The lack of energy transfer between the genetic variants of yellow fluores-cent protein-tagged D2R and cyan fluorescent protein-tagged D1R may result from differing localization of these p
Trang 1possibly involved in electrostatic interactions on the
Sylwia Łukasiewicz1, Agata Faron-Go´recka2, Jerzy Dobrucki3, Agnieszka Polit1
and Marta Dziedzicka-Wasylewska1,2
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 D2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D1 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 D1–D2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D2R, which may suggest that the indicated region of ic3 in D2R might be involved in interactions between two dopa-mine receptors In addition, the subcellular localization of these receptors
in cells expressing both receptors D1–cyan fluorescent protein, D2–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 D2R and cyan fluorescent protein-tagged D1R may result from differing localization of these proteins in the cell rather than from the possible role of the D2R basic domain in the mechanism of
D1–D2 receptor heterodimerization However, we find that the acidic epitope from the C-terminus of the dopamine D1receptor is engaged in the heterodimerization process
Abbreviations
CFP, 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.
Trang 2of 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 quesforma-tion 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 GABAB 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 stabilizaforma-tion 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 D2 receptor and two
adjacent aspartate residues or a phosphorylated serine
residue in the C-terminus of the A2A receptor are
involved in heterodimerization between the
adeno-sine A2A receptor and the dopamine D2 receptor [43]
A similar interaction has also been shown for
D1–NMDA receptor heterodimers [44]
Although the dopamine D1 and D2 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 D1 and
D2 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 D1 and D2 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 D1 and D2 receptors was similar to that of the mesocortical dopamine afferents [48] The dopamine D2 receptor can form homodimers [19] Recently, we have shown that the D2 receptor also forms heterodimers with the dopamine D1 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 D2 receptor, which contains adjacent argi-nine residues (217RRRRKR222), and an acidic epi-tope from the C-terminus of dopamine D1 receptor (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 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 D1 receptor and its mutant indicate that the Kd values for these two receptors were similar; however, the density of the D1MUT (404AA405) was
Table 1 Binding parameters for the dopamine receptors For dopa-mine D2receptor 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 D1 receptor binding, the statistical significance was evaluated using a Student’s t-test;
***P < 0.001.
Species
B max ± SEM (pmolÆmg)1protein)
K d ± SEM (n M)
Trang 3lower than that of wild-type D1R (Fig 1A) Also, all
three genetic variants of dopamine D2R displayed
sim-ilar Kd values, but the density of these receptors
strongly depended on the number of Arg residues still
present within the receptor sequence The D2R1
(217AARRKR222) mutant displayed half of the Bmax
value obtained for D2R, whereas the density of the
D2R2 (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 cellular
membrane This conclusion is further justified by
confocal microscopy analysis of receptor localization
Analysis of the localization of dopamine D1, D2
and their genetic variant fusion proteins
Confocal microscopy was used to visualize HEK293
cells co-expressing the dopamine D1 and D2 receptors,
as well as their genetic variants (D1MUT, D2R1,
D2R2, D2R3) 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 D1, dopamine D2, D1MUT, D2R1, D2R2 and
D2R3 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 D1 and D1MUT recep-tors localize in the plasma membrane, in contrast to the dopamine D2 receptor 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 D2 receptor mutants, the degree of membrane localization depended on the number of mutated resi-dues in the ic3 region (D2217–222)
The dopamine D2R3 receptor location was very interesting and surprising As seen in Fig 2A, which shows 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 localized inside the cell, whereas the D1 receptor was found in the plasma membrane However, when the cell co-expressed both types of D2 receptors, i.e the wild-type and the D2R1, D2R2 as well as D2R3 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 D1 and dopa-mine D2 receptor mutants, the degree of colocalization decreased, which was correlated with number of exchanged residues within the ic3 of D2 receptor When cells were cotransfected with the same type of receptors (D1MUT–CFP⁄ D1–YFP, D2–CFP⁄ D2R1– YFP, D2–CFP⁄ D2R2–YFP, D2–CFP⁄ D2R3–YFP) and with dopamine D2 and genetic variant dopamine D1 receptors (D1MUT–CFP⁄ D2–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 R or D 1 R, respectively, does not alter
the ligand binding constant.
Trang 4very 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 D1
and D2 receptors and their genetic variants
Fluores-cence emission profiles for the HEK293 cell suspension
expressing 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) 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 D1 and D2
receptor fusion proteins (D1–CFP and D2–YFP) in
comparison with emission spectra of the cell
popula-tions that co-express dopamine D1 receptor fusion protein (D1–CFP) and one of the genetic variants of dopamine D2 receptor fusion protein (D2R1, D2R2
or D2R3–YFP) (Fig 3A) In Fig 3B, the results presented are from a cell suspension expressing the dopamine D2–YFP fusion protein and the genetic vari-ant of the dopamine D1 receptor (D1MUT–CFP) fusion protein We observed energy transfer between wild-type dopamine D1 and D2 receptors, but when either the genetic variant of dopamine D1 (D1MUT)
or the D2R3 genetic variant of the dopamine D2 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 (D1 or D2, respec-tively), tagged with different fluorescence proteins,
A
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 D2–YFP, D2R1–YFP, D2R2–YFP, D2R3–YFP or D1MUT–YFP (green and red) Image overlays show extensive colocalization in
D1⁄ D 1 , D1⁄ D 1 MUT, D1⁄ D 2 and D1⁄ D 2 R1 assays and partial colocalization in D1⁄ D 2 R2 assays D1⁄ 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.
Trang 5compared with emission profiles of cells in which one
of 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 panel
of Fig 3 shows that both dopamine receptors, D1 and
D2, 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, aiand as
tagged with CFP with dopamine D1 or D2 receptors,
which were tagged with YFP As seen in Fig 4, the
FRET phenomenon takes place only when the D1
receptor is co-expressed with as or D2 receptor is
co-expressed with ai The interactions are specific
because no energy transfer was observed following
co-transfection of D1–YFP⁄ ai–CFP or D2YFP⁄ asCFP, 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 D1 and D2 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 abspres-ence 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 D1R and D2R 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
D2R3–YFP (black dashed line) (B) Cotransfection of HEK293 with D1MUT–CFP and D2–YFP (gray line) in comparison with D1–CFP and
D2–YFP (black line) (C) Cotransfection of HEK293 with D1MUT–CFP and D1–YFP (gray line) in comparison with D1–CFP and D1–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.
Trang 6Fluorescence 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 D1–CFP bearing donor alone and with
the donor and acceptor D1–CFP⁄ D1MUT–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 D1 and D2 receptor fusion proteins indicated
4% efficiency of energy transfer, with an average donor fluorescence lifetime of 2.27 ns This changed when the dopamine D2 receptor was replaced by a genetic variant (D2R1, D2R2 or D2R3) and also when
D1MUT was used instead of the dopamine D1 recep-tor Transfer efficiency was equal to 2.1% (2.32 ns) for
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 GaS–CFP (black line) fusion protein (excited
at 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 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 D2–YFP and GaI–CFP (gray line) or D2–YFP and GaS–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.
Trang 7D1⁄ D2R1, further decreased to 1.26% (2.34 ns) for
D1⁄ D2R2, and finally reached the value of 0.44%
(2.36 ns) for D1⁄ D2R3
The lowest E value, similar to that obtained for the
D1⁄ D2R3 combination, was observed for D1MUT⁄
D2R3 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 D1 receptor mutant (D1MUT) and
the wild-type dopamine D2 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, D1or D2, and when one of the
appro-priate receptors was replaced by its mutant (D1 by
D1MUT or D2 by D2R3), no change in transfer
effi-ciency was detectable The E value for D1MUT⁄ D1
was estimated to be 7.8% (2.19 ns) versus 8%
(2.18 ns) for D1⁄ D1, while for D2⁄ D2R3, it equaled
3.4% (2.29 ns) versus 3.5% (2.28 ns) for D2⁄ D2
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 D2 and D1 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
D1MUT 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 D1–CFP ⁄ D 2 –YFP.
Species
Average lifetime (ns) Transfer
efficiency ÆEæ (%)
Æs D æ Æs DA æ
D1MUT–CFP ⁄ D 2 R3–YFP g 2.36 ± 0.01 0.40
a Measured in cell expressing CFP coupled to the dopamine D1 receptor. bMeasured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D1–CFP and D2–YFP) c Measured in cell co-expressing dopamine D1 and D2 fusion proteins (D1–CFP and
D 2 R1–YFP – genetic variant of dopamine D 2 receptor).dMeasured
in cell co-expressing dopamine D1and D2fusion protein (D1–CFP and D2R2–YFP – genetic variant of dopamine D2receptor) e Mea-sured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D 1 –CFP and D2R3–YFP – genetic variant of dopamine D2receptor).
f Measured in cell co-expressing dopamine D1and D2fusion pro-teins (D 1 MUT–CFP – genetic variant of dopamine D 1 receptor and
D 2 –YFP). gMeasured in cell co-expressing dopamine D 1 and D 2 fusion proteins (D1MUT–CFP – genetic variant of dopa-mine D 1 receptor and D 2 R3–YFP – genetic variant of dopamine D 2 receptor).
Trang 8experimental 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 A2A and dopamine D2 receptors strongly
depends on an electrostatic interaction between an
Arg-rich epitope from the ic3 of the D2R
(217RRRRKR222) and either the two adjacent Asp
residues (DD 401–402) or a phosphorylated Ser374 in
the C-tail of the A2AR [43]
Because the dopamine D1R contains an acidic
region on the C-terminus, like A2AR, we designed
experiments to determine whether a similar interaction
is responsible for the heterodimerization of the D2
receptor with the D1 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 D1 and D2 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 b2-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 D1 and D2 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 D1 receptor did not inter-act with Gai, and the D2receptor did not interact with
Gas, 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 æ Æs DA æ
a
Measured in cell expressing CFP coupled to dopamine D 1
recep-tor b Measured in cell co-expressing two dopamine D1 receptor
fusion proteins (D1–CFP and D1–YFP) c Measured in cell
co-expressing two dopamine D 1 receptor fusion proteins (D 1 MUT–
CFP – genetic variants of dopamine D1 receptor and D1–YFP).
d Measured in cell expressing dopamine D2 receptor coupled to
CFP (D 2 –CFP).eMeasured in cell co-expressing two dopamine D 2
receptor fusion proteins (D2–CFP and D2–YFP) f Measured in cell
co-expressing two dopamine D2receptor fusion proteins (D2–CFP
and D 2 R3–YFP – genetic variant of dopamine D 2 receptor).
Trang 9show 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 D1
receptor, as well as the Arg-rich region of ic3 of the D2
receptor, do not seem to take part in receptor
homodimerization, but they do influence D1–D2
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 D1–D2receptor 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 D2 receptor The
effi-ciency of energy transfer in the wild-type of the D1and
D2heterodimer 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 D2 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 D1receptor 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 D2receptor and acidic region
of the C-tail of the D1 receptor might be involved in
the interactions between the two dopamine receptors
In addition, the subcellular localization of D1–CFP,
D2–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 D1 and D2 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 D1 receptor did not change the localization of the
receptor because both wild-type D1 and the mutant
were localized in the cell membrane However, the D2
receptor was localized at the cell surface with a consid-erable portion also present within the cell Analysis of cells containing the D1 and D2 receptors, as well as cells expressing D1MUT and D2, showed that the level
of colocalization was very similar This result clearly indicates that the significant decrease in energy transfer observed between D1MUT and D2 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 D2 receptor substantially changed its receptor traf-ficking properties The binding experiments also pointed to a decrease in the density of the D2R vari-ants in the cellular membrane; the number of D2 receptor binding sites decreased with the number of changed Arg residues in the ic3 When compared with wild-type receptor, the binding of [3H]spiperone to
D2R1 and D2R2 showed a significant decrease in the
Bmax, 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 D2 receptor
in the membrane The results obtained by confocal microscopy show that the D2R3 mutant was mainly localized in the cytoplasmic compartments However, cotransfection with wild-type D2R changed the distri-bution of this protein This suggests that wild-type D2 receptor can modulate the localization of the D2R3 mutant receptor We did not observe such an effect in cells expressing the dopamine D1 and D2R3 receptors The D2R3 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 D2–D2R3 homodimers are being created during D2 receptor biosynthesis, whereas that process does not take place in the case of D1-D2R3 co-expression It is probably the direct interactions between the D2 and the D2R3 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 D2 receptor with the wild-type receptor resulted in attenuation of binding and reduced efficiency in the trafficking of the wild-type D2 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 D1–D2receptor 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 D1receptor are engaged in heterodimerization, but
Trang 10not in homodimerization, as the efficiency of energy
transfer is the same for wild-type D1 receptor as for
D1–D1MUT 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 D1 receptor membrane
localization
However, genetically manipulating the Arg-rich
epi-tope in the ic3 of the D2receptor 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 D1–D2 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 A2A–dopamine D2 heterodimerization [43]
However, based on these data, we have to conclude
that the Arg-rich epitope in the ic3 loop of D2 is also
responsible for receptor localization The lack of energy
transfer between the YFP-tagged D2 receptor genetic
variants and CFP-tagged D1 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 D2R,
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 D2R
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 D2R serves as a retention signal, then
replacing adjacent Arg residues should increase the
surface expression of D2R We observed the opposite
effect; the Arg-rich sequence in the cytoplasmic ic3
loop of D2R does not act as an endoplasmic reticulum
retention signal Misfolding of the D2R2 and D2R3
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 D2R to the cell surface by interacting with the N-terminal portion of the ic3 loop
of D2R via its C-terminal domain [66] Truncation analysis localized a region of interaction within resi-dues 211–241 of D2R Because this study used genetic variants of D2R 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 D2R 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 D2 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 D2R after modi-fication within ic3 217–222 region (observed in the cells co-expressing D1R and D2R3) can result from defec-tive interactions with cytoskeletal proteins as well as from impaired heterodimerization with D1R When in the cell both D2R3 mutant and D2R wild-type are present, most likely the D2R may help D2R3 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]