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

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D 1 and D 2 receptor oligomerization Sylwia Łukasiewicz 1 , Agata Faron-Go ´ recka 2 , Jerzy Dobrucki 3 , Agnieszka Polit 1 and Marta Dziedzicka-Wasylewska 1,2 1 Department of Physical Biochemistry, Jagiellonian University, Krako ´ w, Poland 2 Laboratory of Biochemical Pharmacology, Polish Academy of Sciences, Krako ´ w, Poland 3 Division of Cell Biophysics, Jagiellonian University, Krako ´ w, Poland Various 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 be physiologically active as dimers in the plasma mem- brane [1,2]. These molecules can both homo- and heterodimerize. 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 transduction speed and specificity [1,5,9–12], and receptor desensiti- zation [5,13–16]. Interactions between different classes Keywords Arg-rich motif; dopamine D 1 receptor; dopamine D 2 receptor; FRET; GPCR oligomerization Correspondence M. Dziedzicka-Wasylewska, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University 7 Gronostajowa Street, Krakow, Poland Fax: +48 012 664 6902 or +48 012 637 4500 Tel: +48 012 664 6122 or +48 012 662 3372 E-mail: wasyl@if-pan.krakow.pl or marta.dziedzicka-wasylewska@uj.edu.pl (Received 1 August 2008, revised 19 November 2008, accepted 27 November 2008) doi:10.1111/j.1742-4658.2008.06822.x We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D 2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D 1 receptor (404EE405) on the receptors’ localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D 1 –D 2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D 2 R, which may suggest that the indicated region of ic3 in D 2 R might be involved in interactions between two dopa- mine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D 1 –cyan fluorescent protein, D 2 –yellow fluorescent protein, and various mutants was examined by confocal micros- copy. Genetic manipulations of the Arg-rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that 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 D 2 R and cyan fluorescent protein-tagged D 1 R may result from differing localization of these proteins in the cell rather than from the possible role of the D 2 R basic domain in the mechanism of D 1 –D 2 receptor heterodimerization. However, we find that the acidic epitope from the C-terminus of the dopamine D 1 receptor is engaged in the heterodimerization process. Abbreviations CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; GBR, GABA B receptor; 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 FEBS of GPCRs point to a new level of molecular cross-talk among signaling molecules [1,5,11,17]. Structural information about receptor dimer forma- tion is currently limited, and the question of whether receptors dimerize in a similar way or have their own paths of dimerization remains open. In general, either covalent or noncovalent interactions are involved in this 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 been shown that cysteine residues located in the extracellu- lar loops are essential for disulfide-linked m3 musca- rinic receptor (M3R) dimer formation; however this kind of interaction is not the only point of contact [37]. For GABA B receptors (GBR), a coiled-coil inter- action within the C-tail of GBR1 and GBR2 seems to be involved in receptor heterodimerization. However, this motif is not necessary, as deleting the C-tail does not abolish dimerization. Also, hydrophobic interac- tions within the TM of GPCRs are essential for forma- tion and stabilization of the dimers and have been detected for beta-adrenergic, dopamine, muscarinic and angiotensin receptors [38–40]. In earlier studies, the role of certain amino acid resi- dues in the formation of noncovalent complexes between protein molecules was highlighted. Electro- static interactions occur between an epitope containing mainly two or more adjacent arginine residues on one protein fragment and an acidic epitope containing two or more adjacent glutamate or aspartate residues, and ⁄ or a phosphorylated residue, on the other protein [41,42]. Ciruela et al. demonstrated that electrostatic interactions between an arginine-rich epitope from the third intracellular loop of the D 2 receptor and two adjacent aspartate residues or a phosphorylated serine residue in the C-terminus of the A 2A receptor are involved in heterodimerization between the adeno- sine A 2A receptor and the dopamine D 2 receptor [43]. A similar interaction has also been shown for D 1 –NMDA receptor heterodimers [44]. Although the dopamine D 1 and D 2 receptor sub- classes are biochemically distinct, coactivation of both receptors has been shown to be essential for their physi- ological function. The view that these receptors may also function as a physically linked unit is especially important because recent data suggest that the D 1 and D 2 receptors 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 D 1 and D 2 receptors in the caudate and pyramidal cells in the rat frontal cortex [47]. Earlier studies by Vincent et al. have also shown that the lami- nar distribution of medial prefrontal cortex neurons expressing both D 1 and D 2 receptors was similar to that of the mesocortical dopamine afferents [48]. The dopamine D 2 receptor can form homodimers [19]. Recently, we have shown that the D 2 receptor also forms heterodimers with the dopamine D 1 recep- tor [49]; however, the precise role of specific regions of receptor molecule(s) in that process has not yet been elucidated. In this study, we investigated the role of an epitope from the third intracellular loop (ic3) of the dopamine D 2 receptor, which contains adjacent argi- nine residues (217RRRRKR222), and an acidic epi- tope from the C-terminus of dopamine D 1 receptor (404EE405) on the D 1 –D 2 receptor interaction. Fluorescence resonance energy transfer (FRET) occurs between fluorescence donor and acceptor chro- mophores when they are located within 100 A ˚ of each other and are arranged properly in terms of their tran- sition dipole moments [50]. Using this technique, we studied receptor dimer formation using fluorescence lifetime microscopy and time-correlated single photon counting (TCSPC) measurements. The receptor pro- teins were tagged with cyan (CFP; fluorescence donor) and yellow fluorescent proteins (YFP; fluorescence acceptor) and expressed in HEK293 cells. We find FRET to be a very sensitive tool, and measurements are especially useful to quantitatively monitor the physical interactions between receptor proteins [51,52]. Results Radioligand binding assay As shown in Table 1, the binding parameters obtained for dopamine D 1 receptor and its mutant indicate that the K d values for these two receptors were similar; however, the density of the D 1 MUT (404AA405) was Table 1. Binding parameters for the dopamine receptors. For dopa- mine D 2 receptor binding, the statistical significance was evaluated using a one-way ANOVA, followed by a Dunnett’s test for post hoc comparison. *P < 0.05. For dopamine D 1 receptor binding, the statistical significance was evaluated using a Student’s t-test; ***P < 0.001. Species B max ± SEM (pmolÆmg )1 protein) K d ± SEM (n M) D 1 –CFP 14.66 ± 0.13 1.50 ± 0.08 D 1 MUT–CFP 9.85 ± 0.08*** 1.20 ± 0.06 D 2 –YFP 4.88 ± 0.10 0.41 ± 0.06 D 2 R1–YFP 2.53 ± 0.08* 0.44 ± 0.03 D 2 R2–YFP 0.70 ± 0.07* 0.44 ± 0.09 S. Łukasiewicz et al. Dopamine D 1 and D 2 receptors dimerization FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 761 lower than that of wild-type D 1 R (Fig. 1A). Also, all three genetic variants of dopamine D 2 R displayed sim- ilar K d values, but the density of these receptors strongly depended on the number of Arg residues still present within the receptor sequence. The D 2 R1 (217AARRKR222) mutant displayed half of the B max value obtained for D 2 R, whereas the density of the D 2 R2 (217AAAAKR222) mutant was much lower (Fig. 1B). For the D 2 R3 (217AAAAAA222) variant, no binding parameters could be obtained, which indi- cates that there was no receptor protein in the cellular membrane. This conclusion is further justified by confocal microscopy analysis of receptor localization. Analysis of the localization of dopamine D 1 ,D 2 and their genetic variant fusion proteins Confocal microscopy was used to visualize HEK293 cells co-expressing the dopamine D 1 and D 2 receptors, as well as their genetic variants (D 1 MUT, D 2 R1, D 2 R2, D 2 R3). These experiments were performed to determine the influence of the introduced mutations on the localization of the receptor proteins and the degree of their colocalization. Figure 2A,B shows HEK293 cells transiently cotransfected with plasmids encoding the dopa- mine D 1 , dopamine D 2 ,D 1 MUT, D 2 R1, D 2 R2 and D 2 R3 receptors in different combinations. Merged pic- tures with apparent yellow signal indicating overlap of green fluorescent signal (CFP channel) and red fluores- cent signal (YFP channel) show colocalization. As seen from the figures, these receptor proteins were localized differentially in the cell. Cell edge sharp- ness confirms that dopamine D 1 and D 1 MUT recep- tors localize in the plasma membrane, in contrast to the dopamine D 2 receptor and its genetic variants, D 2 R1, D 2 R2, which were localized in the plasma mem- brane and inside the cell. In the case of the dopa- mine D 2 receptor mutants, the degree of membrane localization depended on the number of mutated resi- dues in the ic3 region (D 2 217–222). The dopamine D 2 R3 receptor location was very interesting and surprising. As seen in Fig. 2A, which shows a cell co-expressing both D 1 –CFP and D 2 R3– YFP fusion proteins, these receptors were found in dif- ferent parts of the cell. The D 2 R3 mutant was localized inside the cell, whereas the D 1 receptor was found in the plasma membrane. However, when the cell co-expressed both types of D 2 receptors, i.e. the wild- type and the D 2 R1, D 2 R2 as well as D 2 R3 variant, colo- calization was observed in both the plasma membrane and inside the cell. For a quantitative estimation of the degree of colocalization between the two different pro- teins of interest, Pearson’s correlation coefficients and coefficients of determination were estimated (Fig. 2C). In case of cells co-expressing dopamine D 1 and dopa- mine D 2 receptor mutants, the degree of colocalization decreased, which was correlated with number of exchanged residues within the ic3 of D 2 receptor. When cells were cotransfected with the same type of receptors (D 1 MUT–CFP ⁄ D 1 –YFP, D 2 –CFP ⁄ D 2 R1– YFP, D 2 –CFP ⁄ D 2 R2–YFP, D 2 –CFP ⁄ D 2 R3–YFP) and with dopamine D 2 and genetic variant dopamine D 1 receptors (D 1 MUT–CFP ⁄ D 2 –YFP) the obtained values of coefficients remained approximated. Fluorescence spectroscopy measurements of dopamine receptor dimerization Although steady-state fluorescence spectroscopy mea- surements in cell suspension enable only the qualitative estimation of the FRET phenomenon, this approach is Fig. 1. Saturation binding of [ 3 H]SCH23390 (A) and [ 3 H]-spiperone (B) to human D 1 and D 2 dopamine receptors, respectively. Data are from a single experiment performed in triplicate and are representa- tive of at least three independent experiments. Elimination of the Arg-rich or di-Glu motif in D 2 RorD 1 R, respectively, does not alter the ligand binding constant. Dopamine D 1 and D 2 receptors dimerization S. Łukasiewicz et al. 762 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS very demonstrative and gives a quick answer to whether there is any energy transfer in the examined sample. Therefore, we used this type of measurement to investigate interactions between the dopamine D 1 and D 2 receptors and their genetic variants. Fluores- cence emission profiles for the HEK293 cell suspension expressing fusion proteins in different combinations (D 1 –CFP ⁄ D 2 –YFP, D 1 –CFP ⁄ D 2 R1–YFP, D 1 –CFP ⁄ D 2 R2–YFP, D 1 CFP ⁄ D 2 R3–YFP, D 1 MUT–CFP ⁄ D 2 –YFP, D 1 –CFP ⁄ D 1 –YFP, D 1 MUT–CFP ⁄ D 1 –YFP, D 2 –CFP ⁄ D 2 –YFP and D 2 –CFP ⁄ D 2 R3–YFP) were compared using an excitation wavelength of 434 nm (donor absorption). The upper panel of Fig. 3 shows emission spectra of HEK293 cell populations after cotransfection with plasmids encoding genes for dopamine D 1 and D 2 receptor fusion proteins (D 1 –CFP and D 2 –YFP) in comparison with emission spectra of the cell popula- tions that co-express dopamine D 1 receptor fusion protein (D 1 –CFP) and one of the genetic variants of dopamine D 2 receptor fusion protein (D 2 R1, D 2 R2 or D 2 R3–YFP) (Fig. 3A). In Fig. 3B, the results presented are from a cell suspension expressing the dopamine D 2 –YFP fusion protein and the genetic vari- ant of the dopamine D 1 receptor (D 1 MUT–CFP) fusion protein. We observed energy transfer between wild-type dopamine D 1 and D 2 receptors, but when either the genetic variant of dopamine D 1 (D 1 MUT) or the D 2 R3 genetic variant of the dopamine D 2 recep- tor was present in the sample, there was no visible energy transfer, despite the presence of both fluoro- phores in the sample. Figure 3C,D shows the emission profiles of cells cotransfected with plasmids encoding genes for the same type of dopamine receptor (D 1 or D 2 , respec- tively), tagged with different fluorescence proteins, A B C Fig. 2. Expression of D 1 R and D 2 R and their mutants in HEK293 cells. (A) HEK293 cells were cotransfected with either D 1 –CFP or D 1 MUT– CFP and either D 2 –YFP, D 2 R1–YFP, D 2 R2–YFP, D 2 R3–YFP or D 1 MUT–YFP (green and red). Image overlays show extensive colocalization in D 1 ⁄ D 1 ,D 1 ⁄ D 1 MUT, D 1 ⁄ D 2 and D 1 ⁄ D 2 R1 assays and partial colocalization in D 1 ⁄ D 2 R2 assays. D 1 ⁄ D 2 R3 does not colocalize. (B) HEK293 cells were cotransfected with D 2 –CFP and either D 2 –YFP, D 2 R1–YFP, D 2 R2–YFP or D 2 R3–YFP. Image overlays show extensive colocalization in every case. (C) Bar graph of Pearson‘s correlation coefficient calculated for HEK293 cells cotransfected with different dopamine D 1 and D 2 receptor 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 D 1 with all variants of D 2 versus D 1 ⁄ D 2 . Either D 2 ⁄ D 2 R1, D 2 ⁄ D 2 R2 or D 2 ⁄ D 2 R3 versus D 2 ⁄ D 2 , D 1 MUT ⁄ D 1 versus D 1 ⁄ D 1 , and D 1 MUT ⁄ D 2 versus D 1 ⁄ D 2 combinations are not statistically significant. Values of corresponding coefficients of determination (r 2 ) are reported in brackets. S. Łukasiewicz et al. Dopamine D 1 and D 2 receptors dimerization FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 763 compared with emission profiles of cells in which one of the tagged receptors was its own mutant (D 1 –CFP ⁄ D 1 –YFP and D 1 MUT–CFP ⁄ D 1 –YFP or D 2 –CFP ⁄ D 2 –YFP and D 2 –CFP ⁄ D 2 R3–YFP). The lower panel of Fig. 3 shows that both dopamine receptors, D 1 and D 2 , form homo-oligomeric structures and confirms that both of the investigated epitopes are probably not engaged in the homodimerization process. In both cases, we observed efficient energy transfer, which can be judged by the localization of the appropriate peaks of the spectra. To serve as a control for this experiment, we co-expressed the a subunits of the G protein, a i and a s tagged with CFP with dopamine D 1 or D 2 receptors, which were tagged with YFP. As seen in Fig. 4, the FRET phenomenon takes place only when the D 1 receptor is co-expressed with a s or D 2 receptor is co-expressed with a i . The interactions are specific because no energy transfer was observed following co-transfection of D 1 –YFP ⁄ a i –CFP or D 2 YFP ⁄ a s CFP, despite the identical overexpression level of the proteins in all studied combinations. Fluorescence lifetime microscopy studies of dopamine receptor dimerization Time-correlated single-photon counting experiments were performed on the inverted fluorescence micro- scope. The FRET phenomenon was observed in a single living cell transiently transfected with the dopamine D 1 and D 2 receptors and their genetic variants, tagged with fluorescent proteins. This kind of measurement provides highly quantifiable data because it is independent of any change in fluorophore concen- tration or excitation intensity. To determine FRET efficiency, precise measurement of the donor fluorescence lifetime (CFP), in the pres- ence and absence of the acceptor (YFP), is required. A C B D Fig. 3. Fluorescence emission spectra of HEK293 cells expressing the CFP- and YFP-tagged proteins coupled to D 1 R and D 2 R and their mutants. (A) Cotransfection of HEK293 with D 1 –CFP and either D 2 –YFP (gray dashed line), D 2 R1–YFP (black line), or D 2 R2–YFP (gray line) or D 2 R3–YFP (black dashed line). (B) Cotransfection of HEK293 with D 1 MUT–CFP and D 2 –YFP (gray line) in comparison with D 1 –CFP and D 2 –YFP (black line). (C) Cotransfection of HEK293 with D 1 MUT–CFP and D 1 –YFP (gray line) in comparison with D 1 –CFP and D 1 –YFP (black line). (D) Cotransfection of HEK293 with D 2 –CFP and D 2 R3–YFP (gray line) in comparison with D 2 –CFP and D 2 –YFP (black line). CFP was excited at 434 nm, and fluorescence was detected at 450–550 nm through a double monochromator. The spectral contributions arising from light scattering and nonspecific fluorescence of cells and buffer were eliminated. Dopamine D 1 and D 2 receptors dimerization S. Łukasiewicz et al. 764 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS Fluorescence decays were analyzed as both mono- and multi-exponentials. Analysis of the reduced chi-squared value and residual distribution led to the conclusion that best fit parameters were obtained with two expo- nentials. Adding a third exponential did not signifi- cantly influence the parameters, and the fractional contribution of the additional lifetime was close to zero. Figure 5 shows the typical time-dependent donor decays for the D 1 –CFP bearing donor alone and with the donor and acceptor D 1 –CFP ⁄ D 1 MUT–YFP. The average CFP fluorescence lifetime obtained during TCSPC experiments was 2.37 ns, and the value changed when acceptor was present in a cell. The greatest average fluorescence lifetime decrease (to 1.52 ns), which was regarded as the highest FRET effi- ciency ($ 36%), was detected in our earlier studies for the CFP–YFP hybrid (CFP connected by a short 15 amino acid linker with YFP) [49]. Measurements on the cells co-expressing dopa- mine D 1 and D 2 receptor fusion proteins indicated $ 4% efficiency of energy transfer, with an average donor fluorescence lifetime of 2.27 ns. This changed when the dopamine D 2 receptor was replaced by a genetic variant (D 2 R1, D 2 R2 or D 2 R3) and also when D 1 MUT was used instead of the dopamine D 1 recep- tor. Transfer efficiency was equal to 2.1% (2.32 ns) for A B C D Fig. 4. Representative fluorescence emission spectra of HEK293 cells cotransfected with either D 1 –YFP or D 2 –YFP and Ga–CFP fusion pro- teins. (A) Negative FRET control, spectra from a 1 : 1 mixture of cells individually expressing the Ga S –CFP (black line) fusion protein (excited at 434 nm) and the D 1 –YFP (gray line) fusion protein (excited at 475 nm). (B) Cotransfection of HEK293 cells with D 1 –YFP and Ga S –CFP (gray line) or D 1 –YFP and Ga I –CFP (black line), excited at 434 nm. (C) Negative FRET control, spectra from a 1 : 1 mixture of cells individually expressing Ga I –CFP (black line) fusion protein (excited at 434 nm) and D 2 –YFP (gray line) fusion protein (excited at 475 nm). (D) Cotransfec- tion of HEK293 cells with D 2 –YFP and Ga I –CFP (gray line) or D 2 –YFP and Ga S –CFP (black line), excited at 434 nm. Fluorescence was detected 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 D 1 and D 2 receptors dimerization FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 765 D 1 ⁄ D 2 R1, further decreased to 1.26% (2.34 ns) for D 1 ⁄ D 2 R2, and finally reached the value of 0.44% (2.36 ns) for D 1 ⁄ D 2 R3. The lowest E value, similar to that obtained for the D 1 ⁄ D 2 R3 combination, was observed for D 1 MUT ⁄ D 2 R3 and was equal to 0.4% (2.36 ns). A similar result (0.8%; 2.35 ns) was obtained for cells co-express- ing the dopamine D 1 receptor mutant (D 1 MUT) and the wild-type dopamine D 2 receptor, as donor and acceptor of fluorescence, respectively. However, when the cells were cotransfected with plasmids encoding genes for the same type of dopa- mine receptors, D 1 or D 2 , and when one of the appro- priate receptors was replaced by its mutant (D 1 by D 1 MUT or D 2 by D 2 R3), no change in transfer effi- ciency was detectable. The E value for D 1 MUT ⁄ D 1 was estimated to be 7.8% (2.19 ns) versus 8% (2.18 ns) for D 1 ⁄ D 1 , while for D 2 ⁄ D 2 R3, it equaled 3.4% (2.29 ns) versus 3.5% (2.28 ns) for D 2 ⁄ D 2 combi- nations. The summary of TCSPC results is presented in Tables 2 and 3. The error of the average fluorescence lifetime is the standard error of mean obtained from different cells and independent transfections (we ignored standard deviations derived from fitting of individual fluorescence decay because they were very small). Discussion The data provided from numerous studies indicate that oligomerization may play important roles in receptor trafficking and ⁄ or signaling. In several cases, receptors appear to fold into constitutive dimers early after bio- synthesis, although ligand-promoted dimerization at the cell surface has been also proposed [53]. Many GPCRs have been shown to participate in homo- or heterodimerization [54]. Using a biophysical approach, we had previously shown that the D 2 and D 1 dopa- mine receptors exist as functional homo- and hetero- oligomers in cell lines [49], and similar conclusions can be drawn from biochemical studies [14,19,55,56]. However, the exact sequence motifs responsible for that interaction had not been identified. In family 1 receptors, robust hydrophobic TM interactions have been proposed as the most probable structural ele- ments involved in oligomerization [27,57,58]. Some Fig. 5. Time-dependent fluorescence intensity decays of CFP attached to the D 1 receptor with and without YFP attached to the D 1 MUT receptor. The black dotted curve shows the intensity decay of the donor alone (D), and the dark gray dotted curve shows the intensity decay of the donor in the presence of acceptor (DA). The black solid lines and weighted residuals (lower panels) are for 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 up at 434 nm, and emission was observed through the appropriate interference filters, as described in Experimental procedures. The standard errors of means (obtained from at least 15 single cells) are presented in parentheses. Statistical significance was evaluated using Student’s t-test; *P < 0.05 versus D 1 –CFP ⁄ D 2 –YFP. Species Average lifetime (ns) Transfer efficiency ÆEæ (%)Æs D æÆs DA æ D 1 –CFP a 2.37 ± 0.01 D 1 –CFP ⁄ D 2 –YFP b 2.27 ± 0.02 4.01 D 1 –CFP ⁄ D 2 R1–YFP c 2.32 ± 0.02 2.10* D 1 –CFP ⁄ D 2 R2–YFP d 2.34 ± 0.01 1.26* D 1 –CFP ⁄ D 2 R3–YFP e 2.36 ± 0.01 0.44* D 1 MUT–CFP ⁄ D 2 –YFP f 2.35 ± 0.02 0.80 D 1 MUT–CFP ⁄ D 2 R3–YFP g 2.36 ± 0.01 0.40 a Measured in cell expressing CFP coupled to the dopamine D 1 receptor. b Measured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D 1 –CFP and D 2 –YFP). c Measured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D 1 –CFP and D 2 R1–YFP – genetic variant of dopamine D 2 receptor). d Measured in cell co-expressing dopamine D 1 and D 2 fusion protein (D 1 –CFP and D 2 R2–YFP – genetic variant of dopamine D 2 receptor). e Mea- sured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D 1 –CFP and D 2 R3–YFP – genetic variant of dopamine D 2 receptor). f Measured in cell co-expressing dopamine D 1 and D 2 fusion pro- teins (D 1 MUT–CFP – genetic variant of dopamine D 1 receptor and D 2 –YFP). g Measured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D 1 MUT–CFP – genetic variant of dopa- mine D 1 receptor and D 2 R3–YFP – genetic variant of dopamine D 2 receptor). Dopamine D 1 and D 2 receptors dimerization S. Łukasiewicz et al. 766 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS experimental studies also suggested the participation of C- 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 the adenosine A 2A and dopamine D 2 receptors strongly depends on an electrostatic interaction between an Arg-rich epitope from the ic3 of the D 2 R (217RRRRKR222) and either the two adjacent Asp residues (DD 401–402) or a phosphorylated Ser374 in the C-tail of the A 2A R [43]. Because the dopamine D 1 R contains an acidic region on the C-terminus, like A 2A R, we designed experiments to determine whether a similar interaction is responsible for the heterodimerization of the D 2 receptor with the D 1 receptor. However, a different approach to that mentioned above was used to address this question. The receptor proteins under investigation were tagged with fluorescent proteins and transfected into HEK293 cells; their localization was then observed with the use of a confocal microscope. The degree of receptor dimerization was also judged by changes in fluorescence lifetime, which we find to be the most sensitive technique with which to measure FRET [49]. The results presented here indicate that dopa- mine D 1 and D 2 receptors form homo- and hetero- dimers; results that are in agreement with previously published data [19,49,55]. Measuring receptor dimer- ization by monitoring changes in the fluorescence life- time of probes linked to the receptors of interest seems the best approach in this kind of the study. Although the approach enables only qualitative estimation of FRET phenomenon, steady-state fluorescence spectros- copy measurements in suspension are also useful because they are very demonstrative. In this study, both approaches yield similar conclusions, although we are aware that quantitative results can only be obtained from fluorescence lifetime microscopy. An often-discussed problem when using biophysical techniques to study receptor oligomerization is that these experiments predominantly involve heterologous expression systems, which in most cases have been per- formed in cell lines transfected with the receptors of interest. Receptors are usually epitope-tagged and, in most cases, are overexpressed. Therefore, it has often been suggested that biophysical techniques characterize interaction 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 widely used in resonance energy transfer studies of membrane receptors, because these cells provide an accepted model in which fluorescently tagged receptor protein can be efficiently expressed. As reported by Mercier et al. [59], the extent of dimerization of b 2 -adrenergic receptors (shown by BRET) was unchanged over a 20-fold range of expression levels (from 1.4 to 26.3 pmolÆmg )1 protein). 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 are not merely artifacts derived from the high levels of expression that are often achieved in heterologous sys- tem. Results obtained in this study, concerning the dopamine D 1 and D 2 receptors and their interactions with the appropriate a subunits of G protein, further confirm that the use of advanced fluorescence techni- ques does indeed allow for the observation of true interactions. The dopamine D 1 receptor did not inter- act with Ga i , and the D 2 receptor did not interact with Ga s , although the physical contact of these receptors with their appropriate a subunit partners could indeed have been observed, despite the identical level of over- expression of the proteins in all studied combinations. The experiments described above serve as a control that must always be performed when using FRET to determine if two proteins interact. That control is to express (preferentially using the same expression con- struct in all experiments) two noninteracting fusion proteins that carry CFP and YFP in the same cell and Table 3. Summary of energy transfer measurements obtained by fluorescence lifetime microscopy in HEK293 cells. Excitation was set up at 434 nm, and emission was observed through appropriate interference filters, as described in Experimental procedures. The standard errors of means (obtained from at least 15 single cells) are presented in parentheses. Species Average lifetime (ns) Transfer efficiency ÆEæ (%)Æs D æÆs DA æ D 1 –CFP a 2.37 ± 0.01 D 1 –CFP ⁄ D 1 –YFP b 2.18 ± 0.01 8.00 D 1 MUT–CFP ⁄ D 1 –YFP c 2.19 ± 0.01 7.80 D 2 –CFP d 2.37 ± 0.02 D 2 –CFP ⁄ D 2 –YFP e 2.28 ± 0.02 3.50 D 2 –CFP ⁄ D 2 R3–YFP f 2.29 ± 0.01 3.40 a Measured in cell expressing CFP coupled to dopamine D 1 recep- tor. b Measured in cell co-expressing two dopamine D 1 receptor fusion proteins (D 1 –CFP and D 1 –YFP). c Measured in cell co-expressing two dopamine D 1 receptor fusion proteins (D 1 MUT– CFP – genetic variants of dopamine D 1 receptor and D 1 –YFP). d Measured in cell expressing dopamine D 2 receptor coupled to CFP (D 2 –CFP). e Measured in cell co-expressing two dopamine D 2 receptor fusion proteins (D 2 –CFP and D 2 –YFP). f Measured in cell co-expressing two dopamine D 2 receptor fusion proteins (D 2 –CFP and D 2 R3–YFP – genetic variant of dopamine D 2 receptor). S. Łukasiewicz et al. Dopamine D 1 and D 2 receptors dimerization FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS 767 show that there was no FRET fluorescence after nor- malizing and making corrections for cross-talk. In experiments investigating receptor interactions, that was the case; FRET was observed only when the receptor 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 a CFP-like species during acceptor photobleaching FRET experiments, we, as well as others, can exclude that such photoconversion interferes with FRET measurements under standard conditions. Two acidic residues in the C-terminal end of the D 1 receptor, as well as the Arg-rich region of ic3 of the D 2 receptor, do not seem to take part in receptor homodimerization, but they do influence D 1 –D 2 recep- 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 D 1 –D 2 receptor heterodimerization strongly depended on the number of Arg residues that were replaced by Ala in the Arg-rich region of ic3 (resi- dues 217–222) of the dopamine D 2 receptor. The effi- ciency of energy transfer in the wild-type of the D 1 and D 2 heterodimer was $ 4% and decreased to 2.1% upon replacing the first two Arg. Replacement of an addi- tional two Arg residues in ic3 caused a further decrease in the FRET efficiency by $ 50 to 1.26%. When all res- idues in the basic region of the D 2 receptor were replaced, only a marginal level of energy transfer was observed (0.44%). A similar effect on energy transfer was observed after the replacement of two acidic Glu residues in the C-tail of the D 1 receptor. The efficiency of energy transfer was reduced to 0.8%. A possible interpretation of the data suggests that the indicated basic region of ic3 of the D 2 receptor and acidic region of the C-tail of the D 1 receptor might be involved in the interactions between the two dopamine receptors. In addition, the subcellular localization of D 1 –CFP, D 2 –YFP and all the mutants of both receptors was examined in cells expressing one or both types of receptors using confocal microscopy. In cotransfected cells, both the D 1 and D 2 receptors were found in the plasma membrane, but a portion of both receptors was also present inside the cell. Similar results were obtained by So et al., suggesting that these receptors were assembled as hetero-oligomers in intracellular compartments [14]. Based on the results obtained with confocal micros- copy, we conclude that the mutation in the C-tail of the D 1 receptor did not change the localization of the receptor because both wild-type D 1 and the mutant were localized in the cell membrane. However, the D 2 receptor was localized at the cell surface with a consid- erable portion also present within the cell. Analysis of cells containing the D 1 and D 2 receptors, as well as cells expressing D 1 MUT and D 2 , showed that the level of colocalization was very similar. This result clearly indicates that the significant decrease in energy transfer observed between D 1 MUT and D 2 is the effect of impaired heterodimerization of the dopamine receptors. Moreover, confocal microscopy experiments revealed that modification of the Arg-rich region in the ic3 of the D 2 receptor substantially changed its receptor traf- ficking properties. The binding experiments also pointed to a decrease in the density of the D 2 R vari- ants in the cellular membrane; the number of D 2 receptor binding sites decreased with the number of changed Arg residues in the ic3. When compared with wild-type receptor, the binding of [ 3 H]spiperone to D 2 R1 and D 2 R2 showed a significant decrease in the B max , 50 and 85%, respectively. In the case where the whole region between amino acids 217 and 222 was exchanged, we were unable to detect any D 2 receptor in the membrane. The results obtained by confocal microscopy show that the D 2 R3 mutant was mainly localized in the cytoplasmic compartments. However, cotransfection with wild-type D 2 R changed the distri- bution of this protein. This suggests that wild-type D 2 receptor can modulate the localization of the D 2 R3 mutant receptor. We did not observe such an effect in cells expressing the dopamine D 1 and D 2 R3 receptors. The D 2 R3 receptor was observed only in the cytoplas- mic compartments, similar to the situation when it was expressed alone. The difference might result from the fact that wild-type D 2 –D 2 R3 homodimers are being created during D 2 receptor biosynthesis, whereas that process does not take place in the case of D 1 -D 2 R3 co-expression. It is probably the direct interactions between the D 2 and the D 2 R3 receptor mutant that reduced efficiency in the trafficking of the wild-type receptor to the cell surface. These observations are consistent with data showing that co-expression of a C- or N-terminal-truncated D 2 receptor with the wild- type receptor resulted in attenuation of binding and reduced efficiency in the trafficking of the wild-type D 2 receptor [61]. The construction of genetic variants of the studied dopamine receptors, which were supposed to prove the contribution of the indicated residues to the formation of D 1 –D 2 receptor heterodimers, did not provide a clear answer to the question posed at the beginning of the study. From the FRET experiments, it may be unequiv- ocally concluded that the acidic C-terminal residues of the D 1 receptor are engaged in heterodimerization, but Dopamine D 1 and D 2 receptors dimerization S. Łukasiewicz et al. 768 FEBS Journal 276 (2009) 760–775 ª 2008 The Authors Journal compilation ª 2008 FEBS not in homodimerization, as the efficiency of energy transfer is the same for wild-type D 1 receptor as for D 1 –D 1 MUT. Both of these receptors are localized in the cell membrane, as can be seen with confocal microscopy. Therefore, it can also be concluded that the C-terminal acidic residues are by no means involved in the regulation of D 1 receptor membrane localization. However, genetically manipulating the Arg-rich epi- tope in the ic3 of the D 2 receptor induced alterations in the cellular localization of the resulting receptor pro- teins. If not for confocal microscopy, which allowed for the visualization of receptor localization, the gradual decrease in the degree of D 1 –D 2 receptor (and its vari- ants) heterodimerization that was observed in FRET experiments could have been interpreted as a direct indication of the role of the Arg-rich epitope in the for- mation of heterodimers, as had been done in case of adenosine A 2A –dopamine D 2 heterodimerization [43]. However, based on these data, we have to conclude that the Arg-rich epitope in the ic3 loop of D 2 is also responsible for receptor localization. The lack of energy transfer between the YFP-tagged D 2 receptor genetic variants and CFP-tagged D 1 receptor can result from the different localization of these proteins in the cell. The molecular mechanisms underlying the transport processes of GPCRs from the ER to the cell surface have recently become the subject of extensive studies [62]. The conserved sequences ⁄ motifs in the D 2 R, essential for their exit from the ER, are currently under investigation. ER export is the first step in intra- cellular trafficking of GPCRs and is a highly regulated event in the biogenesis of GPCRs. Sequence motifs play a crucial role in the targeting of polypeptides to the plasma membrane. The Arg-rich motif in D 2 R might also be a potential trafficking signal. Such motifs serve as endoplasmic reticulum retention signals that prevents the export of proteins to the plasma membrane. There are three types of retention motifs identified in the cytosolic domains of various proteins: KDEL, KKXX and RXR motifs [62,63]. The RXR motif (also three or four repeated Arg residues) actively precludes the exit of the protein from the endoplasmic reticulum [62,64,65]. Under normal condi- tions, this motif is masked, and proteins are trans- ported to the cell surface without significant accumulation in the endoplasmic reticulum. If the Arg- rich motif in D 2 R serves as a retention signal, then replacing adjacent Arg residues should increase the surface expression of D 2 R. We observed the opposite effect; the Arg-rich sequence in the cytoplasmic ic3 loop of D 2 R does not act as an endoplasmic reticulum retention signal. Misfolding of the D 2 R2 and D 2 R3 mutants could potentially be responsible for their accu- mulation in the endoplasmic reticulum because only protein that has assumed its native conformation is available for recruitment into the transport vesicles leaving the endoplasmic reticulum. Therefore, the Arg- rich motif might be responsible for interactions with cytoskeletal proteins. Binda et al. have shown that cytoskeletal protein 4.1 N, a member of the 4.1 family, facilitates the transport of D 2 R to the cell surface by interacting with the N-terminal portion of the ic3 loop of D 2 R via its C-terminal domain [66]. Truncation analysis localized a region of interaction within resi- dues 211–241 of D 2 R. Because this study used genetic variants of D 2 R that lacked either 2, 4 or 6 residues from the 217–222 motif of ic3, and the cellular locali- zation of these mutants depended on the number of the basic residues exchanged for Ala, it may be concluded that proper interaction with protein 4.1 N might have been disturbed. Therefore, the D 2 R mutants stay in the endoplasmic reticulum and are not transported to the cell membrane. Intracellular signaling pathway components, such as heteromeric G proteins and adenylate cyclase, are pres- ent in the endoplasmic reticulum and Golgi apparatus [67]. Because the intracellular localization of the dopa- mine D 2 receptor has been also described in the stria- tum [68], it seems that elucidation of the mechanisms responsible for fine tuning of receptor trafficking, as well as its dimerization with other receptor partners, is very important for understanding the rules that govern receptor activity, both in physiological and patholo- gical conditions. Receptor dimerization, which is important for trans- membrane signal generation [54], also plays a role in intracellular trafficking of receptors and controlling their folding status. As suggested by So et al., hetero- oligomerization, by changing the exposure or masking motifs responsible for endoplasmic reticulum retention or export, may be a strong regulator of the cellular distribution of receptors [14]. Incorrect membrane localization of D 2 R after modi- fication within ic3 217–222 region (observed in the cells co-expressing D 1 R and D 2 R3) can result from defec- tive interactions with cytoskeletal proteins as well as from impaired heterodimerization with D 1 R. When in the cell both D 2 R3 mutant and D 2 R wild-type are present, most likely the D 2 R may help D 2 R3 to achieve the cell-surface receptor dimerization. Similar situation has been described by Concepcion et al. They have shown that rhodopsin mutant devoid of traffick- ing signal motif localized in the plasma membrane when it was co-expressed with the wild-type receptor, as a results of both proteins oligomerization [69]. S. Łukasiewicz et al. Dopamine D 1 and D 2 receptors dimerization FEBS 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 D 1 and D 2 receptor oligomerization Sylwia. These experiments were performed to determine the in uence of the introduced mutations on the localization of the receptor proteins and the degree of their