Báo cáo khoa học: Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization pdf

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Studies on the role of the receptor protein motifspossibly involved in electrostatic interactions on thedopamine D1and D2receptor oligomerizationSylwia Łukasiewicz1, Agata Faron-Go´recka2, Jerzy Dobrucki3, Agnieszka Polit1and Marta Dziedzicka-Wasylewska1,21 Department of Physical Biochemistry, Jagiellonian University, Krako´w, Poland2 Laboratory of Biochemical Pharmacology, Polish Academy of Sciences, Krako´w, Poland3 Division of Cell Biophysics, Jagiellonian University, Krako´w, PolandVarious molecular techniques based on biophysical, bio-chemical and pharmacological approaches have dem-onstrated that G protein-coupled receptors (GPCRs),also known as heptahelical receptors, can exist and bephysiologically active as dimers in the plasma mem-brane [1,2]. These molecules can both homo- andheterodimerize. The phenomenon of receptor dimeriza-tion is important in different aspects of receptor biogen-esis and function, such as receptor maturation, folding,plasma membrane expression [3–8], signal transductionspeed and specificity [1,5,9–12], and receptor desensiti-zation [5,13–16]. Interactions between different classesKeywordsArg-rich motif; dopamine D1receptor;dopamine D2receptor; FRET; GPCRoligomerizationCorrespondenceM. Dziedzicka-Wasylewska, Faculty ofBiochemistry, Biophysics andBiotechnology, Jagiellonian University7 Gronostajowa Street, Krakow, PolandFax: +48 012 664 6902 or+48 012 637 4500Tel: +48 012 664 6122 or+48 012 662 3372E-mail: wasyl@if-pan.krakow.pl ormarta.dziedzicka-wasylewska@uj.edu.pl(Received 1 August 2008, revised 19November 2008, accepted 27 November2008)doi:10.1111/j.1742-4658.2008.06822.xWe investigated the influence of an epitope from the third intracellularloop (ic3) of the dopamine D2receptor, which contains adjacent arginineresidues (217RRRRKR222), and an acidic epitope from the C-terminus ofthe dopamine D1receptor (404EE405) on the receptors’ localization andtheir interaction. We studied receptor dimer formation using fluorescenceresonance energy transfer. Receptor proteins were tagged with fluorescenceproteins and expressed in HEK293 cells. The degree of D1–D2receptorheterodimerization strongly depended on the number of Arg residuesreplaced by Ala in the ic3 of D2R, which may suggest that the indicatedregion of ic3 in D2R might be involved in interactions between two dopa-mine receptors. In addition, the subcellular localization of these receptorsin cells expressing both receptors D1–cyan fluorescent protein, D2–yellowfluorescent protein, and various mutants was examined by confocal micros-copy. Genetic manipulations of the Arg-rich epitope induced alterations inthe localization of the resulting receptor proteins, leading to the conclusionthat this epitope is responsible for the cellular localization of the receptor.The lack of energy transfer between the genetic variants of yellow fluores-cent protein-tagged D2R and cyan fluorescent protein-tagged D1R mayresult from differing localization of these proteins in the cell rather thanfrom the possible role of the D2R basic domain in the mechanism ofD1–D2receptor heterodimerization. However, we find that the acidicepitope from the C-terminus of the dopamine D1receptor is engaged in theheterodimerization process.AbbreviationsCFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; GBR, GABABreceptor; GPCRs, G protein-coupled receptors;ic3, third intracellular loop; M3R, m3 muscarinic receptor; TCSPC, time-correlated single photon counting measurements; TM,transmembrane domains of a receptor; YFP, yellow fluorescent protein.760 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBSof GPCRs point to a new level of molecular cross-talkamong signaling molecules [1,5,11,17].Structural information about receptor dimer forma-tion is currently limited, and the question of whetherreceptors dimerize in a similar way or have their ownpaths of dimerization remains open. In general, eithercovalent or noncovalent interactions are involved inthis process; however, the latter seem to be more effec-tive [18–21]. Either the transmembrane domains (TMs)[22–31] of GPCRs or the N- [32–34] or C-tail [35,36]could play a role in dimer formation. It has beenshown that cysteine residues located in the extracellu-lar loops are essential for disulfide-linked m3 musca-rinic receptor (M3R) dimer formation; however thiskind of interaction is not the only point of contact[37]. For GABABreceptors (GBR), a coiled-coil inter-action within the C-tail of GBR1 and GBR2 seems tobe involved in receptor heterodimerization. However,this motif is not necessary, as deleting the C-tail doesnot abolish dimerization. Also, hydrophobic interac-tions within the TM of GPCRs are essential for forma-tion and stabilization of the dimers and have beendetected for beta-adrenergic, dopamine, muscarinicand angiotensin receptors [38–40].In earlier studies, the role of certain amino acid resi-dues in the formation of noncovalent complexesbetween protein molecules was highlighted. Electro-static interactions occur between an epitope containingmainly two or more adjacent arginine residues on oneprotein fragment and an acidic epitope containing twoor more adjacent glutamate or aspartate residues,and ⁄ or a phosphorylated residue, on the other protein[41,42]. Ciruela et al. demonstrated that electrostaticinteractions between an arginine-rich epitope from thethird intracellular loop of the D2receptor and twoadjacent aspartate residues or a phosphorylated serineresidue in the C-terminus of the A2Areceptor areinvolved in heterodimerization between the adeno-sine A2Areceptor and the dopamine D2receptor [43].A similar interaction has also been shown forD1–NMDA receptor heterodimers [44].Although the dopamine D1and D2receptor sub-classes are biochemically distinct, coactivation of bothreceptors has been shown to be essential for their physi-ological function. The view that these receptors mayalso function as a physically linked unit is especiallyimportant because recent data suggest that the D1andD2receptors are co-expressed by a moderate to sub-stantial proportion of striatal neurons [45,46]. Lee et al.provided anatomical evidence suggesting significant col-ocalization of D1and D2receptors in the caudate andpyramidal cells in the rat frontal cortex [47]. Earlierstudies by Vincent et al. have also shown that the lami-nar distribution of medial prefrontal cortex neuronsexpressing both D1and D2receptors was similar tothat of the mesocortical dopamine afferents [48].The dopamine D2receptor can form homodimers[19]. Recently, we have shown that the D2receptoralso forms heterodimers with the dopamine D1recep-tor [49]; however, the precise role of specific regions ofreceptor molecule(s) in that process has not yet beenelucidated. In this study, we investigated the role of anepitope from the third intracellular loop (ic3) of thedopamine D2receptor, which contains adjacent argi-nine residues (217RRRRKR222), and an acidic epi-tope from the C-terminus of dopamine D1receptor(404EE405) on the D1–D2receptor interaction.Fluorescence resonance energy transfer (FRET)occurs between fluorescence donor and acceptor chro-mophores when they are located within 100 A˚of eachother and are arranged properly in terms of their tran-sition dipole moments [50]. Using this technique, westudied receptor dimer formation using fluorescencelifetime microscopy and time-correlated single photoncounting (TCSPC) measurements. The receptor pro-teins were tagged with cyan (CFP; fluorescence donor)and yellow fluorescent proteins (YFP; fluorescenceacceptor) and expressed in HEK293 cells. We findFRET to be a very sensitive tool, and measurementsare especially useful to quantitatively monitor thephysical interactions between receptor proteins [51,52].ResultsRadioligand binding assayAs shown in Table 1, the binding parameters obtainedfor dopamine D1receptor and its mutant indicate thatthe Kdvalues for these two receptors were similar;however, the density of the D1MUT (404AA405) wasTable 1. Binding parameters for the dopamine receptors. For dopa-mine D2receptor binding, the statistical significance was evaluatedusing a one-way ANOVA, followed by a Dunnett’s test for post hoccomparison. *P < 0.05. For dopamine D1receptor binding, thestatistical significance was evaluated using a Student’s t-test;***P < 0.001.SpeciesBmax± SEM(pmolÆmg)1protein)Kd± SEM(nM)D1–CFP 14.66 ± 0.13 1.50 ± 0.08D1MUT–CFP 9.85 ± 0.08*** 1.20 ± 0.06D2–YFP 4.88 ± 0.10 0.41 ± 0.06D2R1–YFP 2.53 ± 0.08* 0.44 ± 0.03D2R2–YFP 0.70 ± 0.07* 0.44 ± 0.09S. Łukasiewicz et al. Dopamine D1and D2receptors dimerizationFEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 761lower than that of wild-type D1R (Fig. 1A). Also, allthree genetic variants of dopamine D2R displayed sim-ilar Kdvalues, but the density of these receptorsstrongly depended on the number of Arg residues stillpresent within the receptor sequence. The D2R1(217AARRKR222) mutant displayed half of the Bmaxvalue obtained for D2R, whereas the density of theD2R2 (217AAAAKR222) mutant was much lower(Fig. 1B). For the D2R3 (217AAAAAA222) variant,no binding parameters could be obtained, which indi-cates that there was no receptor protein in the cellularmembrane. This conclusion is further justified byconfocal microscopy analysis of receptor localization.Analysis of the localization of dopamine D1,D2and their genetic variant fusion proteinsConfocal microscopy was used to visualize HEK293cells co-expressing the dopamine D1and D2receptors,as well as their genetic variants (D1MUT, D2R1,D2R2, D2R3). These experiments were performed todetermine the influence of the introduced mutations onthe localization of the receptor proteins and the degreeof their colocalization.Figure 2A,B shows HEK293 cells transientlycotransfected with plasmids encoding the dopa-mine D1, dopamine D2,D1MUT, D2R1, D2R2 andD2R3 receptors in different combinations. Merged pic-tures with apparent yellow signal indicating overlap ofgreen fluorescent signal (CFP channel) and red fluores-cent signal (YFP channel) show colocalization.As seen from the figures, these receptor proteinswere localized differentially in the cell. Cell edge sharp-ness confirms that dopamine D1and D1MUT recep-tors localize in the plasma membrane, in contrast tothe dopamine D2receptor and its genetic variants,D2R1, D2R2, which were localized in the plasma mem-brane and inside the cell. In the case of the dopa-mine D2receptor mutants, the degree of membranelocalization depended on the number of mutated resi-dues in the ic3 region (D2217–222).The dopamine D2R3 receptor location was veryinteresting and surprising. As seen in Fig. 2A, whichshows a cell co-expressing both D1–CFP and D2R3–YFP fusion proteins, these receptors were found in dif-ferent parts of the cell. The D2R3 mutant was localizedinside the cell, whereas the D1receptor was found inthe plasma membrane. However, when the cellco-expressed both types of D2receptors, i.e. the wild-type and the D2R1, D2R2 as well as D2R3 variant, colo-calization was observed in both the plasma membraneand inside the cell. For a quantitative estimation of thedegree of colocalization between the two different pro-teins of interest, Pearson’s correlation coefficients andcoefficients of determination were estimated (Fig. 2C).In case of cells co-expressing dopamine D1and dopa-mine D2receptor mutants, the degree of colocalizationdecreased, which was correlated with number ofexchanged residues within the ic3 of D2receptor.When cells were cotransfected with the same type ofreceptors (D1MUT–CFP ⁄ D1–YFP, D2–CFP ⁄ D2R1–YFP, D2–CFP ⁄ D2R2–YFP, D2–CFP ⁄ D2R3–YFP) andwith dopamine D2and genetic variant dopamine D1receptors (D1MUT–CFP ⁄ D2–YFP) the obtained valuesof coefficients remained approximated.Fluorescence spectroscopy measurements ofdopamine receptor dimerizationAlthough steady-state fluorescence spectroscopy mea-surements in cell suspension enable only the qualitativeestimation of the FRET phenomenon, this approach isFig. 1. Saturation binding of [3H]SCH23390 (A) and [3H]-spiperone(B) to human D1and D2dopamine receptors, respectively. Data arefrom a single experiment performed in triplicate and are representa-tive of at least three independent experiments. Elimination of theArg-rich or di-Glu motif in D2RorD1R, respectively, does not alterthe ligand binding constant.Dopamine D1and D2receptors dimerization S. Łukasiewicz et al.762 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBSvery demonstrative and gives a quick answer towhether there is any energy transfer in the examinedsample. Therefore, we used this type of measurementto investigate interactions between the dopamine D1and D2receptors and their genetic variants. Fluores-cence emission profiles for the HEK293 cell suspensionexpressing fusion proteins in different combinations(D1–CFP ⁄ D2–YFP, D1–CFP ⁄ D2R1–YFP, D1–CFP ⁄D2R2–YFP, D1CFP ⁄ D2R3–YFP, D1MUT–CFP ⁄D2–YFP, D1–CFP ⁄ D1–YFP, D1MUT–CFP ⁄ D1–YFP,D2–CFP ⁄ D2–YFP and D2–CFP ⁄ D2R3–YFP) werecompared using an excitation wavelength of 434 nm(donor absorption).The upper panel of Fig. 3 shows emission spectra ofHEK293 cell populations after cotransfection withplasmids encoding genes for dopamine D1and D2receptor fusion proteins (D1–CFP and D2–YFP) incomparison with emission spectra of the cell popula-tions that co-express dopamine D1receptor fusionprotein (D1–CFP) and one of the genetic variants ofdopamine D2receptor fusion protein (D2R1, D2R2or D2R3–YFP) (Fig. 3A). In Fig. 3B, the resultspresented are from a cell suspension expressing thedopamine D2–YFP fusion protein and the genetic vari-ant of the dopamine D1receptor (D1MUT–CFP)fusion protein. We observed energy transfer betweenwild-type dopamine D1and D2receptors, but wheneither the genetic variant of dopamine D1(D1MUT)or the D2R3 genetic variant of the dopamine D2recep-tor was present in the sample, there was no visibleenergy transfer, despite the presence of both fluoro-phores in the sample.Figure 3C,D shows the emission profiles of cellscotransfected with plasmids encoding genes for thesame type of dopamine receptor (D1or D2, respec-tively), tagged with different fluorescence proteins,ABCFig. 2. Expression of D1R and D2R and their mutants in HEK293 cells. (A) HEK293 cells were cotransfected with either D1–CFP or D1MUT–CFP and either D2–YFP, D2R1–YFP, D2R2–YFP, D2R3–YFP or D1MUT–YFP (green and red). Image overlays show extensive colocalization inD1⁄ D1,D1⁄ D1MUT, D1⁄ D2and D1⁄ D2R1 assays and partial colocalization in D1⁄ D2R2 assays. D1⁄ D2R3 does not colocalize. (B) HEK293 cellswere cotransfected with D2–CFP and either D2–YFP, D2R1–YFP, D2R2–YFP or D2R3–YFP. Image overlays show extensive colocalization inevery case. (C) Bar graph of Pearson‘s correlation coefficient calculated for HEK293 cells cotransfected with different dopamine D1and D2receptor protein construct combination. Data are mean ± SE, and statistical significance was evaluated using Student’s t-test and Mann–Whitney test. ***P < 0.001 for combinations D1with all variants of D2versus D1⁄ D2. Either D2⁄ D2R1, D2⁄ D2R2 or D2⁄ D2R3 versus D2⁄ D2,D1MUT ⁄ D1versus D1⁄ D1, and D1MUT ⁄ D2versus D1⁄ D2combinations are not statistically significant. Values of corresponding coefficientsof determination (r2) are reported in brackets.S. Łukasiewicz et al. Dopamine D1and D2receptors dimerizationFEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 763compared with emission profiles of cells in which oneof the tagged receptors was its own mutant (D1–CFP ⁄D1–YFP and D1MUT–CFP ⁄ D1–YFP or D2–CFP ⁄D2–YFP and D2–CFP ⁄ D2R3–YFP). The lower panelof Fig. 3 shows that both dopamine receptors, D1andD2, form homo-oligomeric structures and confirmsthat both of the investigated epitopes are probably notengaged in the homodimerization process. In bothcases, we observed efficient energy transfer, which canbe judged by the localization of the appropriate peaksof the spectra.To serve as a control for this experiment, weco-expressed the a subunits of the G protein, aiand astagged with CFP with dopamine D1or D2receptors,which were tagged with YFP. As seen in Fig. 4, theFRET phenomenon takes place only when the D1receptor is co-expressed with asor D2receptor isco-expressed with ai. The interactions are specificbecause no energy transfer was observed followingco-transfection of D1–YFP ⁄ ai–CFP or D2YFP ⁄ asCFP,despite the identical overexpression level of theproteins in all studied combinations.Fluorescence lifetime microscopy studies ofdopamine receptor dimerizationTime-correlated single-photon counting experimentswere performed on the inverted fluorescence micro-scope. The FRET phenomenon was observed in asingle living cell transiently transfected with thedopamine D1and D2receptors and their geneticvariants, tagged with fluorescent proteins. This kind ofmeasurement provides highly quantifiable data becauseit is independent of any change in fluorophore concen-tration or excitation intensity.To determine FRET efficiency, precise measurementof the donor fluorescence lifetime (CFP), in the pres-ence and absence of the acceptor (YFP), is required.ACBDFig. 3. Fluorescence emission spectra of HEK293 cells expressing the CFP- and YFP-tagged proteins coupled to D1R and D2R and theirmutants. (A) Cotransfection of HEK293 with D1–CFP and either D2–YFP (gray dashed line), D2R1–YFP (black line), or D2R2–YFP (gray line) orD2R3–YFP (black dashed line). (B) Cotransfection of HEK293 with D1MUT–CFP and D2–YFP (gray line) in comparison with D1–CFP andD2–YFP (black line). (C) Cotransfection of HEK293 with D1MUT–CFP and D1–YFP (gray line) in comparison with D1–CFP and D1–YFP (blackline). (D) Cotransfection of HEK293 with D2–CFP and D2R3–YFP (gray line) in comparison with D2–CFP and D2–YFP (black line). CFP wasexcited at 434 nm, and fluorescence was detected at 450–550 nm through a double monochromator. The spectral contributions arising fromlight scattering and nonspecific fluorescence of cells and buffer were eliminated.Dopamine D1and D2receptors dimerization S. Łukasiewicz et al.764 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBSFluorescence decays were analyzed as both mono- andmulti-exponentials. Analysis of the reduced chi-squaredvalue and residual distribution led to the conclusionthat best fit parameters were obtained with two expo-nentials. Adding a third exponential did not signifi-cantly influence the parameters, and the fractionalcontribution of the additional lifetime was close tozero. Figure 5 shows the typical time-dependent donordecays for the D1–CFP bearing donor alone and withthe donor and acceptor D1–CFP ⁄ D1MUT–YFP.The average CFP fluorescence lifetime obtainedduring TCSPC experiments was 2.37 ns, and the valuechanged when acceptor was present in a cell. Thegreatest average fluorescence lifetime decrease (to1.52 ns), which was regarded as the highest FRET effi-ciency ($ 36%), was detected in our earlier studies forthe CFP–YFP hybrid (CFP connected by a short 15amino acid linker with YFP) [49].Measurements on the cells co-expressing dopa-mine D1and D2receptor fusion proteins indicated$ 4% efficiency of energy transfer, with an averagedonor fluorescence lifetime of 2.27 ns. This changedwhen the dopamine D2receptor was replaced by agenetic variant (D2R1, D2R2 or D2R3) and also whenD1MUT was used instead of the dopamine D1recep-tor. Transfer efficiency was equal to 2.1% (2.32 ns) forABCDFig. 4. Representative fluorescence emission spectra of HEK293 cells cotransfected with either D1–YFP or D2–YFP and Ga–CFP fusion pro-teins. (A) Negative FRET control, spectra from a 1 : 1 mixture of cells individually expressing the GaS–CFP (black line) fusion protein (excitedat 434 nm) and the D1–YFP (gray line) fusion protein (excited at 475 nm). (B) Cotransfection of HEK293 cells with D1–YFP and GaS–CFP(gray line) or D1–YFP and GaI–CFP (black line), excited at 434 nm. (C) Negative FRET control, spectra from a 1 : 1 mixture of cells individuallyexpressing GaI–CFP (black line) fusion protein (excited at 434 nm) and D2–YFP (gray line) fusion protein (excited at 475 nm). (D) Cotransfec-tion of HEK293 cells with D2–YFP and GaI–CFP (gray line) or D2–YFP and GaS–CFP (black line), excited at 434 nm. Fluorescence wasdetected at 450–550 nm through a double monochromator. The spectral contributions arising from light scattering and nonspecific fluores-cence of cells and buffer were eliminated.S. Łukasiewicz et al. Dopamine D1and D2receptors dimerizationFEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 765D1⁄ D2R1, further decreased to 1.26% (2.34 ns) forD1⁄ D2R2, and finally reached the value of 0.44%(2.36 ns) for D1⁄ D2R3.The lowest E value, similar to that obtained for theD1⁄ D2R3 combination, was observed for D1MUT ⁄D2R3 and was equal to 0.4% (2.36 ns). A similarresult (0.8%; 2.35 ns) was obtained for cells co-express-ing the dopamine D1receptor mutant (D1MUT) andthe wild-type dopamine D2receptor, as donor andacceptor of fluorescence, respectively.However, when the cells were cotransfected withplasmids encoding genes for the same type of dopa-mine receptors, D1or D2, and when one of the appro-priate receptors was replaced by its mutant (D1byD1MUT or D2by D2R3), no change in transfer effi-ciency was detectable. The E value for D1MUT ⁄ D1was estimated to be 7.8% (2.19 ns) versus 8%(2.18 ns) for D1⁄ D1, while for D2⁄ D2R3, it equaled3.4% (2.29 ns) versus 3.5% (2.28 ns) for D2⁄ D2combi-nations.The summary of TCSPC results is presented inTables 2 and 3. The error of the average fluorescencelifetime is the standard error of mean obtained fromdifferent cells and independent transfections (weignored standard deviations derived from fitting ofindividual fluorescence decay because they were verysmall).DiscussionThe data provided from numerous studies indicate thatoligomerization may play important roles in receptortrafficking and ⁄ or signaling. In several cases, receptorsappear to fold into constitutive dimers early after bio-synthesis, although ligand-promoted dimerization atthe cell surface has been also proposed [53]. ManyGPCRs have been shown to participate in homo- orheterodimerization [54]. Using a biophysical approach,we had previously shown that the D2and D1dopa-mine receptors exist as functional homo- and hetero-oligomers in cell lines [49], and similar conclusions canbe drawn from biochemical studies [14,19,55,56].However, the exact sequence motifs responsible forthat interaction had not been identified. In family 1receptors, robust hydrophobic TM interactions havebeen proposed as the most probable structural ele-ments involved in oligomerization [27,57,58]. SomeFig. 5. Time-dependent fluorescence intensity decays of CFPattached to the D1receptor with and without YFP attached to theD1MUT receptor. The black dotted curve shows the intensity decayof the donor alone (D), and the dark gray dotted curve showsthe intensity decay of the donor in the presence of acceptor (DA).The black solid lines and weighted residuals (lower panels) arefor the best double exponential fits. The gray dotted curve repre-sents the excitation pulse diode laser profile, set up at 434 nm.Table 2. Summary of energy transfer measurements by fluores-cence lifetime microscopy in HEK293 cells. Excitation was set upat 434 nm, and emission was observed through the appropriateinterference filters, as described in Experimental procedures. Thestandard errors of means (obtained from at least 15 single cells)are presented in parentheses. Statistical significance was evaluatedusing Student’s t-test; *P < 0.05 versus D1–CFP ⁄ D2–YFP.SpeciesAverage lifetime (ns)TransferefficiencyÆEæ (%)ÆsDæÆsDAæD1–CFPa2.37 ± 0.01D1–CFP ⁄ D2–YFPb2.27 ± 0.02 4.01D1–CFP ⁄ D2R1–YFPc2.32 ± 0.02 2.10*D1–CFP ⁄ D2R2–YFPd2.34 ± 0.01 1.26*D1–CFP ⁄ D2R3–YFPe2.36 ± 0.01 0.44*D1MUT–CFP ⁄ D2–YFPf2.35 ± 0.02 0.80D1MUT–CFP ⁄ D2R3–YFPg2.36 ± 0.01 0.40aMeasured in cell expressing CFP coupled to the dopamine D1receptor.bMeasured in cell co-expressing dopamine D1and D2fusion proteins (D1–CFP and D2–YFP).cMeasured in cellco-expressing dopamine D1and D2fusion proteins (D1–CFP andD2R1–YFP – genetic variant of dopamine D2receptor).dMeasuredin cell co-expressing dopamine D1and D2fusion protein (D1–CFPand D2R2–YFP – genetic variant of dopamine D2receptor).eMea-sured in cell co-expressing dopamine D1and D2fusion proteins(D1–CFP and D2R3–YFP – genetic variant of dopamine D2receptor).fMeasured in cell co-expressing dopamine D1and D2fusion pro-teins (D1MUT–CFP – genetic variant of dopamine D1receptor andD2–YFP).gMeasured in cell co-expressing dopamine D1and D2fusion proteins (D1MUT–CFP – genetic variant of dopa-mine D1receptor and D2R3–YFP – genetic variant of dopamine D2receptor).Dopamine D1and D2receptors dimerization S. Łukasiewicz et al.766 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBSexperimental studies also suggested the participation ofC- and N-terminal regions and the ic3 in this process[16,32,43]. Using pull-down and MS experiments, Ciru-ela et al. postulated that heterodimerization of theadenosine A2Aand dopamine D2receptors stronglydepends on an electrostatic interaction between anArg-rich epitope from the ic3 of the D2R(217RRRRKR222) and either the two adjacent Aspresidues (DD 401–402) or a phosphorylated Ser374 inthe C-tail of the A2AR [43].Because the dopamine D1R contains an acidicregion on the C-terminus, like A2AR, we designedexperiments to determine whether a similar interactionis responsible for the heterodimerization of the D2receptor with the D1receptor. However, a differentapproach to that mentioned above was used to addressthis question. The receptor proteins under investigationwere tagged with fluorescent proteins and transfectedinto HEK293 cells; their localization was thenobserved with the use of a confocal microscope. Thedegree of receptor dimerization was also judged bychanges in fluorescence lifetime, which we find to bethe most sensitive technique with which to measureFRET [49].The results presented here indicate that dopa-mine D1and D2receptors form homo- and hetero-dimers; results that are in agreement with previouslypublished data [19,49,55]. Measuring receptor dimer-ization by monitoring changes in the fluorescence life-time of probes linked to the receptors of interest seemsthe best approach in this kind of the study. Althoughthe approach enables only qualitative estimation ofFRET phenomenon, steady-state fluorescence spectros-copy measurements in suspension are also usefulbecause they are very demonstrative. In this study,both approaches yield similar conclusions, although weare aware that quantitative results can only beobtained from fluorescence lifetime microscopy.An often-discussed problem when using biophysicaltechniques to study receptor oligomerization is thatthese experiments predominantly involve heterologousexpression systems, which in most cases have been per-formed in cell lines transfected with the receptors ofinterest. Receptors are usually epitope-tagged and, inmost cases, are overexpressed. Therefore, it has oftenbeen suggested that biophysical techniques characterizeinteraction artifacts that occur due to high nonphysio-logical protein expression. However, GPCRs oligomer-ization is difficult to analyze in native cells, therefore,the human embryonic kidney cell line has been widelyused in resonance energy transfer studies of membranereceptors, because these cells provide an acceptedmodel in which fluorescently tagged receptor proteincan be efficiently expressed. As reported by Mercieret al. [59], the extent of dimerization of b2-adrenergicreceptors (shown by BRET) was unchanged over a20-fold range of expression levels (from 1.4 to26.3 pmolÆmg)1protein). While studying the homodi-merization of neuropeptide Y receptors, Dinger et al.[60] also demonstrated that the FRET effect was inde-pendent of the level of receptor expression. These find-ings imply that examples of GPCR dimerization arenot merely artifacts derived from the high levels ofexpression that are often achieved in heterologous sys-tem. Results obtained in this study, concerning thedopamine D1and D2receptors and their interactionswith the appropriate a subunits of G protein, furtherconfirm that the use of advanced fluorescence techni-ques does indeed allow for the observation of trueinteractions. The dopamine D1receptor did not inter-act with Gai, and the D2receptor did not interact withGas, although the physical contact of these receptorswith their appropriate a subunit partners could indeedhave been observed, despite the identical level of over-expression of the proteins in all studied combinations.The experiments described above serve as a controlthat must always be performed when using FRET todetermine if two proteins interact. That control is toexpress (preferentially using the same expression con-struct in all experiments) two noninteracting fusionproteins that carry CFP and YFP in the same cell andTable 3. Summary of energy transfer measurements obtained byfluorescence lifetime microscopy in HEK293 cells. Excitation wasset up at 434 nm, and emission was observed through appropriateinterference filters, as described in Experimental procedures. Thestandard errors of means (obtained from at least 15 single cells)are presented in parentheses.SpeciesAverage lifetime (ns)TransferefficiencyÆEæ (%)ÆsDæÆsDAæD1–CFPa2.37 ± 0.01D1–CFP ⁄ D1–YFPb2.18 ± 0.01 8.00D1MUT–CFP ⁄ D1–YFPc2.19 ± 0.01 7.80D2–CFPd2.37 ± 0.02D2–CFP ⁄ D2–YFPe2.28 ± 0.02 3.50D2–CFP ⁄ D2R3–YFPf2.29 ± 0.01 3.40aMeasured in cell expressing CFP coupled to dopamine D1recep-tor.bMeasured in cell co-expressing two dopamine D1receptorfusion proteins (D1–CFP and D1–YFP).cMeasured in cellco-expressing two dopamine D1receptor fusion proteins (D1MUT–CFP – genetic variants of dopamine D1receptor and D1–YFP).dMeasured in cell expressing dopamine D2receptor coupled toCFP (D2–CFP).eMeasured in cell co-expressing two dopamine D2receptor fusion proteins (D2–CFP and D2–YFP).fMeasured in cellco-expressing two dopamine D2receptor fusion proteins (D2–CFPand D2R3–YFP – genetic variant of dopamine D2receptor).S. Łukasiewicz et al. Dopamine D1and D2receptors dimerizationFEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 767show that there was no FRET fluorescence after nor-malizing and making corrections for cross-talk. Inexperiments investigating receptor interactions, thatwas the case; FRET was observed only when thereceptor was co-expressed with the appropriate a sub-unit of the G protein and not in the other case.Although there is discussion in the literature concern-ing the possibilities of photoconversion of YFP into aCFP-like species during acceptor photobleachingFRET experiments, we, as well as others, can excludethat such photoconversion interferes with FRETmeasurements under standard conditions.Two acidic residues in the C-terminal end of the D1receptor, as well as the Arg-rich region of ic3 of the D2receptor, do not seem to take part in receptorhomodimerization, but they do influence D1–D2recep-tor heterodimerization. Replacing the C-tail Glu resi-dues with Ala significantly decreased the FRET signal,as measured by changes in the fluorescence lifetimes.Also, the degree of D1–D2receptor heterodimerizationstrongly depended on the number of Arg residues thatwere replaced by Ala in the Arg-rich region of ic3 (resi-dues 217–222) of the dopamine D2receptor. The effi-ciency of energy transfer in the wild-type of the D1andD2heterodimer was $ 4% and decreased to 2.1% uponreplacing the first two Arg. Replacement of an addi-tional two Arg residues in ic3 caused a further decreasein the FRET efficiency by $ 50 to 1.26%. When all res-idues in the basic region of the D2receptor werereplaced, only a marginal level of energy transfer wasobserved (0.44%). A similar effect on energy transferwas observed after the replacement of two acidic Gluresidues in the C-tail of the D1receptor. The efficiencyof energy transfer was reduced to 0.8%. A possibleinterpretation of the data suggests that the indicatedbasic region of ic3 of the D2receptor and acidic regionof the C-tail of the D1receptor might be involved inthe interactions between the two dopamine receptors.In addition, the subcellular localization of D1–CFP,D2–YFP and all the mutants of both receptors wasexamined in cells expressing one or both types ofreceptors using confocal microscopy. In cotransfectedcells, both the D1and D2receptors were found in theplasma membrane, but a portion of both receptorswas also present inside the cell. Similar results wereobtained by So et al., suggesting that these receptorswere assembled as hetero-oligomers in intracellularcompartments [14].Based on the results obtained with confocal micros-copy, we conclude that the mutation in the C-tail ofthe D1receptor did not change the localization of thereceptor because both wild-type D1and the mutantwere localized in the cell membrane. However, the D2receptor was localized at the cell surface with a consid-erable portion also present within the cell. Analysis ofcells containing the D1and D2receptors, as well ascells expressing D1MUT and D2, showed that the levelof colocalization was very similar. This result clearlyindicates that the significant decrease in energy transferobserved between D1MUT and D2is the effectof impaired heterodimerization of the dopaminereceptors.Moreover, confocal microscopy experiments revealedthat modification of the Arg-rich region in the ic3 ofthe D2receptor substantially changed its receptor traf-ficking properties. The binding experiments alsopointed to a decrease in the density of the D2R vari-ants in the cellular membrane; the number of D2receptor binding sites decreased with the number ofchanged Arg residues in the ic3. When compared withwild-type receptor, the binding of [3H]spiperone toD2R1 and D2R2 showed a significant decrease in theBmax, 50 and 85%, respectively. In the case where thewhole region between amino acids 217 and 222 wasexchanged, we were unable to detect any D2receptorin the membrane. The results obtained by confocalmicroscopy show that the D2R3 mutant was mainlylocalized in the cytoplasmic compartments. However,cotransfection with wild-type D2R changed the distri-bution of this protein. This suggests that wild-type D2receptor can modulate the localization of the D2R3mutant receptor. We did not observe such an effect incells expressing the dopamine D1and D2R3 receptors.The D2R3 receptor was observed only in the cytoplas-mic compartments, similar to the situation when it wasexpressed alone. The difference might result from thefact that wild-type D2–D2R3 homodimers are beingcreated during D2receptor biosynthesis, whereas thatprocess does not take place in the case of D1-D2R3co-expression. It is probably the direct interactionsbetween the D2and the D2R3 receptor mutant thatreduced efficiency in the trafficking of the wild-typereceptor to the cell surface. These observations areconsistent with data showing that co-expression of aC- or N-terminal-truncated D2receptor with the wild-type receptor resulted in attenuation of binding andreduced efficiency in the trafficking of the wild-type D2receptor [61].The construction of genetic variants of the studieddopamine receptors, which were supposed to prove thecontribution of the indicated residues to the formationof D1–D2receptor heterodimers, did not provide a clearanswer to the question posed at the beginning of thestudy. From the FRET experiments, it may be unequiv-ocally concluded that the acidic C-terminal residues ofthe D1receptor are engaged in heterodimerization, butDopamine D1and D2receptors dimerization S. Łukasiewicz et al.768 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBSnot in homodimerization, as the efficiency of energytransfer is the same for wild-type D1receptor as forD1–D1MUT. Both of these receptors are localized inthe cell membrane, as can be seen with confocalmicroscopy. Therefore, it can also be concluded thatthe C-terminal acidic residues are by no meansinvolved in the regulation of D1receptor membranelocalization.However, genetically manipulating the Arg-rich epi-tope in the ic3 of the D2receptor induced alterations inthe cellular localization of the resulting receptor pro-teins. If not for confocal microscopy, which allowed forthe visualization of receptor localization, the gradualdecrease in the degree of D1–D2receptor (and its vari-ants) heterodimerization that was observed in FRETexperiments could have been interpreted as a directindication of the role of the Arg-rich epitope in the for-mation of heterodimers, as had been done in case ofadenosine A2A–dopamine D2heterodimerization [43].However, based on these data, we have to concludethat the Arg-rich epitope in the ic3 loop of D2is alsoresponsible for receptor localization. The lack of energytransfer between the YFP-tagged D2receptor geneticvariants and CFP-tagged D1receptor can result fromthe different localization of these proteins in the cell.The molecular mechanisms underlying the transportprocesses of GPCRs from the ER to the cell surfacehave recently become the subject of extensive studies[62]. The conserved sequences ⁄ motifs in the D2R,essential for their exit from the ER, are currentlyunder investigation. ER export is the first step in intra-cellular trafficking of GPCRs and is a highly regulatedevent in the biogenesis of GPCRs. Sequence motifsplay a crucial role in the targeting of polypeptides tothe plasma membrane. The Arg-rich motif in D2Rmight also be a potential trafficking signal. Suchmotifs serve as endoplasmic reticulum retention signalsthat prevents the export of proteins to the plasmamembrane. There are three types of retention motifsidentified in the cytosolic domains of various proteins:KDEL, KKXX and RXR motifs [62,63]. The RXRmotif (also three or four repeated Arg residues)actively precludes the exit of the protein from theendoplasmic reticulum [62,64,65]. Under normal condi-tions, this motif is masked, and proteins are trans-ported to the cell surface without significantaccumulation in the endoplasmic reticulum. If the Arg-rich motif in D2R serves as a retention signal, thenreplacing adjacent Arg residues should increase thesurface expression of D2R. We observed the oppositeeffect; the Arg-rich sequence in the cytoplasmic ic3loop of D2R does not act as an endoplasmic reticulumretention signal. Misfolding of the D2R2 and D2R3mutants could potentially be responsible for their accu-mulation in the endoplasmic reticulum because onlyprotein that has assumed its native conformation isavailable for recruitment into the transport vesiclesleaving the endoplasmic reticulum. Therefore, the Arg-rich motif might be responsible for interactions withcytoskeletal proteins. Binda et al. have shown thatcytoskeletal protein 4.1 N, a member of the 4.1 family,facilitates the transport of D2R to the cell surface byinteracting with the N-terminal portion of the ic3 loopof D2R via its C-terminal domain [66]. Truncationanalysis localized a region of interaction within resi-dues 211–241 of D2R. Because this study used geneticvariants of D2R that lacked either 2, 4 or 6 residuesfrom the 217–222 motif of ic3, and the cellular locali-zation of these mutants depended on the number ofthe basic residues exchanged for Ala, it may beconcluded that proper interaction with protein 4.1 Nmight have been disturbed. Therefore, the D2Rmutants stay in the endoplasmic reticulum and are nottransported to the cell membrane.Intracellular signaling pathway components, such asheteromeric G proteins and adenylate cyclase, are pres-ent in the endoplasmic reticulum and Golgi apparatus[67]. Because the intracellular localization of the dopa-mine D2receptor has been also described in the stria-tum [68], it seems that elucidation of the mechanismsresponsible for fine tuning of receptor trafficking, aswell as its dimerization with other receptor partners, isvery important for understanding the rules that governreceptor activity, both in physiological and patholo-gical conditions.Receptor dimerization, which is important for trans-membrane signal generation [54], also plays a role inintracellular trafficking of receptors and controllingtheir folding status. As suggested by So et al., hetero-oligomerization, by changing the exposure or maskingmotifs responsible for endoplasmic reticulum retentionor export, may be a strong regulator of the cellulardistribution of receptors [14].Incorrect membrane localization of D2R after modi-fication within ic3 217–222 region (observed in the cellsco-expressing D1R and D2R3) can result from defec-tive interactions with cytoskeletal proteins as well asfrom impaired heterodimerization with D1R. When inthe cell both D2R3 mutant and D2R wild-type arepresent, most likely the D2R may help D2R3 toachieve the cell-surface receptor dimerization. Similarsituation has been described by Concepcion et al. Theyhave shown that rhodopsin mutant devoid of traffick-ing signal motif localized in the plasma membranewhen it was co-expressed with the wild-type receptor,as a results of both proteins oligomerization [69].S. Łukasiewicz et al. Dopamine D1and D2receptors dimerizationFEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 769[...]... Construction of genetic variants of the dopamine receptors The following genetic variants of the dopamine receptors were constructed: three variants of the dopamine D2 receptor in which six amino acid residues (two each) from the arginine-rich epitope (217RRRRKR222) of the third intra- 770 cellular loop were exchanged (D2R1: 217AARRKR222, D2R2: 217AAAAKR222, D2R3: 217AAAAAA222), as well as one variant of the. .. fluorescently tagged D1 and D2 receptor protein) The average intensity of the fluorescence signal was measured for every image in a determined individual area of interest free of cell culture and subtracted as a background For analysis these regions were used of which fluorescence intensities were correlated For each combination of proteins, a minimum of 20 individual regions from different, independently transfected... concentration of 20 and 40 lgÆtube)1 for the D1 and D2 dopamine receptor, respectively) using concentrations of [3H]SCH23390 ranging from 0.06 to 6 nm or concentrations of [3H]spiperone ranging from 0.01 to 4 nm Nonspecific binding was assessed by the addition 10 lm cis-(Z)-flupentixol (Lundbeck, Copenhagen, Denmark) for the dopamine D1 receptor or 50 lm butaclamol (Research Biochemicals Inc., Natick,... min [3H]SCH23390 (specific activity of 86 CiÆmmol)1; NEN, Boston, MA, USA) was used as the dopamine D1 receptorspecific radioligand, and [3H]spiperone (specific activity of 15.7 CiÆmmol)1; NEN) was used as the dopamine D2 receptor- specific radioligand Binding assays were performed in a total volume of 500 lL Saturation studies were carried out on a fresh membrane preparation (final protein concentration... respectively, were used as the mold for the PCRQuik reaction Incorporating the oligonucleotide primers, each complementary to the opposite strand of the vector and containing the desired mutations, generated a mutated plasmid The resulting product was treated with endonuclease DpnI, specific for methylated and hemimethylated DNA, in order to select synthesized DNA containing the introduced mutations E coli DH5a... PCR-amplified The forward primer was universal for pcDNA3.1(+), and the reverse primers removed the stop codons and introduced a unique restriction site, XhoI, for both dopamine receptors and SacI for stimulatory and inhibitory G protein subunit The resulting fragments were inserted, in- frame, into the NheI ⁄ XhoI (dopamine receptors) or NheI ⁄ SacI (Ga subunits) sites of the pECFP–N1 and pEYFP–N1 vectors Construction... George SR (2003) D2 dopamine receptor homodimerization is mediated by multiple sites of interaction, including an intermolecular interaction involving transmembrane domain 4 Biochemistry 42, 11023–11031 20 Nemoto W & Toh H (2005) Prediction of interfaces for oligomerizations of G -protein coupled receptors Proteins 58, 644–660 21 Romano C, Yang WL & O’Malley KL (1996) Metabotropic glutamate receptor 5 is... muscarinic receptor dimers J Biol Chem 274, 19487–19497 Bai M (2004) Dimerization of G -protein- coupled receptors: roles in signal transduction Cell Signal 16, 175–186 Bouvier M (2001) G protein- coupled receptor oligomerization: implications for G protein activation and cell signaling Nat Rev Neurosci 2, 274–286 Hebert TE & Bouvier M (1998) Structural and functional aspects of G protein- coupled receptor oligomerization. .. Kong MM, Alijaniaram M, Ji X, Nguyen T, O’Dowd BF & George SR (2005) D1 and D2 dopamine receptors form heterooligomers and cointernalize after selective activation of either receptor Mol Pharmacol 68, 568–578 15 Terrillon S & Bouvier M (2004) Roles of G-proteincoupled receptor dimerization EMBO Rep 5, 30–34 16 Cvejic S & Devi LA (1997) Dimerization of the delta opioid receptor: implication for a role. .. measurements and binding assays or on glass cover slips in 30 mm dishes at a density of 1 · 106 cells per dish for fluorescence lifetime measurements and confocal imaging They were transfected with 12 lg of DNA per 100 mm dish and 2 lg of DNA per 30 mm dish The ratio of DNA coding donor to DNA coding acceptor was 1 : 1 or 1 : 2 Membrane preparation and radioligand binding assay For binding experiments, the transfected . Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization Sylwia. These experiments were performed todetermine the in uence of the introduced mutations on the localization of the receptor proteins and the degree of their
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