(Received for publication, December 6, 1994)
From the
Two isoforms of the dopamine D2 receptor have been
characterized, D2L (long) and D2S (short), generated by alternative
splicing from the same gene. They differ by an in-frame insert of 29
amino acids specific to D2L within the putative third intracytoplasmic
loop of the receptor. We have previously demonstrated (Montmayeur,
J.-P., Guiramand, J., and Borelli, E.(1993) Mol. Endocrinol. 7, 161-170) that D2S and D2L, although presenting very
similar pharmacological profiles, couple differently to the
-subunit of guanine nucleotide-binding regulatory proteins
(G-proteins). In particular, D2L, but not D2S, requires the presence of
the
-subunit of the inhibitory G-protein (G
i2) to elicit
greater inhibition of adenylyl cyclase activity. The insert present in
D2L must therefore confer the specificity of interaction with G
i2.
Thus, we introduced substitution mutations within the D2L insert. These
mutant receptors were expressed in JEG3 cells, a G
i2-deficient
cell line, scoring for those presenting an increased inhibition of
adenylyl cyclase by dopamine. Our analysis identified two mutants,
S259/262A and D249V, with these properties. These results clearly show
that the insert present in D2L plays a critical role in the selectivity
for the G-proteins interacting with the receptor.
Dopamine mediates its effect in vivo through the
activation of five different receptors, which show distinct
pharmacological profiles(1) . All dopamine receptors belong to
the large family of seven-transmembrane domain
G-protein()-coupled receptors (7TM receptors).
Interestingly, the receptor with a pharmacology corresponding to the D2
subtype is represented by two isoforms generated by alternative
splicing of the same
gene(2, 3, 4, 5, 6, 7, 8, 9, 10) .
These isoforms, named D2L and D2S, are identical except for an insert
of 29 amino acids present in the putative third intracellular loop of
D2L. The pharmacological profiles of these isoforms are very similar in
transfected cells. Ligand activation of both receptors lowers
intracellular cAMP
levels(2, 3, 4, 5, 6, 7, 8, 9, 10) .
Thus, it appears that both isoforms have very similar functional
properties. However, the D2L insert confers a different specificity for
the coupling to G-proteins. Our previous studies have shown that D2L
and D2S couple differently to G-proteins(11) . Specifically,
D2L requires the presence of the
-subunit of the inhibitory
G-protein (G
i2) to inhibit adenylyl cyclase more
potently(12) . Similar results have also been obtained in other
systems(13) . These observations underline the functional
significance of the 29-amino acid insert in the third loop of D2L. In
general, the third intracytoplasmic domain of 7TM receptors appears to
direct the interaction of the receptor with the appropriate G-proteins.
For example, swapping of this region from the
- to the
-adrenergic receptor creates a chimeric receptor with
the signal transduction characteristics of an
-adrenergic receptor(14) . Similarly, swapping
experiments performed between the m1- and m2-muscarinic receptors also
demonstrate the importance of this loop in the selective coupling to
specific G-protein/effector systems(15) .
Analysis of the
amino acid composition of the third loop of the known 7TM receptors has
shown the presence of highly charged residues in the N- and C-terminal
regions of the loop. It is known that an alternation of
hydrophobic/hydrophilic amino acids can influence the secondary
structure of proteins inducing an amphipathic -helical structure.
Thus, the N- and C-terminal regions of the third loop are postulated to
adopt such a structure. Moreover, these highly charged regions of the
third loop are strikingly conserved between many different 7TM
receptors. Point mutations or deletions affecting these regions disrupt
the normal signal transduction by these receptors by altering their
binding to
G-proteins(16, 17, 18, 19) .
An
interesting feature of the D2 receptor is that the D2L-specific insert
is located outside of the regions mentioned above. Furthermore, the
insert interrupts a putative -helical structure present in the D2S
third loop and incorporates a novel stretch of alternating
hydrophobic/hydrophilic residues.
To establish the importance of
this region in determining the D2L coupling characteristics, we
generated amino acid substitution mutations in the 29-amino acid
insert. We took advantage of the observed D2L requirement for Gi2
in JEG3 cells to score for mutants that display a higher potency of
adenylyl cyclase inhibition and, in this respect, that behave similarly
to D2S.
Amino acid mutations were generated in the D2L insert in order to identify the residues playing a role in the coupling to G-proteins. Charged amino acids were substituted with valine, a nonpolar amino acid. In addition, proline 264 was substituted with glycine, and serines 259 and 262 with alanines. These mutant receptors were analyzed for their ability to bind D2-specific ligands and to transduce the signal at the cAMP level. We have identified mutants with an associated increase in the inhibition of cAMP levels. This clearly demonstrates that the D2L-specific insert determines the selectivity of coupling of this isoform to G-proteins.
Figure 1: Construction of the mutant D2L receptors. A, nucleotide sequence of the mouse D2L receptor cDNA from nucleotides 715 to 834 and the corresponding amino acid sequence. The cleavage sites for Bsu36I, BspHI, and SacI used for the construction of the various mutants are indicated by arrows. The D2L-specific insert is indicated by the blackdiamonds located beneath the amino acid sequence. B, sequences of the oligonucleotides used to construct mutant D2L receptors. The nucleotides that differ from the wild-type sequence are indicated in boldface. C, amino acid sequence from positions 239 to 278 of the D2L receptor compared with the various mutants. The mutagenized amino acids for each mutant are indicated. The blackdiamonds above the sequence indicate the 29-amino acid D2L-specific insert.
To perform [H]NPA binding (21, 22) to transfected JEG3 cell membranes, cells
were first harvested in phosphate-buffered saline and pelleted at 500
g. After homogenization in 10 mM Tris-HCl (pH
7.5) and 5 mM EDTA with 10 strokes of a Dounce homogenizer,
membranes were isolated by centrifugation at 1000
g for 10 min. The supernatant was recovered and centrifuged at
45,000
g for 40 min. The pellet containing the
membranes was resuspended in the same buffer and centrifuged again at
the same speed. The final pellet was resuspended in 50 mM Tris-HCl (pH 7.7) and stored in aliquots at -80 °C.
[
H]NPA binding was performed in buffer containing
40 mM Tris-HCl (pH 7.7), 96 mM NaCl, 4 mM KCl, 1.6 mM CaCl
, 0.8 mM MgCl
, and 0.1% ascorbic acid. Membranes were incubated
in this buffer in the presence of increasing concentrations of
[
H]NPA ranging from 0.08 to 5 nM for 30
min at 37 °C. Nonspecific binding was determined in the presence of
1 µM (+)-butaclamol. Incubations were terminated by
rapid filtration at 0-4 °C through Whatman GF/B filters using
a Brandel harvester apparatus, followed by three washes with 2 ml of
ice-cold 50 mM Tris-HCl (pH 7.7). Binding data were analyzed
with the EBDA-LIGAND program (Elsevier-Biosoft) using the one-site
fitting model, allowing the determination of dissociation constant (K
) and maximal binding capacity (B
) values for each experiment.
The K of
[
H]SPI for the D2L receptor was in agreement with
that observed in vivo(28, 29) . As described
previously(12) , these binding characteristics are not
significantly different from those of the D2S isoform (Table 1).
Among the different mutants studied, we noticed that mutations
affecting the positively charged residues (i.e. K251V, K3R-V,
and K5R-V) resulted in B
values lower than those
for the wild-type D2L receptor (Table 1) for equal amounts of
transfected vectors. This effect becomes more evident as the number of
mutated residues increases (Table 1). Indeed, with the K3R-V
receptor, in which 4 residues (1 lysine and 3 arginines) were
substituted with valine, the B
value is about
half that for D2L. For the K5R-V receptor, in which 6 residues (1
lysine and 5 arginines) were mutated, B
values
are 40% lower with respect to D2L. In addition, we also observed a
slightly higher affinity of [
H]SPI for the K5R-V
mutant receptor than for the wild-type D2L receptor (Table 1). No
significant differences in the [
H]SPI binding to
the other mutants have been observed (Table 1).
Figure 2:
Dose-response curves for inhibition of
cAMP formation by DA in D2L-transfected () and
S259/262A-transfected (
) JEG3 cells. Cells were cotransfected with
0.4 µg of plasmid expressing wild-type or mutant D2 receptors and
0.4 µg of a plasmid expressing the
-adrenergic
receptor. 18 hours after transfection, the cells were incubated for 20
min with 500 µM isobutylmethylxanthine and then stimulated
for an additional 45 min with 10 µM(-)-IPR and
increasing concentrations of DA ranging from 0.1 nM to 10
µM. After extraction, the cAMP was quantified by
radioimmunoassay. The results are expressed as percentages of the cAMP
concentration. The values of cAMP obtained by stimulation of the
transfected cells with(-)-IPR, in the absence of dopamine, were
taken as 100%. The fittings of the curves were calculated using the
EBDA program. This figure is representative of one experiment performed
at least three times, each in duplicate. In this experiment, we
obtained the following values: IC
= 5 nM and I
= 64% for D2L and IC
= 3 nM and I
= 65%
for S259/262A.
Figure 3:
Dose-response curves for inhibition of
cAMP formation by DA in D2L-transfected () and D249V-transfected
(
) JEG3 cells. The experiments were performed as described for Fig. 2. These data, expressed as percentages of
the(-)-IPR-evoked cAMP concentration, are representative of one
experiment performed in duplicate and repeated at least three times.
Estimated values corresponding to the experiment presented in this
figure are as follows: IC
= 14 nM and I
= 71% for D2L and IC
= 3 nM and I
= 74%
for D249V.
Noticeably, all the D2L mutants present an unaltered
efficacy (I) of dopamine inhibition of
(-)-IPR-induced cAMP formation, which is comparable to that of
the wild-type D2 receptors. This indicates that the D2L insert does not
directly participate in the recruitment of the G-proteins to the
receptor, in agreement with findings showing that the regions
responsible for such function reside elsewhere in the third loop (16, 17, 18, 19) . However, the
lower IC
suggests that the insert is implicated in the
establishment of the interactions of the D2L receptor with specific
G-proteins. Mutants P264G and D271V were not significantly different
from the wild-type receptor in the inhibition of cAMP levels (Table 1).
These data show that the increased potencies observed in the cAMP tests for the mutant receptors correlate, as expected, with a gain in the affinity of these receptors for the agonists. They also indicate that these receptors can interact better, with respect to D2L, with the G-proteins available in JEG3 cells, in a manner similar to the D2S isoform.
The dopamine D2 receptor represents an interesting paradigm
among the characterized 7TM receptors to understand the
structure/function relationships of these proteins. Two isoforms of
this receptor have been isolated with comparable pharmacological
characteristics and anatomical distribution. This is in spite of the
structural difference located at the level of the third
intracytoplasmic loop. This loop plays a central role in the coupling
of this class of receptors to G-proteins(23, 24) . In
particular, the regions flanking each extremity of the loop, adjacent
to transmembrane domains V and VI, are fundamental to the coupling of
the receptor with
G-proteins(16, 17, 25, 26) . The
insert present in D2L does not affect these regions, while it adds 29
amino acids at a position 31 residues C-terminal from transmembrane
domain V. Nevertheless, both receptors transduce the signal correctly
and are consequently able to interact with G-proteins. Interestingly,
in previous reports, we have demonstrated that the D2L isoform requires
the presence of Gi2 for maximum inhibition of adenylyl
cyclase(12) . This indicates that the insert plays a role in
specifying the interaction of D2L with G
i2, although even in its
absence, this receptor is able to interact less efficiently with other
G-protein(s). The results presented in this paper support this
hypothesis. Indeed, none of the amino acid substitutions tested creates
mutant D2L receptors that are unable to inhibit cAMP levels in
transfected cells. In contrast, we show that S259/262A and in
particular D249V display a lower IC
( Fig. 2and Fig. 3and Table 1), demonstrating that these mutants
acquire an increased potency with respect to D2L in inhibiting cAMP
levels. This is of particular interest given that the wild-type D2L
receptor works less efficiently in these cells than in other cell types
due to the lack of the
-subunit of Gi2. This indicates that the
mutations generate receptors able to interact more efficiently with
G-proteins other than G
i2. Indeed, mutants S259/262A and D249V
present IC
values similar to and even higher than that of
the D2S isoform, respectively (Table 1). We believe that the
29-amino acid insert of D2L generates a structure that confers
interaction selectivity for G
i2. Computer analysis of the sequence
of the D2L insert, using the method of Garnier et
al.(27) , predicts an
-helical structure. This type
of structure has been previously shown to be important for
receptor/G-protein
interaction(30, 31, 32, 33) . The
substitution of a negatively charged amino acid, such as aspartate 249,
with a nonpolar amino acid might modify the structure of the
neighboring region of the protein, and in this way, the mutant receptor
may acquire novel G-protein specificities. This could also apply for
mutant S259/262A. Alternatively, serines 259 and 262 might represent
phosphorylation target sites. Phosphorylation is believed to be
involved in mechanisms of G-protein-coupled receptor
desensitization(19, 34) ; therefore, mutant S259/262A
could be more efficient because it is not desensitized as efficiently
as the wild-type receptor. However, by sequence comparison, serines 249
and 262 do not constitute consensus substrates for known kinases. While
consensus phosphorylation sites for 7TM receptor kinases are not well
defined, it seems that acidic residues are required in the proximity of
the target Ser or Thr residues(35) . This is not the case for
either serine 259 or 262. Future experiments will be required to assess
whether these serines are phosphorylation sites for known or still
uncharacterized kinases. In conclusion, our experiments reinforce the
notion of the importance of the D2L-specific insert in determining the
functional properties of this isoform with regard to coupling to
G-proteins.
Comparison of the dopamine D2 receptor gene in human and mouse demonstrates that its sequence and splicing events have been highly conserved through evolution. This might indicate that the presence of two isoforms and their selective interaction with different G-proteins represent an essential feature of dopamine D2 receptor function in vivo. It is tempting to speculate that in vivo, these receptors might serve different functions by activating different G-proteins and possibly different transduction pathways. The simultaneous activation of different G-proteins might be a cellular mechanism to amplify the cellular response to the dopaminergic signal.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X55674[GenBank].