REGULAR ARTICLE

Evidence for a role of caveolin-1 in neurokinin-1 receptorplasma-membrane localization, efficient signaling,and interaction with β-arrestin 2

Valentina Kubale & Zrinka Abramović &

Azra Pogačnik & Anders Heding & Marjeta Šentjurc &

Milka Vrecl

Received: 17 April 2007 /Accepted: 4 July 2007 /Published online: 23 August 2007# Springer-Verlag 2007

Abstract This study was focused on the relationship betweenthe plasma-membrane localization of neurokinin-1 receptor(NK1-R) and its endocytic and signaling properties. First, weemployed electron paramagnetic resonance (EPR) to study thedomain structure of HEK-293 cells and NK1-R microlocal-ization. EPR spectra and the GHOST condensation routinedemonstrated that NK1-R was distributed in a well-ordereddomain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolaechanged the NK1-R plasma-membrane distribution. Internal-ization and second messenger assays combined with biolu-minescence resonance energy transfer were employedsubsequently to evaluate the functional importance of theNK1-R microlocalization in lipid raft/caveolae microdo-

mains. The internalization pattern was delineated throughthe use of dominant-negative mutants (DNM) of caveolin-1S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and β-arrestin (β-arr 319–418) and by means of cell lines thatexpressed various endogenous levels of β-arrestins. NK1-Rdisplayed rapid internalization that was substantially reducedby DNMs of dynamin-1 and β-arrestin and even moreprofoundly in cells lacking both β-arrestin1 and β-arrestin2.These internalization data were highly suggestive of thepredominant use of the clathrin-mediated pathway by NK1-R,even though NK1-R tended to reside constitutively in lipidraft/caveolae microdomains. Evidence was also obtained thatthe proper clustering of the receptor in these microdomainswas important for effective agonist-induced NK1-R signalingand for its interaction with β-arrestin2.

Keywords 7TM receptors . Neurokinin receptor type 1 .

Caveolin-1 . Internalization . Signaling .

Lipid raft/caveolae microdomains .

Electron paramagnetic resonance . Cell culture

Introduction

Tachykinins constitute a large family of peptides that arewidely distributed within the central and peripheral nervoussystems and in non-neuronal and non-innervated tissues(Pennefather et al. 2004). The effects of tachykinins aremediated via three types of neurokinin receptors (NK-Rs)designated NK1-R, NK2-R, and NK3-R and belonging tothe family of seven transmembrane-spanning receptors(7TM receptors). Tachykinins have been recognized toexert a variety of effects under physiological and patholog-ical conditions (Almeida et al. 2004; Maggi and Schwartz1997). Thus, the study of NK-Rs is important for the

Cell Tissue Res (2007) 330:231–245DOI 10.1007/s00441-007-0462-y

Electronic supplementary material The online version of this article(doi:10.1007/s00441-007-0462-y) contains supplementary material,which is available to authorized users.

This work was supported by the Slovenian Ministry of HigherEducation, Science, and Technology (grant number 0406-007) and aSlovenian-Danish collaboration grant (BI-DK/06-07-007).

V. Kubale :A. Pogačnik :M. Vrecl (*)Institute of Anatomy, Histology & Embryology,Veterinary Faculty, University of Ljubljana,Gerbičeva 60,SI-1000 Ljubljana, Sloveniae-mail: milka.vrecl@vf.uni-lj.si

Z. Abramović :M. ŠentjurcEPR Center, Jozef Stefan Institute,Jamova 39,SI-1000 Ljubljana, Slovenia

A. Heding7TM Pharma,Fremtidsvej 3,DK-2970 Hørsholm, Denmark

identification of novel 7TM receptor modulators (Menzaghiet al. 2002). Compartmentalization of 7TM receptors inplasma-membrane microdomains probably plays an impor-tant role in regulating various receptor functions includingtheir endocytic trafficking and signaling and interactionswith protein partners (Insel et al. 2005).

Studies addressing the plasma-membrane localization ofthe NK1-R are rare; however, recent reports have proposedits localization in lipid rafts and caveolae (Meyer et al. 2006;Monastyrskaya et al. 2005). During the past decade, it hasbecome evident that lipids in the plasma membrane areorganized in liquid-ordered domains that contain elevatedlevels of cholesterol and glycosphingolipids and that arereferred to as lipid rafts in comparison with the bulk of theplasma membrane, which is enriched in phosphatidylcho-line. The tight packing of the saturated acyl chains of thesphingolipids and the presence of cholesterol appears toresult in a domain that is less fluid than the surroundingplasma membrane. Caveolae are regarded as a subset oflipid rafts that possess the protein caveolin-1 (Nichols 2003).Electron paramagnetic resonance (EPR) studies are highlyinformative in distinguishing different plasma-membranedomains in living cells by using phospholipid moleculeswith nitroxide spin labels attached to selected positions inthe fatty acyl chains. The presence of rigid proteins (e.g.,7TM receptors) in the plasma membrane can reduce theextent of motional fluctuations of the lipid fatty acyl chains,thus leading to changes in domain structure (Lee 2005).

In contrast to the data concerning the plasma-membranelocalization of NK1-R, several studies have provided strongevidence suggesting that NK1-R is internalized via aclathrin-mediated pathway. Rapid internalization of theNK1-R, which has high affinity for the main tachykininSubstance P (SP), has been reported in various cultured celllines (Grady et al. 1995; Sanders et al. 1996; Schmidlin etal. 2002) and in dorsal horn and mesenteric neurons (Gradyet al. 1996; Southwell et al. 1998; Wang and Marvizon2002). This process has been shown to be mediated both byβ-arrestin 1 and 2 (β-arr1 and 2; McConalogue et al. 1998,1999) and by dynamin (Schmidlin et al. 2001), indicative ofthe clathrin-mediated pathway. NK1-R belongs to the ClassB 7TM receptors, based on its ability to recruit bothnonvisual β-arrestins (β-arr1 and 2) equally well, andremains in complex with them for a prolonged time periodafter internalization (Oakley et al. 2000). Direct interactionof the NK1-R with β-arr2 has also been confirmed bybioluminescence resonance energy transfer (BRET; Vrecl etal. 2004). These observations have raised questions aboutthe relationship between the compartmentalization ofreceptor-caveolae/lipid rafts and the mode of endocyticentry. In this regard, the following paradigms have beendemonstrated for different 7TM receptors: (1) receptorscluster to the caveolae/lipid rafts, but after agonist binding,

the internalization is clathrin-dependent (Rybin et al. 2000),(2) receptors translocate to the caveolae/lipid rafts afteragonist binding and then internalize via either caveolae/lipid-rafts (Krisch et al. 1998; Mentlein et al. 2001) or aclathrin-dependent pathway (Wyse et al. 2003), and (3)receptors mainly reside in the caveolae/lipid rafts and enterthis pathway by default (Okamoto et al. 2000).

Membrane microdomains of lipid rafts and caveolaecould therefore represent the functional platform thatenables interaction between receptors and numerous sig-naling and scaffolding proteins implicated in signal trans-duction and intracellular trafficking (Conner and Schmid2003; Le Roy and Wrana 2005). The caveolae/lipid raftsmicrodomain has previously been established as beingimportant for proper agonist-induced NK1-R activation, inparticular for its interaction with Gαq (Monastyrskaya et al.2005), whereas subsequent desensitization resulting fromreceptor uncoupling from the G-protein is mediated by β-arr1 and 2 (McConalogue et al. 1998). This suggests thatNK1-R/arrestin complex formation occurs in caveolae/lipidrafts microdomain so that the β-arrestins can fulfill theirrole in receptor desensitization and trafficking. We havetherefore studied the NK1-R plasma-membrane distributionand addressed the role of the lipid raft/caveolae micro-domains in various aspects of NK1-R regulation, includingplasma-membrane distribution, mode of endocytic entry,agonist-induced Ca2+ signaling, and interaction with β-arrestin 2 (β-arr2). For this purpose, we have appliedvarious methodological approaches. Conventional EPRspectroscopy with methyl ester of 5-doxyl-palmitic acid(MeFASL(10,3)) as a spin probe and computer simulationof the EPR spectra (Strancar et al. 2005) have been used toevaluate the NK1-R plasma-membrane distribution. Themode of endocytic entry has been tested by the use ofdominant-negative mutants (DNM) of caveolin-1 (Cav1S80E), dynamin-1 (Dyn K44A), and β-arrestin (β-arr 319–418), and in cell lines that endogenously express variouslevels of β-arrestins (Kohout et al. 2001; Menard et al.1997). As specific membrane microdomains could providesites of local enrichment of molecules that need to interactwith each other or to be transported to specific locationswithin the cell, we have assessed second messenger Ca2+

signaling and inositol phosphate accumulation andemployed the BRET method to obtain data of relevancefor receptor/β-arr2 interactions.

Materials and methods

Materials

Molecular biology reagents were obtained from Invitrogen(Carlsbad, Calif., USA) or Qiagen (Crawley, West Sussex,

232 Cell Tissue Res (2007) 330:231–245

UK). All tissue culture media and reagents were purchasedeither from Gibco Invitrogen (Carlsbad, Calif., USA) orSigma-Aldrich (St. Louis, Mo., USA), unless otherwisestated. In addition, 125I-labeled SP (125I-SP), [3H]-myo-inositol, and isoproterenol were obtained from AmershamBiosciences (Little Chalfont, UK), anti-mouse horseradishperoxidase (HRP)-conjugated antibody from Pierce Bio-technology (Rockford, Ill., USA), and heat-inactivated FCS(fetal calf serum) from Euroclone (Milano, Italy). Humanembryonic kidney (HEK-293) cells and African greenmonkey SV40-transformed fibroblasts (COS-7 cells) wereobtained from the European Collection of Cell Cultures(Salisbury, UK). Mouse embryonic fibroblasts from knock-out (KO) mice that lack both β-arrestin1 and 2 (β-arr 1/2-KO) were kindly provided by Prof. Robert J. Lefkowitz(Duke University Medical Center, Durham, N.C., USA;Kohout et al. 2001). Methylester of 5-doxyl-palmitic acid(MeFASL(10,3)) was synthesized by Prof. S. Pečar (Facul-ty of Pharmacy, University of Ljubljana, Slovenia); thisspin probe is distributed in the cell plasma membrane. Oxy-redoxy systems in cell organelles and in cytoplasm reducespin probes to hydoxylamines, which are no longerdetectable by EPR.

Expression constructs

N-terminally hemagglutin (HA)-tagged human neurokinin-1receptor (HANK1-R) in the vector pcDNA3 and C-terminallyRenilla luciferase (Rluc)-tagged NK1-R (NK1-R/Rluc) andβ2-adrenergic receptor (β2-AR/Rluc) constructs were madeby using standard molecular biology techniques and wereverified by sequencing. All constructs were as previouslycharacterized (Martini et al. 2002; Vrecl et al. 2004). Humanβ-arr2 N-terminally tagged with the green fluorescentprotein (GFP) variant GFP2 (GFP2/β-arr2) was purchasedfrom Biosignal Packard, Canada. Mutation Arg393,395→Gluintroduced into human GFP2/β-arr2 (GFP2/β-arr2 R393E,R395E) by using QuickChange site-directed mutagenesiswas as previously described and characterized (Vrecl et al.2004). The commercial use of the β-arr2 R393E, R395Emutant required a license from 7TM Pharma. Caveolin-1, β-arrestin, and dynamin constructs were kindly provided byProf. J.E. Pessin (Department of Physiology and Biophysics,University of Iowa, Iowa, USA), Prof. V.V. Gurevich(Vanderbilt University Medical Center, Nashville, Tenn.,USA), and Prof. M.G. Caron (Duke University MedicalCenter, N.C., USA), respectively.

Cell culture and transfection

Cells were routinely maintained and passaged in Dulbecco’smodified Eagle’s Medium (DMEM) containing 10% heat-inactivated FCS, L-glutamine (0.3 mg/ml), penicillin (100 U/

ml), and streptomycin (100 μg/ml) at 37°C in a humidifiedatmosphere of 5% (v/v) CO2. For transient transfection,COS-7 and β-arr 1/2-KO cells were seeded at a density of7.5×105 and 1.5×106 cells per 60 mm and 100 mm dishes,respectively, and transfections were performed the followingday by using Lipofectamine.

HEK-293 cells were stably transfected with eitherHANK1-R alone, HANK1-R and the dominant negativecaveolin-1 mutant (Cav1 S80E), or GFP2/β-arr2 R393E,R395E alone by using Lipofectamine. Clonal cell linesdesignated as HEK-NK1-R, HEK-NK1-R + Cav1 S80E,and HEK-GFP2/β-arr2 R393E,R395E were selected bymeans of 1 mg/ml Geneticin. The β-arr2 R393E, R395Emutant is deficient in its ability to interact with thecomponents of the clathrin-coated pits thus prolonging thelifetime of the receptor/β-arrestin complex (Vrecl et al.2004). The Cav1 S80E mutant mimics the phosphorylationstate of caveolin-1 and is retained in the endoplasmicreticulum in a complex with caveolin-2 and consequentlydisrupts caveolar organization (Shigematsu et al. 2003).

Quantitative real-time reverse transcription/polymerasechain reaction

Total RNAwas extracted from HEK-293 cells, HEK-NK1-R,and HEK-NK1-R + Cav1 S80E stable cell lines and fromCOS-7 cells according to the instructions for the use of theQiagen SV Total RNA Isolation System (Promega, Madison,Wis., USA). Of each RNA sample, 10 μg was reverse-transcribed into cDNA by using the High-capacity cDNAArchive Kit (Applied Biosystems, Foster City, Calif., USA) ina final reaction volume of 50 μl. The Assay-by-DesignService was used to design primers and fluorescent 6-FAMdye-labeled minor-groove-binder probes. Sequences of theprimer pairs and probes used in this study were as follows:WT Cav1 [5-GAAGATGTGATTGCAGAACCAGAAG(sense); 5-CACAGTGAAGGTGGTGAAGCT (antisense),5-CACACAGTTTTGACGGCATT-3 (probe)]; Cav1 S80E[(5-ACTTTGAAGATGTGATTGCAGAACCA (sense), 5-CACAGTGAAGGTGGTGAAGCT (antisense), 5-AAGGGACACACGAATTT (probe)]. The eukaryotic ribosomal (r)18s RNA (ABI PRISM TaqMan Pre-developed AssayReagent Ribosomal RNA control PN 4310893E) was usedas a endogenous control. Real-time polymerase chain reaction(PCR) was then performed with the TaqMan Universal PCRMaster Mix (Applied Biosystems) in a final reaction volumeof 20 μl in an AbiPrism 7000 Sequence Detection Systemaccording to the manufacturer’s protocol. PCR amplificationwas carried out for 40 cycles. The results were expressed asthreshold cycle (Ct) defined as the cycle number at which thePCR product crosses the threshold of detection. Relativequantification of target transcripts (WT Cav1 and Cav1 S80E)normalized to endogenous control 18s rRNAwas performed

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by the comparative Ct method (ΔCt) and calculated accordingto the manufacturer’s protocol (User Bulletin No. 2, AppliedBiosystems). The 2−ΔΔCt method was used to analyze therelative changes in gene expression between tested cell lines.Control experiments with RNA samples without reversetranscription (RT) were performed to demonstrate theabsence of possible contamination with the genomic DNA.

Receptor-binding assay

Self-competition radioligand-binding assays were carriedout on stable cell lines expressing either HANK1-R (HEK-NK1-R) or HANK1-R and Cav1 S80E (HEK-NK1-R +Cav1 S80E) in 24-well plates. Cells were incubated with125I-SP tracer in the presence of increasing concentrationsof unlabeled SP (10−12–10−6 M final concentration) in 0.5-ml assay medium for 3 h at 4°C. By using solubilizationsolution, samples were collected into tubes, and γ radioac-tivity was counted on a 1275 Minigamma Counter (LKBWallac, Turku, Finland). Determinations were performed intriplicates. Binding parameters were determined fromdisplacement curves generated by sigmoidal dose-response-curve fitting (GraphPad Prism 4.0). Receptor density (Bmax)was then expressed in femtomoles per 100,000 cells aspreviously described (Ramsay et al. 2002).

Enzyme-linked immunosorbent assay of surface expressedNK1-R

Enzyme-linked immunosorbent assay (ELISA) was per-formed as described previously (Holst et al. 2004; Vrecl etal. 1998). Briefly, stable cell lines expressing eitherHANK1-R (HEK-NK1-R) or HANK1-R and Cav1 S80E(HEK-NK1-R + Cav1 S80E) were seeded out at a density1×105 cells per well in 48-well plate. After 24 h, cells werewashed three times, fixed, and incubated in blocking solution(3% dry milk, 50 mMTRIS HCl pH 7.5 in PBS) for 60 min atroom temperature. The cells were kept at room temperaturefor all subsequent steps. First, cells were incubated for 2 hwith anti-HA antibody (1:300 dilution), washed three times,incubated with anti-mouse HRP-conjugated antibody(1:1,250 dilution), and extensively washed. Immunoreactivitywas revealed by the addition of HRP-TMB substrate. Thereaction was terminated by the addition of 0.2 M sulfuricacid, and absorbance was measured at 450 nm. Determina-tions were replicated six times.

EPR spectroscopy

EPR measurements were performed as described previously(Koklic et al. 2005; Strancar et al. 2005). A thin film oflipophilic spin probe MeFASL(10,3) in 25 μl ethanolsolution (C=10−4 mol/l) was prepared on the wall of a

glass tube by rotary evaporation of ethanol. HEK-293 cellsor stable cell lines expressing either HANK1-R (HEK-NK1-R) or HANK1-R and Cav1 S80E (HEK-NK1-R +Cav1 S80E) were suspended at 5×106 in 3 ml HEPES-buffered DMEM, added to a glass tube, and agitatedmanually for 10 min enabling the spin probe to penetrateinto the cell membranes. Subsequently, the sample wascentrifuged at 120g for 3 min at room temperature; thesupernatant was decanted, and spin-labeled cells from thepellet were transferred in a 1 mm diameter glass capillarytube for EPR measurement. Measurements were performedon an X-band EPR spectrometer Bruker ESP 300 at roomtemperature, with a microwave frequency of 9.59 GHz,power set at 20 mW, modulation frequency at 100 kHz, andamplitude at 0.2 mT.

Computer simulation of EPR spectra and GHOSTcondensation procedure

Computer simulation of the shape of the EPR spectra lineof the spin probe distributed mainly in cell plasmamembranes (Swartz et al. 1986) in combination with theGHOST condensation procedure allows information aboutthe membrane domain structure and the properties of themembrane domains to be obtained (Filipic and Strancar2001). To describe the EPR spectra of spin probes, thestochastic Liouville equation is used (Budil et al. 1996;Robinson et al. 1985; Schneider and Freed 1989). Themodel takes into account that the local rotational motion isrestricted but fast with respect to the EPR time scale, andthat the EPR spectrum is the superposition of severalspectral components reflecting different modes of rotationalmotion of spin probe molecules in different membraneregions. Each spectral component is described with differ-ent sets of spectral parameters including the order param-eter (S), rotational correlation time (τc), polarity correctionfactors of hyperfine A and g tensors (pA and pg), and thebroadening constant (W; Strancar et al. 2005). The differ-ences in the EPR spectra depend mostly on the orderparameter, correlation time, and relative proportions be-tween the spectral components (d); therefore, we haveconcentrated our attention on these parameters. Withrespect to the order parameter, the time-averaged deviationof the spin probe acyl chain from the normal to the bilayerplane is 1 for perfectly ordered structures and 0 for the non-ordered isotropic motion of molecules. The rotationalcorrelation time describes the rate of motion of moleculesin the membrane. The relative proportion of each spectralcomponent describes the relative amount of the spin probeswith a particular motional mode and depends on thedistribution of the spin probe between different membraneregions. Of note, the lateral motion of the spin probe withinthe membrane is slow on the time scale of the EPR spectra

234 Cell Tissue Res (2007) 330:231–245

(Johnson et al. 1996; Sackmann and Trauble 1972).Therefore, each spin probe molecule reflects the motionalproperties of its nearest surrounding on the nanometer scale.EPR spectral contributions of all spin probe molecules locatedin membrane regions with the same properties give onespectral component. These membrane regions are referred ascertain types of membrane domain, with dimensions of theorder of magnitude of several nanometers.

The computer simulation procedure is implemented inthe software package EPRSIM (http://www.ijs.si/ijs/dept/epr/). To obtain the best fit of the calculated EPR spectrum tothe experimental spectrum, the stochastic and population-based genetic algorithm is used in combination with SimplexDownhill (HEO; Filipic and Strancar 2001). According tothis procedure, one still has to define the number of spectralcomponents before applying the optimization. To resolvethis problem, a multirun HEO optimization is used togetherwith a newly developed GHOST condensation procedure,which enables the determination of the number of differentdomain types in the membrane. According to this method,200 independent HEO simulation runs for each EPRspectrum are applied. The simulation procedure starts withthe prediction that the experimental EPR spectrum is asuperimposition of four spectral components, which corre-sponds to four coexisting membrane domain types (spinlabels exhibit four different modes of motion). Filipic andStrancar (2001) have previously established that, eventhough the simulation starts with four domain types, thefinal number of groups in GHOST diagrams is properlyreduced when there are less than four domain types. On theother hand, if there are more than four different domaintypes within the membrane, these are found by simulationprocedure. However, the groups in GHOST diagrams arenot well defined but are merged together to give a group ofsolutions with continuous distributions of parameters. Theparameters of the best fits are presented by three two-dimensional cross-section plots: S-τc, S-W, and S-pAdiagrams, the two other parameters of each diagram beingdefined by the intensity of the colors: red, green, and bluefor τc, W, and pA, respectively (GHOST diagrams; Strancaret al. 2003, 2005). From these plots, information about themembrane domain types, dynamic of motion, and orderingwithin the domain types and about the polarity of spinprobe surrounding them can be obtained.

Receptor internalization assay

NK1-R internalization assay based on the acid-washmethod was performed as described previously (Vrecl etal. 2004). The effect of DNMs of dynamin (Dyn K44A),caveolin-1 (Cav1 S80E), and β-arrestin (β-arr 319–418) onNK1-R internalization was assessed by using 125I-SP as thetracer. HEK-NK1-R stably expressing NK1-R or appropri-

ately transfected HEK-293, COS-7, and β-arr 1/2-KO cellswere plated at a density of ~2×105 cells per well in poly-L-Lysine-coated 24-well plates, and the assay was performed48 h after transfection. All time points were recorded intriplicate in three experiments. The amount of internalizedreceptors as a function of time was fitted by using a one-site-binding (hyperbola) curve fit (GraphPad Prism 4.0) toestimate the half-time of internalization (t1/2).

Ca2+ assay

Changes in the intracellular free Ca2+ concentration afterNK1-R agonist activation in stable cell lines expressingeither HANK1-R (HEK-NK1-R) or HANK1-R and Cav1S80E (HEK-NK1-R + Cav1 S80E) and in HEK-NK1-Rcells transiently co-transfected with Cav1 S80E weremeasured by using the NovoStar microplate reader aspreviously described (Heding et al. 2002). Briefly, cellswere seeded in 96-well plates at 30,000 cells per well andgrown overnight in a humidified incubator at 37°C and 5%CO2. On the day of the experiment, the cells were removedfrom the incubator, and 100 μl loading buffer, containingcalcium-sensitive fluorophore, was added (FLIPR calciumassay; Molecular Devices). The cells were then incubated for1 h at 37°C and 1 h at 25°C before the assay was performedat room temperature under the following parameters:excitation filter at 485 nm and emission filter at 520 nm.

Inositol phosphate assay

Inositol phosphate assay (based on the principles of immobi-lized metal ion affinity chromatography) and scintillationproximity assay (SPA) were performed as described previ-ously (Liu et al. 2003). NK1-R agonist-induced inositolphosphate (IP) accumulation was determined in stablecell lines expressing either HANK1-R (HEK-NK1-R)or HANK1-R and Cav1 S80E (HEK-NK1-R + Cav1S80E) and in HEK-NK1-R cells transiently co-transfectedwith Cav1 S80E. The amount of [3H]inositol phosphatesgenerated in the cells was determined by measurement of theradioactivity on SPA beads with a TopCount microplatereader (Packard BioScience). The obtained data weretransferred to GraphPad Prism and EC50 values (nM±SEM)generated by using sigmoidal dose-response-curve fitting(GraphPad Prism).

BRET2 assay

BRET2 measurements with the Mithras LB 940 plate reader(Berthold Technologies) were performed as previouslydescribed (Vrecl et al. 2004). Briefly, following harvesting,100 μl resuspended HEK-293 and COS-7 cells containing~200,000 cells were distributed to wells in 96-well micro-

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plates (white Optiplate) in the presence of increasingconcentrations of the NK1-R agonist SP (10−11–10−5 M,final concentration) or the β2-AR agonist isoproterenol(10−5 M). The substrate DeepBlueC (Packard Bioscience)was added at a final concentration of 5 μM. The signalsdetected at 395 nm and 515 nm were measured sequential-ly, and the 515/395 ratios calculated and expressed as amiliBRET level (mBU; BRET ratio×1,000). Data wereanalyzed by the two-tailed unpaired Student t-test. Differ-ences were considered statistically significant at P<0.001.

Luminescence and fluorescence measurement

Expression levels of Rluc- and GFP2-tagged constructswere monitored by total luminescence and fluorescencemeasurement in the NovoStar microplate reader (BMGLabTech) and the TopCount microplate reader (PackardBioScience), respectively. For a luminescence measurement,cells resuspended in glucose-supplemented Dulbecco’sphosphate-buffered saline were plated in 96-well microplatesat a density of ~5×104 cells per well. After a 1-h incubationand before measurement, DeepBlueC was added at a finalconcentration of 5 μM, and total luminescence wasmeasured by using the TopCount microplate reader. For afluorescence measurement, the cells from the same trans-fections were plated in a black microplate with a clearbottom at a density of ~5×104 cells per well. After a 1hincubation in darkness, the total fluorescence was measuredby using the NovoStar microplate reader with the excitationline at 485 nm and an emission filter at 520 nm.Background values obtained with untransfected cells weresubtracted from both measurements, and the means oftriplicate wells per sample were then calculated.

Results

Characteristics of generated stable cell lines

Stable cell lines designated HEK-NK1-R and HEK-NK1-R +Cav1 S80E were characterized by radioactive ligand-binding

assay and real-time RT-PCR. Characterization of HEK-NK1-R stably expressing the HANK1-R revealed a calculatedreceptor number (Bmax) of ~150 fmol/100,000 cells. In self-competition assays, SP competed for binding with 125I-SPwith an affinity of 0.38±0.10 nM. This IC50 value iscomparable with those previously published for wild-typehuman NK1-R (Holst et al. 1998). Calculated Bmax and theIC50 values obtained with the stable cell lines designatedHEK-NK1-R + Cav1 S80E stably expressing HANK1-Rand DNM caveolin-1 were ~105 fmol/100,000 cells and0.59±0.13, respectively.

Quantitative RT-PCR analysis was used to assess themRNA expression levels of WT Cav1 and the dominant-negative Cav1 S80E mutant in cell lines used in the study.The expression level of the WT Cav1 in both generatedstable cell lines (HEK-NK1-R and HEK-NK1-R + Cav1S80E) was comparable with that obtained in control HEK-293 cells. WT Cav1 expression in COS-7 was approxi-mately 2.2-fold higher compared with that of HEK-293cells. Cav1 S80E expression was detected only in the HEK-NK1-R + Cav1 S80E stable cell line; its expression was ~6-fold higher compared with the expression of WT Cav1 (seeTable 1). The expression level of the GFP2/β-arr2 R393E,R395E construct in the stable cell line (GFP2/β-arr2R393E,R395E) assessed by fluorescence measurementwas approximately 12,000 arbitrary units per 5×104 cells.

Properties of plasma membranes

Conventional EPR spectroscopy with MeFASL(10,3) as aspin probe was used to assess the effect of NK1-Rexpression on the plasma-membrane domain structure ofHEK-293 cells and to evaluate the possible involvement ofcaveolin-1 on the plasma-membrane properties and receptorlocalization. EPR spectra of MeFASL(10,3) in the plasmamembrane of HEK-NK1-R and of HEK-NK1-R + Cav1S80E stable cell lines were compared with controluntransfected HEK-293 cells (Fig. 1a,b). The shoulderobserved in the spectrum of the non-treated HEK-NK1-Rcell line (arrow in Fig. 1a) indicated that, in the spectrum ofthe HEK-NK1 cell line, one component exhibited a more

Table 1 Relative quantification of Cav1 and Cav1 S80E dominant-negative mutant expression by real-time polymerase chain reaction

Cell line ΔCt (WT Cav1−18s) 2−ΔΔCt ΔCt (Cav1 S80E−18s) 2−ΔΔCt

HEK-293 15.24±0.23 1.00 – –HEK-NK1-R 15.80±0.31 0.68 – –HEK-NK1-R + Cav1 S80E 15.50±0.26 0.83 12.93±0.12 5.94COS-7 14.10±0.21 2.20 – –

Relative quantification of target transcripts (WT Cav1 and Cav1 S80E) normalized to endogenous control 18s rRNA was carried out by thecomparative Ct method (ΔCt). The 2−ΔΔCt method was used to analyze the relative changes in gene expression between tested cell lines [18srRNAwas used as an internal control for each cDNA sample, Ct cycle threshold calculated as the cycle at which fluorescence signal passes a fixedthreshold line, ΔCt Ct(target gene)−Ct(18s), 2−ΔΔCt fold change].

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restricted motion of its phospholipid alkyl chains comparedwith that of the other cell lines. The EPR spectra of agonist-stimulated HEK-NK1 cell line and control HEK-293 cellswere largely overlapping indicating that, after agoniststimulation of the NK1-R-expressing cells, the membranestructure became comparable with that observed in thecontrol HEK-293 cells. This effect was less pronounced forthe HEK-NK1 + Cav1 S80E cell line. After treatment withSP, no changes in the shape of the spectral line were observed(Fig. 1b).

By computer simulation of the EPR spectra in combinationwith the GHOST condensation procedure (Filipic andStrancar 2001), a more detailed insight was obtained intothe domain structure of the plasma membrane of these celllines. A good correlation was observed between the

experimental and computer-simulated EPR spectrum of thespin probe in the plasma membrane of the HEK-293 stablecell lines (Fig. 1c).

In GHOST diagrams, the results of several runs of com-puter simulation for one and the same spectrum are collectedinto groups of solutions in which each group represents amode of spin label motion in a certain membrane domaintype. The parameters, which describe the motional character-istics of spin probes in a certain domain type, are obtainedfrom the groups in GHOST diagrams in which the density ofthe solutions is maximal (red points in GHOST diagram inFig. 2) and are presented in Table 2.

The results of the GHOST condensation routine, whichwas applied to the EPR spectra of HEK-293 cells andunstimulated and agonist-stimulated HEK-293 stable cell

a

b

1 mT

1 mT

HEK-293 HEK-NK1-R + SP HEK-NK1-R

HEK-293 HEK-NK1-R + Cav1 S80E + SPHEK-NK1-R + Cav1 S80E

experimental EPR spectrum computer-simulated EPR spectrum

HEK-293 HEK-NK1-R

c

Fig. 1 Experimental EPR spec-tra of the lipophilic spin probe5-doxylpalmitate methyl ester[MeFASL(10,3)] in the plasmamembrane of untransfectedHEK-293 cells and theHEK-293 stable cell lines. aExperimental EPR spectra ofHEK-NK1-R stably expressingthe NK1-R in the absence (blueline) or presence (1 μM SP; redline) of agonist. The EPR spec-trum of control untransfectedHEK-293 cells is shown forcomparison (black line). Thearrow indicates the portion ofthe spectrum obtained with theuntreated HEK-NK1-R cell lineand corresponding to a morerestricted motion of the spinprobe. b EPR spectra of HEK-NK1-R + Cav1 S80E cells sta-bly expressing the NK1-R andCav1 S80E dominant-negativemutant (DNM) in the absence(blue line) or presence (1 μMSP; red line) of agonist. TheEPR spectrum of controluntransfected HEK-293 cells isshown for comparison (blackline). c Comparison of experi-mental EPR spectrum ofMeFASL(10,3) (red lines) andbest fits (black lines) obtainedby computer simulation

Cell Tissue Res (2007) 330:231–245 237

lines, are shown in Fig. 2. In GHOST diagrams, orderparameters versus the rotational correlation times (τc) areplotted. Three modes of spin probe motion in untransfectedHEK-293 cells with order parameters S=0.18, S=0.37, andS=0.63 can be seen; these correspond to three types ofmembrane domains. However, in the HEK-293 cells stablyexpressing NK1-R (HEK-NK1-R), only two separategroups are resolved in the GHOST diagram, indicatingthat, in cells stably transfected with HANK1-R, the domainwith the intermediate order parameter (S=0.37) mergeswith the most ordered domain (see also Fig. 2b). Thehighest order parameter in domain 1, which is characterizedby slow lateral diffusion; more restricted acyl chain motion

is indicative of lipid raft/caveolae microdomains, since itincludes a large portion of cholesterol associated withhighly saturated lipids (Simons and Ilkonen 2000). How-ever, the discrimination between caveolar and non-caveolarrafts is not possible by our methods. An increase in theportion of domain 1 observed in the HEK-NK1-R cells issuggestive for partitioning of the NK1-R in this particulardomain. After agonist stimulation of the HEK-NK1-R cellline, three domain types appear again, approaching thesituation of untransfected HEK-293 cells (cf. Fig. 2c,a).Similar changes are not observed in the plasma-membraneproperties of the HEK-NK1-R + Cav1 S80E cell line inwhich the separation of the three domains is still well

Table 2 Order parameters (S), rotational correlation times (τc), and portions (d) of different domain types in the membrane of control HEK-293cells and HEK-NK1-R and HEK-NK1-R + Cav1 S80E cell lines in the absence (Untreated) and presence (+) of the agonist Substance P (SP)

Domain Order parameters HEK-293 HEK-NK1-R HEK-NK1-R + Cav1 S80E

Untreated + SP Untreated + SP

Domain 1 S 0.63±0.06 0.58±0.09 0.59±0.07 0.62±0.04 0.61±0.04τc 0.17±0.06 0.19±0.09 0.15±0.06 0.20±0.10 0.20±0.10d 0.69 0.86 0.71 0.58 0.65

Domain 2 S 0.37±0.03 – 0.36±0.05 0.41±0.04 0.38±0.03τc 0.70±0.20 – 0.70±0.30 0.60±0.30 0.70±0.20d 0.20 – 0.18 0.27 0.23

Domain 3 S 0.18±0.02 0.17±0.01 0.16±0.03 0.01±0.01 0.03±0.01τc 1.00±0.20 0.90±0.20 1.00±0.30 2.50±0.10 2.30±0.20d 0.11 0.14 0.11 0.15 0.12

Data were obtained by computer simulation of the EPR spectra as the best fit of the calculated spectra to the experimental one. The parameters ofdifferent domain types are taken from the groups in GHOST diagrams in which the density of solutions is maximal (red points in GHOSTdiagram) and are given as the average±SD.

Fig. 2 Comparison of the do-main structure of the plasmamembrane of untransfected HEK-293 cells with that of HEK-293stable cell lines. S-τc (order pa-rameter − rotational correlationtime) GHOST diagrams of EPRspectral parameters of motionalmodes of spin probe in theplasma membrane of controluntransfected HEK-293 cells (a)and untreated (b) and agonist-stimulated (1 μM SP; c) HEK-NK1-R stable cell lines. GHOSTdiagrams of untreated (d) andagonist-stimulated (1 μM SP; e)HEK-NK1-R + Cav1 S80E sta-ble cell line are also shown. Notethe fusion of membrane domainsin the HEK-NK1-R cell line

238 Cell Tissue Res (2007) 330:231–245

distinguished (Fig. 2d,e). The two domains with the higherorder parameter (S=0.62, S=0.41) have similar motionalcharacteristics to those of the HEK-293 cells, whereas theorder parameter of the less ordered domain is significantlylower (S=0.01). The stimulation with SP does not changethe ordering and dynamics of membrane molecules signif-icantly; only the portion of the most ordered domainincreases (Fig. 2, Table 2).

Computer simulation of the EPR spectra and the resultsof the GHOST condensation routine suggested the parti-tioning of NK1-R into lipid raft/caveolae microdomains andprovided evidence for a role of Cav1 in NK1-R plasma-membrane microlocalization. Therefore, we next investi-gated whether caveolin-1 also had a role in the NK1-Rinternalization pattern and the signaling and interactionwith β-arr2.

Internalization pattern of NK1-R

Agonist-induced NK1-R endocytic entry into HEK-293cells was tested by the use of the DNM of dynamin 1 K44A(Dyn K44A), caveolin-1 S80E (Cav1 S80E), and β-arrestin(β-arr 319–418; see Electronic Supplementary Material,Fig. S1). The NK1-R displayed extensive and rapid agonist-induced internalization with an estimated half-time (t1/2) ofless than 1.5 min. The Dyn K44A mutant known to inhibitendocytic entry through caveolae and clathrin-coatedvesicles (Conner and Schmid 2003) reduced the internaliza-tion of the NK1-R by up to 50%. The effect of the dominant-negative β-arr 319–418 mutant, a marker of the involvementof a clathrin-mediated pathway, inhibited the internalizationof the NK1-R to a lesser extent (around 25%).

To assess the specific role that caveolae might play in NK1-R internalization, we used a caveolin-1 mutant that mimicsphosphorylation on serine 80 (Cav1 S80E). In the unpho-sporylated state, Cav1 assembles into plasma-membranecaveolae together with caveolin-2. When phosphorylated onserine 80, it is retained in the endoplasmic reticulum in acomplex with caveolin-2 and consequently disrupts plasma-membrane caveolae organization; this phenotype is recapit-ulated by the Cav1 S80E mutant (Shigematsu et al. 2003).The effect of the Cav1 S80E mutant on NK1-R internaliza-tion was less obvious. Accordingly, Dyn K44A and β-arr319–418 substantially prolonged the t1/2 of internalization(t1/2>10 min versus t1/2<1.5 min), whereas the effect ofCav1 S80E on the internalization kinetics was only moderate(t1/2≤2.5 min). The effect of dynamin and β-arrestin mutantson NK1-R internalization largely agreed with the results ofMcConalogue et al. (1998, 1999).

The importance of β-arrestins and the clathrin-mediatedpathway in NK1-R internalization was further assessed byperforming internalization assays in cell lines that endoge-nously expressed different β-arrestin levels (HEK-293, COS-7,

and β-arr 1/2-KO; Fig. 3). The proportion of internalizedNK1-Rs and the t1/2 of internalization (t1/2≤1.8 min) were onlyslightly reduced in COS-7 cells compared with HEK-293cells. However, the proportion of internalized NK1-Rs wasreduced by around 70% in β-arr 1/2-KO cells, suggesting thatNK1-R internalization is, to a large extent, β-arr-dependentand therefore mainly proceeds via a clathrin-dependentpathway.

Agonist induced Ca2+ release

Next, we addressed the role of caveolin-1 in NK1-Rsignaling by following SP-induced changes in the intracel-lular free Ca2+ concentration and inositol phosphateproduction (Fig. 4). The obtained EC50 values for SP-induced Ca2+ response in the HEK-NK1-R and HEK-NK1-R + Cav1 S80E cell lines were 0.77±0.17 nM and 1.88±0.31 nM, respectively. The HEK-NK1-R stable cell linetransiently co-transfected with Cav1 S80 displayed asimilar EC50 (1.53±0.73 nM) as the HEK-NK1-R + Cav1S80E cell line. Maximal SP-induced Ca2+ release wasdecreased by approximately 20% in the HEK-NK1-R +Cav1 S80 cell line compared with the HEK-NK1-R cellline (Fig. 4a1). A similar reduction was also observed in theHEK-NK1-R stable cell line transiently co-transfected withCav1 S80 (Fig. 4b1). We also investigated whether Cav1S80E caused time-dependent changes in the intracellularCa2+ concentration, which was monitored over a period of90 s. The Ca2+ signal peaked within 15 s after the additionof the agonist and subsequently rapidly decreased duringthe following 45 s. The measured values on the time scalebetween time points 15 s and 60 s were adjusted to the

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Cell Tissue Res (2007) 330:231–245 239

linear function y=a+bx, and the slope (s) of the fitted linewas calculated. The fitted curve for the HEK-NK1-R stablecell line was steeper compared with the HEK-NK1-R +Cav1 S80E cell line curve (−1.048±0.011 versus −0.753±0.003; R2 fit of 0.9943 and 0.9993). Transient co-transfection of the HEK-NK1-R cell line with Cav1 S80gave comparable results (s=−1.346±0.0288 versus−0.9078±0.046; R2 fit of 0.8739 and 0.5781). These datasuggested that Cav1 S80E affected the NK1-R desensitiza-tion rate. Cells from the same experiments were alsolabeled with [3H]-inositol, and we measured the dose-dependent SP-stimulated production of [3H]-inositol phos-phates over 45 min (Fig. 4a2,b2). Expression of Cav1 S80Ereduced [3H]-labeled inositol phosphates by 25% in theHEK-NK1-R + Cav1 S80E cell line and by ~50% in HEK-NK1-R cells transiently co-transfected with Cav1 S80E incomparison with the response observed in the HEK-NK1-Rcell line. EC50 values for SP-induced inositol phosphateproduction varied from 1.43 nM to 2.59 nM agreeing well

with previously published values for wild-type humanNK1-R (Holst et al. 1998). Receptor surface expressionwas monitored by ELISA (Fig. 4a3,b3).

Agonist-induced functional interaction between NK1-Rand β-arr2

To evaluate the role of caveolin-1 in agonist-inducedfunctional interaction between NK1-R and β-arr2, we useda proximity based BRET2 assay that measured energytransfer from an RLuc-tagged protein to a GFP-taggedprotein upon addition of the RLuc substrate Deep Blue C(Heding 2004; Mercier et al. 2002; Vrecl et al. 2004). Theeffects of the DNM of caveolin 1 on the BRET2 signal wasexamined in HEK-293 cells transiently transfected withNK1-R/Rluc, GFP2/β-arr2, and Cav1 S80E. In parallel, theeffects of the DNMs of dynamin 1 and β-arr on receptor/β-arr2 interaction was also examined. In the presence of Cav1S80E, the maximal BRET2 signal (BRETmax) was reduced

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Fig. 4 Effect of dominant neg-ative caveolin-1 mutant on ago-nist-induced Ca2+ release,agonist-induced IP accumula-tion, and receptor surface ex-pression. Intracellular free Ca2+

mobilization (a1, b1), IP accu-mulation (a2, b2), and receptorsurface expression (a3, b3) weredetermined in stable cell lines(HEK-NK1-R and HEK-NK1-R + Cav1 S80E; a1–a3)and in the HEK-NK1-R stablecell line transientlyco-transfected with Cav1 S80E(b1–b3). Surface expression ofstably transfected HA-taggedWT NK1-R was tested by theaddition of anti-HA antibodies.SP (100 nM) was used to followtime-dependent changes in in-tracellular Ca2+ release. Data aremean±SEM of three indepen-dent experiments performed intriplicate (pcDNA3empty vector)

240 Cell Tissue Res (2007) 330:231–245

by approximately 55% compared with the control cells andthe cells co-transfected with either Dyn K44A or β-arr319–418; this was reflected in a BRETmax of ≈80 mBUversus 170, 174, and 164 mBU obtained in control cells andcells co-transfected either with Dyn K44A or β-arr 319–418 (Fig. 5a). To provide additional evidence for theinvolvement of the lipid raft/caveolae microdomains forefficient receptor/β-arr interaction, a NK1-R/β-arr recruit-ment assay was also performed in dimethyl-beta-cyclodex-trin (MβCD) cholesterol-depleted cells. Similarly to Cav1S80E, pretreatment with MβCD reduced the BRETmax byapproximately 50% (BRETmax of ≈70 mBU versus140 mBU). The addition of cholesterol improved thepotency of SP-induced receptor/β-arr2 interaction, whereasthe BRETmax was not restored (Fig. 5c).

The effect of Cav1 S80E on the receptor/β-arrestininteraction was also tested in COS-7 cells and with anothermember of the G-protein-coupled receptor (GPCR) family,viz., β2-AR. β2-AR creates a relative short-lived lowBRET2 signal in the β-arr2 recruitment assay; therefore,we used our previously described β-arrestin mutant, β-arr2R393E,R395E, which is deficient in its ability to interactwith the components of the clathrin-coated pits, thusprolonging the lifetime of the receptor/β-arrestin complexand yielding a higher BRET signal (Vrecl et al. 2004). Theresults obtained in COS-7 cells were comparable with thoseobtained in the HEK-293 cells, suggesting that the Cav1 S80Eeffect is neither cell- nor receptor-specific (Fig. 6a). Theexpression levels of Rluc- and GFP2-tagged constructs weremonitored in all BRET2 experiments by total luminescenceand fluorescence measurements (Figs. 5b, 6b). As thepresence of Cav1 S80E reduced the expression levels ofinteracting partners, we performed additional control experi-ments to validate our observations (see Electronic Supple-mentary Material, Fig. S2). Control experiments wereperformed in the stable cell line expressing the β-arr2R393E,395E mutant (HEK-GFP2/β-arr2 R393E,R395E). Wehad previously demonstrated that the β-arr2 R393E,R395Emutant displayed comparable affinity and efficacy for classB NK1-R in β-arr2 recruitment assay as the WT β-arr2(Vrecl et al. 2004). This stable cell line provided us with anexperimental system in which the expression level of theGFP2-tagged β-arr2 R393E,395E was sufficiently high andconstant to enable us to assess the importance of receptorexpression levels on the BRET2 signal. We first showed thatthe BRET2 signal was relatively constant over a five-foldrange for NK1-R expression; however, for the optimalBRET2 signal, the GFP2-arrestin level should exceed thereceptor expression as determined by total fluorescence andluminescence measurements (see Electronic SupplementaryMaterial, Fig. S2a,c). Secondly, we co-transfected the HEK-GFP2/β-arr2 R393E,R395E cell line with a constant amountof Rluc-taged NK1-R while increasing the amount of Cav1

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signal. HEK-293 cells were transiently co-transfected with a total of3 μg GFP2/β-arr2 together with 1 μg NK1-R/Rluc and 3 μg emptypcDNA3 vector, Cav1 S80E, Dyn K44A, or β-arr 319–418. Cellswere treated with increasing concentrations of SP, and the BRET2 wasmeasured. Data are the mean±SEM of duplicate observations fromthree experiments. b Expression levels of NK1-R/Rluc and GFP2/β-arr2 constructs. HEK-293 cells were transfected as shown. Totalluminescence (open bars) was measured after addition of the Rlucsubstrate (DeepBlueC) to assess the expression of NK1-R/Rluc. Totalfluorescence (gray bars) was measured with excitation at 485 nm andemission at 520 nm to assess GFP2/arrestin construct expression level.Data are the mean±SEM of triplicate observations from three experi-ments. c HEK-293 cells were transiently co-transfected with a total of4.5 μg GFP2/β-arr2 together with 1.5 μg NK1-R/Rluc. The BRET2

signal was measured as described in untreated cells (open squares),cholesterol-extracted cells with 2% dimethyl-beta-cyclodextrin(MβCD) for 1 h at 37°C (open triangles), or cells incubated for 1 hwith 0.2 mM cholesterol/2% MβCD mixture to replenish cholesterol(open circles), and the BRET2 signal was calculated. Data are themean±SEM of triplicate observations from at least three independentexperiments

Cell Tissue Res (2007) 330:231–245 241

S80E. As shown in the Electronic Supplementary Material(Fig. S2b), the BRET2 signal decreased with an increasingamount of Cav1 S80E, and even 0.5 μg Cav1 cDNAsignificantly reduced the BRET2 signal compared with thecontrol pcDNA3-co-transfected cells (P>0.01). The reducedBRET2 signal was obtained at the receptor/β-arr2 expressionlevels that, under control conditions, resulted in a BRETmax

of ≈125 mBU (Fig. S2d). The applicability of a stable cellline expressing GFP2/β-arr2 for monitoring the activation of

various transiently expressed 7TM receptors has previouslybeen demonstrated (Bertrand et al. 2002). BRET signal wasalso shown to be largely independent of receptor densitywhen performing dimerization experiments (Mercier et al.2002).

Discussion

This study has sought to investigate the relationshipbetween NK1-R plasma-membrane localization and itsendocytic and signaling properties. EPR membrane spec-troscopy with spin labeling is a method by which thephysical characteristics of the membrane can be followed,and information about the domain structure of the mem-brane can be gathered (Sentjurc and Koklic 2002). The lipidcomposition and biophysical interactions between differentconstituents are highly complex in membranes; therefore,EPR spectra are composed of several superimposed spectralcomponents. To obtain biologically meaningful informationfrom the EPR spectra, fitting procedures are required(Strancar et al. 2005). A novel method called GHOST hasbeen utilized that condenses solutions into groups andprovides a means for quantitative characterization ofcomplex biomembrane systems (Strancar et al. 2003).Computer simulation of the EPR spectra and the GHOSTcondensation routine has shown that NK1-R expressionchanges the membrane domain organization of the HEK-NK1-R stable cell line, as shown by the obvious fusion ofmembrane domains on the GHOST diagram. Partitioning ofNK1-R into a well-ordered domain is suggestive of itslocalization in lipid raft/caveolae microdomains, an obser-vation that correlates well with previous biochemical andimmunoflurescence data on NK1-R localization (Meyer etal. 2006; Monastyrskaya et al. 2005). Constitutive lipid raftlocalization has also been described for mammalian type Iand non-mammalian gonadotropin-releasing hormonereceptor (GnRH-R; Navratil et al. 2003, 2006), whereassome other GPCR members (such as the m2 mAchR andthe B2 bradykinin receptor) translocate from the bulkmembrane to lipid rafts upon agonist stimulation (de Weerdand Leeb-Lundberg 1997; Feron et al. 1997; Navratil et al.2003). The EPR spectra of the agonist-stimulated HEK-NK1-R cell line and control HEK-293 cells are largelyoverlapping indicating that, after agonist stimulation ofNK1-R, the membrane structure becomes comparable withthat observed in the control HEK-293 cells. A previousstudy has indicated that cholesterol depletion by MβCDdoes not affect NK1-R plasma-membrane localization(Monasteriska 2005). However, the DNM of caveolin-1changes the microlocalization of NK1-R as observed by itspartitioning into a less ordered plasma-membrane domainand by the absence of plasma-membrane domain fusion.

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Fig. 6 Effect of the caveolin-1 mutant on the BRET2 signal generatedby agonist-induced GFP2/β-arr2 interaction with both NK1-R and β2-AR in COS-7 cells. a BRET2 signal. COS-7 cells were transientlytransfected with a total of 3 μg GFP2/β-arr2 R393E,R395E togetherwith either NK1-R/Rluc or β2-AR/Rluc and 5 μg of the indicatedexpression constructs. The BRET2 signal was measured as describedin untreated cells (open bars), cells treated with the appropriateagonist SP (1 μM) or isoproterenol (10 μM; gray bars) and thespecific BRET2 signal (solid bars) was calculated by subtracting thevalues obtained with untreated cell from the values obtained afteragonist treatment. Data are the mean±SEM of triplicate observationsfrom at least three independent experiments. b Expression levels ofthe receptor/Rluc and GFP2/arrestin constructs. COS-7 cells weretransfected as shown. Total luminescence (open bars) was measuredafter addition of the Rluc substrate (DeepBlueC) to assess theexpression of the NK1-R/Rluc or the β2-AR/Rluc construct. Totalfluorescence (gray bars) was measured with excitation at 485 nm andemission at 520 nm to assess the GFP2/arrestin construct expressionlevel. Data are the mean±SEM of triplicate observations from at leastthree independent experiments

242 Cell Tissue Res (2007) 330:231–245

The Cav1 S80E mutant probably affects the functionalintegrity and microarchitecture of lipid rafts/cavealae, sinceit heterodimerizes with an integral protein of lipid rafts,viz., caveolin-2, and retains it in the endoplasmic reticulum(Shigematsu et al. 2003).

Having demonstrated constitutive localization of NK1-Rin the lipid raft/caveolae microdomains, we then sought todetermine the relationship between the receptor-caveolae/lipid raft compartmentalization and the mode of endocyticentry. Internalization data showed rapid internalization ofNK1-R, as previously reported in various cultured cell lines(Grady et al. 1995; Li et al. 1999; Schmidlin et al. 2002)and in dorsal horn and mesenteric neurons (Grady et al.1996; Southwell et al. 1998; Wang and Marvizon 2002).The t1/2 of less than 2 min obtained in HEK-293 cells is ingood agreement with previously published time-courses forNK1-R expressed in rat kidney epithelial cells (Schmidlinet al. 2001, 2002) and dorsal horn neurons (Wang andMarvizon 2002). A comparable t1/2 has also been obtainedfor the thyrotropin-releasing hormone type 1 receptor(TRH1-R) expressed in HEK-293 cells (Vrecl et al. 1998;Yu and Hinkle 1998). No major differences have beenobserved between the NK1-R internalization kinetics inHEK-293 and COS-7 cells, even though they endogenouslyexpress high and low level of β-arrestins, respectively(Menard et al. 1997). Such rapid internalization in cellswith low endogenous level of β-arrestins, as observed forNK1-R in COS-7 cells, has also been observed for chickenGnRH-R expressed in COS-1 cells (Pawson et al. 2003).Results obtained with the DNMs have revealed thatinternalization of NK1-R requires dynamin, β-arrestin,and to a lesser extent, caveolin-1. Previous reports havealso shown the importance of β-arrestins and dynamin forNK1-R internalization (McConalogue et al. 1998, 1999).We have found that internalization is substantially reducedin β-arr 1/2-KO cells compared with wild-type HEK-293and COS-7 cells. However, some internalization of NK1-Roccurs in cells devoid of β-arrestins. The same has alsobeen previously noted for TRH1-R (Jones and Hinkle2005). Based on these internalization results, we canpostulate that the agonist-induced NK1-R internalizationmainly proceeds through a clathrin-dependent pathway.However, a β-arrestin-independent pathway might also beemployed by the NK1-R, but to a lesser extent. A similarcorrelation between the constitutive receptor plasma-mem-brane microlocalization and internalization patterns haspreviously been reported for β2-AR (Rybin et al. 2000).

Considerable evidence indicates that caveolae and/orlipid rafts serve as attachment platforms for variousreceptors including GPCRs and for a wide array ofsignaling and scaffolding proteins involved in the regula-tion of signal transduction (Conner and Schmid 2003; LeRoy and Wrana 2005). Therefore, we next evaluated the

importance of the functional integrity of caveolae/lipid raftscomprising the membrane microdomains for functionalintegrity of NK1-R with respect to Ca2+ signaling, inositolphosphate accumulation, and receptor interaction withβ-arr2. Cav1 S80E exerted a substantial effect on theSP-induced Ca2+ signaling and inositol phosphate accumu-lation. NK1-R-mediated Ca2+ signaling was also abolishedin cholesterol-depleted cells (Monastyrskaya et al. 2005).The differences in microlocalization of the NK1-R betweenthe HEK-NK1-R and HEK-NK1-R + Cav1 S80S stable celllines observed by EPR and after the GHOST condensationroutine might lead to a reduction in Ca2+ signaling, asmembrane microlocalization seems to play an importantrole in the activation of the NK1-R signaling pathway. Inaddition, the disappearance of a membrane domain in theHEK-NK1-R cell line might promote cluster formation andprovide contacts between membrane proteins that wouldotherwise be separated in their own domains (Koklic et al.2005). This demonstrates that NK1-R microlocalizationwithin the well-ordered domain 1 is important for theefficient signaling that leads to inositol phosphate genera-tion and intracellular Ca2+ release. Based on the Ca2+ signaltime-course, we might assume that Cav1 S80E also reducesthe NK1-R desensitization rate; this agrees with the lowerreceptor/arrestin interaction in the presence of Cav1 S80E.However, the efficacy of receptor interaction with effectormolecules is also probably affected. In addition to efficientreceptor coupling with effector molecules such as Gproteins, lipid raft/caveolae microdomains might be impor-tant for receptor interaction with adaptor proteins. Consid-ering the NK1-R microlocalization, internalization, andsecond messenger data, we postulate that NK1-R/arrestincomplex formation precedes receptor internalization andoccurs within lipid raft/caveolae microdomains. The Cav1S80E mutant substantially reduces the efficacy of theagonist-induced receptor/β-arr2 interaction as measuredby BRET2. The DNM effect of caveolin-1 in the β-arr2recruitment assay has also been observed with other 7TMmembers and is additionally supported in cholesterol-depleted cells. The observed effect could be attributable tothe disruption of the liquid-ordered phase, by the removalof cholesterol with MβCD leading to increased solubility ofcavealae/raft-associated proteins or by a change in themicrolocalization of NK1-R in the presence of Cav1 S80EDNM, which does not favor efficient receptor/arrestininteraction. The absence of an effect in β-arr 319–418DNM, which have been previously characterized as retainingclathrin binding but lacking receptor-binding activity(Krupnick et al. 1997), and in Dyn K44A DNM on agonist-induced receptor/β-arrestin interaction is thus not surprising,as our data and previously published evidence (Xue et al.2004) suggest that the receptor/arrestin complex formationmost likely occurs in the caveolae/lipid rafts, and both

Cell Tissue Res (2007) 330:231–245 243

DNMs (β-arr 319–418 and Dyn K44A) are involved inevents subsequent to receptor/β-arrestin formation.

In summary, evidence is provided for NK1-R localiza-tion in lipid raft/caveolae microdomains and for theimportance of Cav1 for plasma-membrane microlocaliza-tion, second messenger signaling, and efficient couplingwith the β-arr2. Upon agonist stimulation, NK1-R internal-izes mainly via a β-arrestin-dependent mechanism that isindicative of the involvement of the clathrin-mediatedpathway, although the use of an alternative β-arrestin-independent pathway cannot be completely excluded. In thisregard, our findings provide a new framework to study thefunctional importance of caveolae/lipid rafts in the context of7TM receptors and their interacting protein partners.

Acknowledgments The authors thank Magdalena Dobravec forexcellent technical assistance, Laura Storjohann for English proof-reading, and Dr. Birgitte Holst and Professor Mette Rosenkilde forcritical evaluation of the manuscript.

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