PdbViewer

v3.7 [26]. The ligand docking started with manual docking ofthe melatonin molecule into the predicted binding site of

Proline residues in human MT2 function

3

hMT2R on the basis of homology with bovine rhodopsin.The melatonin topology files were prepared using thePRODRG server [27]. Automated docking of melatonin

molecule into the predicted hMT2R ligand-binding site usingthe AutoDock 3.0 program [28] was used to refine thestructure of hMT2R–melatonin complex.The hMT2R model was then energy minimized with the

frozen protein backbone using GROMACS package withthe parameter set ffgmx [29]. The quality of model waschecked by means of PROCHECK 3.5.4 [30]. The final

model of hMT2R (without any water molecules) was thenembedded into the equilibrated hydrated palmitoyloleoyl-phosphatidylethanolamine (POPE) bilayer [31] and posi-

tioned across the bilayer, while seeking to match thehydrophobic protein segments with the layer formed by thelipid hydrocarbon tails. Lipids overlapping with the proteincomplex were deleted. The receptor was oriented in a way

that allows TM4 to be approximately parallel, helix H8almost perpendicular to the membrane normal and the endsof TM segments located approximately at the water–lipid

interface [22, 24]. To make sure that the cytoplasmic andextracellular loops do not interact, the width of solventlayers on both sides of lipid bilayer was increased and

additional SPC water molecules were added [32]. Chlorideions were added at positions with the most favorableelectrostatic potential using the program GENION (GRO-

MACS package) to compensate for the net positive chargeof the protein [29]. The resulting system comprised of 73791atoms (in the case of hMT2RWT) and contained onemolecule of hMT2R, 255 molecules of POPE, 19,006

molecules of water, and 13 Cl) ions. The obtained systemwas allowed to equilibrate for 3 ns with positionalrestraints on the protein atoms. All MD simulations were

carried out with the GROMACS v3.1.2 package [29]. Allcovalent bond lengths were constrained using LINCSmethod [33] allowing an integration step of 2 fs. Protein,

lipids, solvent and counter-ions were independently weaklycoupled to a reference temperature bath [34] with acoupling constant of sT 0.1 ps. The pressure was controlledwith anisotropic pressure coupling with coupling constant

sP 10 ps. Electrostatic interactions were calculated with theparticle-mesh Ewald method [35]. The cut-off for van derWaals interactions was set to 9 A. The unrestrained

simulation length was 4 ns. The first nanosecond wasconsidered as an additional equilibration, and all analyseswere performed over the remaining part of simulated

trajectory. The same procedure was used for both WT andall studied hMT2R mutants (Table 1). The structure ofhMT2R was found to be stable in all simulations. The root-mean-square deviation (RMSD) of hMT2RWT from the

starting structure (homology model) after 4 ns of unre-strained MD simulation for all backbone heavy atoms andbackbone heavy atoms of TM segments were 3.61 and

1.93 A, respectively. Trajectories were analyzed using theGROMACS package. Cluster analysis was performed usingthe GROMOS method [36]. Representative structure was

calculated as an energy-minimized central structure of themost populated cluster obtained using the cluster analysiswith RMSD cut-off of 1.0 A.

Numbering of GPCRs

In addition to standard amino-acid numbering based on

their position in the receptor sequence, the residues in TMdomains are labeled using scheme proposed by Ballesterosand Weinstein [10]. This scheme designates the most highly

Table 1. Expression and pharmacological properties of hMT2 receptors transiently expressed in CHO-K1 cells

ReceptorcDNA per107 of cells

Comparedwith

Bmax

(fmol/mg) KD (pm)

EC50 (pm) Emax % (over basal)

Melatonin I-mel Melatonin I-mel

WThigh 24 – 7770 ± 920 145 ± 14 570 ± 80 130 ± 40 216 ± 7 220 ± 20WTmed 12 – 5147 ± 257 113 ± 32 770 ± 30 230 ± 130 178 ± 8 184 ± 9WTlow 6 – 1661 ± 330 104 ± 14 350 ± 260 240 ± 170 134 ± 6 138 ± 13P41A 24 WThigh 9135 ± 635 173 ± 8 350 ± 250 400 ± 310 195 ± 15 208 ± 23P93A 24 WThigh 8400 ± 609 164 ± 22 810 ± 400 450 ± 360 194 ± 9 211 ± 15P95A 24 WThigh 7330 ± 521 136 ± 16 610 ± 100 900 ± 500 210 ± 11 213 ± 24P158A 24 WTlow 1496 ± 55 93 ± 18 750 ± 250 490 ± 41 130 ± 18 140 ± 15P174A 24 – No binding – – – – –P174G 24 – No binding – – – – –P212A 24 WTmed 4750 ± 550 125 ± 17 – – 104 ± 7* 102 ± 10*P212G 24 WTlow 1555 ± 755 131 ± 17 4750 ± 2750* 4160 ± 1170* 138 ± 14 142 ± 18P266A 24 – No binding – – – – –P266G 24 – No binding – – – – –A305P 24 – No binding – – – – –A305V 24 WTmed 5075 ± 875 99 ± 22 6750 ± 750* 5130 ± 1870* 138 ± 11* 135 ± 13*

125I-mel- and [35S]GTPcS-binding experiments on membrane isolates of CHO-K1 cells transiently expressing FLAG-tagged hMT2RWT andpoint-mutated receptors were performed as described under �Materials and methods�. Emax values in [35S]GTPcS binding assays weredetermined by addition of melatonin and I-mel at a concentration of 1 lm. In EC50 experiments, both drugs were applied at concentrationsfrom 0.1 pm to 1 lm. Data obtained for membrane preparations of mutant receptors were always compared with preparation expressingWT receptors at similar densities. Values represent means ±S.E.M. of three to five experiments per receptor construct performed on at leasttwo batches of transfected cells. *Significantly different (P < 0.05, Student�s t-test) from value obtained for hMT2RWT-matching expressionof respective mutant receptor (see also section �Results and discussion�). Receptors studied by MD simulation are marked in bold. Bmax, themaximum binding capacity; KD, equilibrium dissociation constant; EC50, drug concentration producing half-maximal response; Emax,maximal response.

Mazna et al.

4

conserved residue in each helix as X.50, where X representsthe helix number.

Results and discussion

To study functional roles of proline residues in the hMT2Rmolecule, cDNA sequences coding hMT2RWT and its

mutants with N-terminally fused FLAG epitope weretransiently expressed in CHO-K1 cells. After 48 hr ofincubation, cell membranes expressing receptor constructs

were isolated and assayed for ability to bind 125I-mel and toshow agonist-promoted [35S]GTPcS binding. To assess thepotential impact of studied mutations on hMT2R structure,

the unconstrained MD simulations of hMT2R embedded ina fully hydrated POPE lipid bilayer were performed.

Initial experiments with non-transfected CHO-K1 cellsshowed no specific binding activity but transfection of the

cells with FLAG epitope-containing hMT2RWT cDNAresulted in high levels of 125I-mel binding with pharmaco-logical characteristics similar to previous reports [37]

(Table 1, Fig. 2). The level of hMT2RWT expression inCHO-K1 cells was dependent on the cDNA amount usedin transfection reaction, and reached top Bmax val-

ues � 8 pmol/mg of protein when 24 lg of hMT2RWT

cDNA was used per 1 · 107 cells. Samples of cell mem-

branes isolated from CHO-K1 cells expressing hMT2RWT

receptor in these amounts were denoted as hMT2RWThigh.Addition of melatonin to hMT2RWThigh membranes stim-

ulated [35S]GTPcS binding in a dose-dependent manner(EC50 of 570 ± 80 pm) reaching 216 ± 7% increase (Emax)over the basal values (basal = 100%) at the maximumeffective concentration of 1 lm. Bmax values similar to

hMT2RWT (7–9 pmol/mg of protein) were obtained formembranes expressing hMT2RP41A, hMT2RP93A andhMT2RP95A mutants, whereas hMT2RP212A and

hMT2RA305V mutants showed medium Bmax (�5 pmol/mg), and hMT2RP158A and hMT2RP212G mutants relativelylow Bmax values (�1.5 pmol/mg) under the same transfec-

tion conditions (=24 lg of hMT2R cDNA/107 cells). Inorder to allow the comparison of functional responsesbetween hMT2RWT and its mutant versions expressed atlower levels (due to the reduced expression of receptors per

cell), we prepared isolates of cell membranes expressinghMT2RWT in medium and low quantities termedhMT2RWTmed (Bmax � 5 pmol/mg; 12 lg of WT cDNA/

107 cells) and hMT2RWTlow (Bmax � 1.5 pmol/mg; 6 lg ofWT cDNA/107 cells), respectively, by decreasing theamount of hMT2RWT cDNA in transfection reactions. In

these preparations, EC50 values of melatonin-induced[35S]GTPcS binding did not significantly differ from thoseobserved for hMT2RWThigh membranes (770 ± 30 pm for

hMT2RWTmed and 350 ± 260 for hMT2RWTlow). How-ever, the reduced receptor expression in the case ofhMT2RWTmed and hMT2RWTlow membranes decreasedmelatonin-stimulated Emax of [35S]GTPcS binding

to 178 ± 8% and 134 ± 6% over basal, respectively(Table 1). Therefore, data obtained from the analysis ofmembrane preparations of mutant receptors were always

compared with the preparations expressing hMT2RWT

receptors at similar density: hMT2RP41A, hMT2RP93A

and hMT2RP95A mutants with hMT2RWThigh;

hMT2RP212A and hMT2RA305V with hMT2RWTmed; andhMT2RP158A and hMT2RP212G with hMT2RWTlow prepa-rations. EC50 and Emax values of [35S]GTPcS binding

obtained after the incubation of hMT2RWThigh,hMT2RWTmed and hMT2RWTlow membranes with I-melwere similar to those obtained with melatonin and aresummarized in Table 1.

Mutant receptors displaying no detectable radioligandbinding (P174A, P174G, P266A, P266G and A305P) weresubjected to immunocytochemical analysis using specific

anti-FLAG monoclonal antibody. Examination underconfocal microscope (Fig. 3) revealed that all these recep-tors were localized in the cell membrane and produced

fluorescence signal comparable to hMT2RWT (see alsobelow).To assess potential structural effects of the studied

mutations, we built models of unliganded hMT2RWT and

its selected mutated versions as well as a model ofhMT2RWT with bound melatonin (hMT2RWT+MEL) allembedded in a realistic explicit membrane environment.

Initial model of hMT2RWT was generated using the crystalstructure of bovine rhodopsin as a template [22]. ResultinghMT2RWT model was then inserted into the fully hydrated

and pre-equilibrated phospholipid bilayer [31]. After theinitial energy minimization and 3-ns-long restrained MD

Fig. 2. Melatonin-induced stimulation of [35S]GTPcS binding inhMT2RWT and P212G and A305V mutants. Representativeresponses obtained after incubation of membrane isolates fromCHO-K1 cells expressing hMT2 receptors (WThigh, WTmed andWTlow membranes) or hMT2RP212G and hMT2RA305V mutantswith melatonin (0.1 pm to 1 lm) are shown. Data are expressed asa percentage of basal stimulation of [35S]GTPcS binding (=100%).Each data set represents one of three to five similar experimentswith every point determined in triplicate. Vertical lines indicate thestandard error of mean. The fitted curve through each data set wasobtained by fitting the data to a one-site sigmoidal model usingnonlinear regression analysis (PRISM 4.0 program). Curves of WTreceptors are shown as dotted lines.

Proline residues in human MT2 function

5

Fig. 3. Cellular localization of hMT2RWT

and its mutant versions where completeloss of specific binding was observed.Nonpermeabilized CHO-K1 cells wereimmunostained with an anti-FLAG M2monoclonal antibody and a horse anti-mouse fluorescein-conjugated antibody(green) and viewed using Leica TCS 1-Bconfocal microscope. Nuclei were stainedwith dye DRAQ 5 (red). No specific flu-orescence signal was detected in non-transfected cells as well as in the CHO-K1cells transfected with an empty pFLAG-CMV-2 vector (not shown). Scale barcorresponds to 20 lm (WT, P174A andP174G) or to 10 lm (P266A, P266G andA305P).

Fig. 4. (A) Side-view of the simulation box. Phospholipids and water are shown in orange and blue, respectively; embedded hMT2R proteinis depicted in green. (B) The superimposition of representative conformation of hMT2RWT with the crystal structure of bovine rhodopsin.Main-chain Ca atom RMSD between the representative conformation of hMT2RWT and the crystal structure of bovine rhodopsin (onlyTM regions included) was 2.17 A using 163 Ca atoms. The overall position of all TM segments of hMT2RWT remained relatively unchangedcompared to rhodopsin, but significant alterations were observed in certain parts of TM2, TM5, TM7 and in the orientation of helix 8(marked by red arrows). (C) Time evolution of root-mean-square deviations (RMSDs) of Ca atoms during MD simulation of hMT2RWT

model. Every frame from the trajectory was first aligned with the starting hMT2RWT model with a least squares fit to the startingconformation, and the RMSD was calculated over either the entire hMT2RWT (black) or the TM segments only (gray). (D) Comparison ofthe positional root-mean-square fluctuations of Ca atoms of hMT2RWT and the crystallographic B-factors of bovine rhodopsin crystalstructure (PDB code 1U19) [24]. Black rectangles show the position of TM segments of hMT2R. (A) and (B) were generated using PyMolv0.99 (http://www.pymol.org).

Mazna et al.

6

simulation (to relax lipid molecules in close vicinity of thereceptor), the unrestrained 4-ns-long production trajectorywas calculated. Fig. 4A shows the side view of the

simulation box with the position of hMT2RWT moleculewithin the phospholipid bilayer. The time course of RMSDof backbone heavy atoms of hMT2RWT (both wholereceptor and TM segments only) is shown in Fig. 4C. The

hMT2RWT structure relaxed quickly from its startingconformation and remained stable throughout the simu-lated time. While the secondary structure of the a-helixbundle was well preserved, the regions of both the intra-and extracellular loops exhibited significant flexibility. Thefirst nanosecond of a simulation was always considered as

an additional equilibration, and all analyses were per-formed over the remaining part of simulated trajectory (1–4 ns). The comparison of the positional fluctuations ofhMT2RWT Ca atoms and crystallographic B-factors of

rhodopsin crystal structure (PDB access code 1U19) isgiven in Fig. 4D. Although the magnitude of the fluctua-tions from X-ray structure and MD simulation were

different, the overall fluctuation pattern was similar. Rep-resentative structure of hMT2RWT was calculated as acentral conformation of the most populated cluster

obtained using a cluster analysis of simulated trajectorywith a RMSD cut-off of 1 A. The comparison of represen-tative conformations of unliganded hMT2RWT with mela-

tonin-bound form (hMT2RWT+MEL) revealed minorchanges in the putative ligand-binding site but no signifi-cant changes in overall receptor structure (see below) These

two structures (only TM regions included) can be super-imposed with an RMSD of 1.57 A over 163 Ca positions.The superimposition of representative conformation of

hMT2RWT with the crystal structure of bovine rhodopsin isshown in Fig. 4B. Main-chain Ca atom RMSD between therepresentative conformation of hMT2RWT and the rho-dopsin molecule (only TM regions included) was 2.17 A

using 163 Ca atoms. The two structures significantlydiffered mainly in the conformation of flexible intra- andextracellular loops. The overall position of all TM segments

of hMT2RWT remained relatively unchanged compared torhodopsin, but significant alterations were observed incertain parts of TM2, TM5, TM7 and in the orientation of

helix 8 (red arrows in Fig. 4B). Our MD-based model ofhMT2RWT showed helical distortion in TM2 regionsurrounding residues P93 (P2.57) and P95 (P2.59) thatwere not observed in the rhodopsin structure. This could be

due to the presence of two prolines that are absent in thecorresponding positions of rhodopsin TM2 (rhodopsincontains in this region flexible GGxTT motiv) [22, 24, 38].

In the rhodopsin molecule, the region around residue H5.46(H208 in hMT2R) forms a bulge and local unwinding thatdistorts the arrangement of TM5 [22, 24, 38]. Histidine

H5.46 was shown to be linked via an ionic interaction ora strong H-bond to the negatively charged E122 (3.37)in TM3, and such interaction can perturb the back-

bone conformation in the manner observed in thecrystal structure [38, 39]. In our model of hMT2RWT,the corresponding region of TM5 displays similar but

Fig. 5. Effects of A305P and A305V mutations on helicity of hMT2R TM7 segment. The rhodopsin crystal structure (shown in orange)exhibits irregular helicity between residues 295–301 [22], whereas the corresponding segment of hMT2RWT model (in green) shows regulara-helical structure. Our simulations suggest that TM7 of hMT2RA305P (in red) loses its helical arrangement between residues Y298 and P305.Minor changes in the helicity of TM7 were also observed in the simulation of the receptor containing mutation A305V (in blue). Changes inhelical arrangement of TM7 predicted by MD simulations are in agreement with PSIPRED secondary structure predictions [57, 58],suggesting that the replacement of alanine with proline at position 305 lowers the propensity of this region to form regular a-helix (right-hand side of the figure). Conf, confidence of the prediction (0 = low, 9 = high); Pred, predicted type of secondary structure (H, helix, E,strand, C, coil); AA, target amino-acid sequence.

Proline residues in human MT2 function

7

less-pronounced distortion (Fig. 4B). As far as TM7 is con-cerned, the rhodopsin structure exhibits irregular helicitybetween residues 295 and 301 [22, 24], whereas our model of

hMT2RWT displays regular a-helix in this segment (Figs 4Band 5). Such difference could be explained by the fact thatthis part of hMT2R contains a sequence highly favoringa-helical arrangement (Fig. 5 and below), whereas the

corresponding region in rhodopsin does not favor a-helicalstructure as suggested by PSIPRED secondary structureprediction (Fig. 5). The position of the helix H8 is slightly

changed in modeled hMT2RWT compared to that inrhodopsin structure. This helix is located at the membraneinterface perpendicular to the membrane normal [22, 24],

and the predicted change in its orientation in hMT2Rmodel likely arises from different interactions with lipidsurroundings in our simulations compared to rhodopsincrystal structure.

The TM7 of majority of rGPCRs contains conservedNPxxY motif that is known to play an important role inreceptor signaling [12, 40–44]. The crystal structure of

rhodopsin revealed that the residue N302 (N304 inhMT2R), a key residue at the beginning of the NPxxY

motif, interacts with several residues in TMs 2, 3, 4 and 7(Fig. 6A) [22, 24]. In particular, a highly conserved residueD83 (D86 in hMT2R) seems to be linked through a water

bridge to this conserved asparagine. These interactionscreate inter-helical constrains and their disruption is prob-ably involved in the receptor activation process [45–47].However, TM7 of all melatonin receptors contain NAxxY

instead of NPxxY motif. To investigate this interestingdifference and the importance of the second residue in thismotif, we replaced alanine with proline to generate NPxxY

sequence in TM7 of hMT2R. The resulting hMT2RA305P

failed to bind 125I-mel, despite being localized in the cellmembrane (Table 1, Fig. 3). The introduction of valine

(residue structurally closer to alanine) completely rescuedreceptor binding (Table 1 and Fig. 2). However, bothEC50s and maximal responses of [35S]GTPcS bindingstimulated by both melatonin and I-mel were significantly

reduced in hMT2RA305V compared to hMT2RWTmed.MD simulations of hMT2RWT, hMT2RA305P and

hMT2RA305V suggest that mutations at position 305 affect

helicity of TM7 in the region N-terminal to the mutationsite. While TM7 of hMT2RWT forms regular a-helix, the

Fig. 6. Predicted effects of mutations within NPxxY motif on hMT2R structure. (A) Crystal structure of bovine rhodopsin [22], (B) modelof hMT2RWT, (C) model of hMT2RA305P and (D) model of hMT2RA305V. Compared to rhodopsin structure, the model of hMT2RWT

predicts very similar orientation of TM1, TM2 and TM7. Our models suggest that the mutation A305P may affect interactions betweenTM7 and TM6. WAT denotes a water molecule; Rhod denotes rhodopsin. This figure was generated using PyMol v0.99 (http://www.pymol.org).

Mazna et al.

8

TM7 of hMT2RA305P loses its helical arrangement betweenresidues Y298 and P305 (Fig. 6B,C). Minor changes in thehelicity of TM7 were also observed in the case of receptor

containing mutation A305V. Changes in helical arrange-ment of TM7 predicted by MD simulations are in agree-ment with secondary structure predictions, indicating thatthe replacement of alanine with proline at position 305

lowers the propensity of this region to form regular a-helix(Fig. 5). Our models suggest that changes in helicity ofTM7 induced by A305P mutation affect interactions

between residues from TM1, TM2, TM6 and TM7. Themodel of hMT2RWT shows very similar orientation ofTM1, TM2 and TM7 compared to rhodopsin structure

(Fig. 6A,B). On the other hand, the important residueN304 (see above) in the model of inactive mutanthMT2RA305P is rotated toward TM1 likely as a result ofchanged helicity of TM7 (Fig. 6C). In the case of active

mutant hMT2RA305V, the orientation of residue N304toward space between TM3 and TM6 resembles that seen inthe model of hMT2RWT and rhodopsin structure (Fig. 6D).

We also noticed that mutation A305P, but not A305V,affects the structure of the bottom part of putative hMT2Rligand-binding site [19, 25, 48] compared to hMT2RWT

(Figs 6B–D and 7A). These observations are in agreementwith our binding experiments and could explain the loss ofligand binding in the case of A305P mutation (Table 1).

Recently, it has been shown that the NPxxY and E/DRY(very highly conserved sequence in TM3 of rGPCRs) motifsprovide, in concert, a dual control of the activatingstructural changes in the rhodopsin [43]. In addition, Fritze

et al. [43] showed that mutant E134Q in ERY motif canefficiently restore the activity of Ala replacement mutants inthe NPxxY motif. This observation could explain why

melatonin receptors tolerate the alanine instead of prolinein NPxxY motif and asparagine instead of glutamate oraspartate in E/DRY motif. However, more work is needed

to verify this hypothesis.As summarized in Table 1, alanine substitution of both

highly conserved P174 (P4.59) in the C-terminal part of

TM4 and totally conserved P266 (P6.50) in TM6 resulted ina complete loss of radioligand binding to hMT2R. Theintroduction of flexible glycine (mutations P174G andP266G) did not rescue receptor binding, indicating low

tolerance for changes at these positions. Neither of thesetwo mutants seemed to suffer from trafficking defects, asconfocal microscopy performed on nonpermeabilized

CHO-K1 cells expressing corresponding constructs revealedthat all receptors reached the cell membrane and producedfluorescence signal comparable to hMT2RWT (Fig. 3).

These data are consistent with previous studies on otherrGPCRs where proline residues at positions 4.59 and 6.50were shown to play important roles in receptor signaling[12, 14, 42]. For example in the bovine rhodopsin, muta-

tions occurring at equivalent sites are known to causeretinal neurodegenerative disorder retinitis pigmentosa [49].In both rhodopsin structure and our hMT2RWT model, the

C-terminal part of TM4 interacts with N-terminal parts ofTM3 and TM5 and with residues in ECL2, thus formingthe wall of the ligand-binding site (Fig. 7A) [19, 24]. One of

the interacting residues, N175, was previously shown to beimportant for ligand binding to hMT2R [50]. Therefore, it

is reasonable to speculate that despite the fact that our MDsimulation of P174A mutant did not show any largestructural changes in the region around mutationsite compared to the hMT2RWT (data not shown), the

substitutions of P174 may affect interactions in this part ofreceptor and thus inhibit ligand binding.In the crystal structure of bovine rhodopsin, the TM6 is

significantly bent around residue P267 (P6.50) [24] and suchproline kink is expected to exist also in other GPCRs [38].

(A)

(B)

(C)

Fig. 7. Structure of the region surrounding residue P6.50 in ourmodels of hMT2R. (A) Model hMT2RWT+MEL showing theputative ligand-binding site of hMT2R with docked melatoninmolecule (shown in magenta). (B) Model of hMT2RWT in theabsence of ligand. (C) Model of hMT2RP266A suggests that P266Amutation affects helical arrangement in the upstream part of TM6around the residue W264. This figure was generated using PyMolv0.99 (http://www.pymol.org).

Proline residues in human MT2 function

9

It was proposed for b2-adrenergic receptor that alteringconfiguration of this proline kink through concerted changein the rotamer state of W6.48 (W264 in hMT2R) together

with other highly conserved residues in TM6 may trigger

conformational events ultimately leading to the receptoractivation [38, 47, 51]. It is possible that this mechanismmight be shared by other rGPCRs as well [38, 47]. Indeed,mutations of this completely conserved proline were

reported to profoundly affect receptor binding and/oractivation as well as the expression of several otherrGPCRs including m3 muscarinic, prostacyclin and B2

bradykinin receptors [11, 12, 14, 52, 53]. The alaninesubstitution of corresponding proline in hMT1R producedreceptors with reduced expression levels [54]. Fig. 7 shows

the structure of the region surrounding residue P6.50 in ourmodels of hMT2R (WT with bound melatonin, WT andP266A mutation). Our modeling suggests that the mutation

of P266A affects the helical arrangement in the upstreampart of TM6 around the residue W264 (Fig. 7). The P266Amutation in TM6 may affect the packing with neighboringhelices that contain important residues that are either part

of, or in the close vicinity of the putative ligand-binding site(e.g. V204, H208 and N268) (Fig. 7) [19, 25, 50, 54–56].These changes can be responsible for the observed loss of

binding (Table 1).In TM5, a mutation of highly conserved P212 (P5.50) did

not alter ligand binding to hMT2R but led to a complete loss

of receptor signaling (Table 1). As demonstrated by the[35S]GTPcS-binding assay, the receptor function was par-tially rescued by the introduction of flexible glycine (muta-tion P212G). P212Gmutant produced Emax response similar

to hMT2RWTlow but significantly increased EC50s of bothstudied agonists compared to hMT2RWTlow (Table 1 andFig. 2). In our model of hMT2RP212A, the part of TM5

a-helix downstream of A212 is distorted and forms a bulge-like structure not found in hMT2RWT (Fig. 8A,B). Thischange in TM5 secondary structure may affect interactions

between important residues surrounding the mutation site(e.g. H208 and W264). For example, it has been shown thatmutation of residueH208 decreases both agonist binding and

melatonin-mediated receptor activation in the MT1R [55].Considering the importance of residues H208 and W264 forthe function of hMT2R and other rGPCRs [38, 47, 51], wehypothesize that the signaling defects observed for

hMT2RP212A/G mutants might be connected with conforma-tional changes in TM5 and TM6 around the mutation site.Unlike the mutations described above, replacements of

proline residues at positions 41 (TM1), 93 and 95 (bothTM2) did not decrease signaling competence of hMT2R.All three mutants were expressed at hMT2RWThigh-like

levels and gave both binding and functional responses com-parable to hMT2RWThigh. Mutation P158A (TM4) reducedreceptor�s Bmax but other pharmacological parameters (KD,Emax and EC50) did not significantly differ from

hMT2RWTmed. As these mutations had no significant effecton function of hMT2R and considering computational

(A)

(B)

(C)

Fig. 8. Structure of the region surrounding residue P212 in modelsof hMT2RWT (A), hMT2RP212A (B) and hMT2RP212G (C). In themodel of hMT2RP212A, the part of TM5 a-helix downstream ofA212 is distorted and forms a bulge-like structure not found inhMT2RWT. This change in TM5 secondary structure affectsinteractions between residues surrounding the mutation site (e.g.H208 and W264). This figure was generated using PyMol v0.99(http://www.pymol.org).

Mazna et al.

10

costs of MD simulations of such a large system, weperformed simulation only for one of the mutants(hMT2RP93A) to have a trajectory of hMT2R bearing

harmless mutation. This simulation revealed that P93Areplacement does not have any substantial effect onconformation of TM2 compared to hMT2RWT (data notshown) in good agreement with our experimental data.

In conclusion, we investigated the role of proline residuesin the structure and function of hMT2R. All prolines in TMsegments of hMT2R were individually replaced with alanine

and/or glycine. In addition, the unusual NAxxY motiflocated in TM7 was mutated to generate highly conservedNPxxY motif found in the majority of rGPCR proteins.

Following transient expression in CHO-K1 cells, bindingproperties of the mutant receptors and their ability totransduce signal were analyzed using 125I-mel- and[35S]GTPcS-binding assays, respectively. The impact of

performed mutations on the receptor structure was assessedby MD simulations of MT2 receptor embedded in the fullyhydrated phospholipid bilayer. Our results indicate that

residues P174, P212 and P266 are important for the ligandbinding and/or signaling of human MT2 receptor. We alsoshow that changes within unusual NAxxY sequence in the

TM7 (mutations A305P and A305V) produce defectiveMT2 receptors indicating an important role of this motif inthe function of melatonin receptors.

Acknowledgment

This research was supported by Grants 309/04/0496, 305/

07/0681, 303/07/0915 and 204/06/0565 of the Grant Agencyof the Czech Republic, by Grant IAA600110701 of theGrant Agency of the Academy of Sciences of the Czech

Republic, by research project AVOZ 50110509 of theAcademy of Sciences of the Czech Republic, and by Centreof Neurosciences LC554 and Research Project

MSM0021620857 of the Ministry of Education, Youthand Sport of the Czech Republic.

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