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. 2011 Jan 28;404(4):928-34.
doi: 10.1016/j.bbrc.2010.12.083. Epub 2010 Dec 22.

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions

Dasiel O Borroto-Escuela  1 , Kathleen Van Craenenbroeck, Wilber Romero-Fernandez, Diego Guidolin, Amina S Woods, Alicia Rivera, Guy Haegeman, Luigi F Agnati, Alexander O Tarakanov, Kjell Fuxe
Affiliations

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions

Dasiel O Borroto-Escuela et al. Biochem Biophys Res Commun. .

Abstract

Dopamine D(2) and D(4) receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D(2)R-D(4.)(x)R (D(4.2)R, D(4.4)R or D(4.7)R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET(1) techniques with the D(2)R and D(4.7)R receptors being the least effective in forming heteromers. Allosteric receptor-receptor interactions in D(2)R-D(4.2)R and D(2)R-D(4.4) R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor-receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor-receptor interaction interfaces.

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Figures

Fig. 1.
Fig. 1.
Dopamine D2R and the different polymorphic variants of dopamine D4.xR form constitutive heteromers. Representative images were obtained in co-immunoprecipitation experiments and in situ PLA. (A) Co-immunoprecipitation studies of FLAGD2SR and HAD4.2/4.4/4.7R were performed in HEK293T cells. Immunoprecipitation (IP) was made with mouse anti-FLAG (M2) antibody (2 μg). Proteins were visualized with HRP-coupled anti-FLAG M2 or rabbit anti-HA (lysates) or mouse anti-HA (16B12) (IP blot) antibody (1/1000) and HRP-coupled anti-mouse (1/2000). Signal denoting anti-FLAG antibody is indicated with (*). (B) Schematic representation of in situ PLA detection. Blue and green is indicate the primary and oligo-conjugated secondary antibodies. The long curved black line represents the circular DNA template that resulted from proximity-dependent ligation and was amplified by rolling circle amplification. The fluorescent-oligonucleotide detection probes are represented by red-asterisk. (C) HEK293T cells were transiently transfected with D2LR and D4.xR and grown as described in Section 2. After fixation, in situ PLA was performed with D2R and D4R-specific antibodies, followed by PLA reagents. The detected heterodimers are represented by the fluorescent rolling circle products (red clusters). Nuclei are shown in blue (DAPI). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2.
Fig. 2.
Quantitative analysis of D2LR and D4.xR heterodimerization. BRET1 donor saturation curves were performed by transfecting HEK293T cells with a constant DNA concentration of acceptor receptor-Rluc and increasing concentrations of donor receptor-YFP constructs. BRET1 ratio, total fluorescence, and total luminescence as well as transformed values into receptor numbers were determined as described under supplementary material. The curves represent 8 saturation curves that were fitted using a non-linear regression equation assuming a single binding site.
Fig. 3.
Fig. 3.
(A) Bioinformatic analysis and sequence alignment of D4.xR and D2R receptor homologies containing the triplet homologies ALQ (in the IL1 in both receptors) and LRA (in the IL3 and C-tail of D2R and D4.xR, respectively) in the receptor interface. The following amino acid residues are marked by a color code as the basic elements of leucine-rich motifs. Red bold, (Leu). Orange bold, (Ile) and (Val) that may also occupy a position of Leu in leucine-rich motifs. Green, (Asn) and (Cys). Black bold, (Ser) and (Thr) where agonist-regulated phosphorylation may occur. White letters are charged amino acids: negatively charged (dark blue background) (Glu), (Asp) or positively charged (dark red background) (Arg), (Lys), (His). Color-shaded are two-letter homologies which include leucine and seem rather typical for ligand-receptor interactions: LL (green), LI or IL and LV or VL (blue), LN (pink), together with ‘leucine-serine zipper’ LSS, LS or SL (yellow). (B) Per-residue analysis of the intrinsic disorder in D4.xR. The molecular model of D4.4R is represented (in red the lateral and cytosolic views of IL3) Each amino acids (aa) was assigned a Disorder Index value as the ratio between the number of predictors estimating it as belonging to a disordered sequence and the total number of predictors used. Thus, this value ranges between 0 (no predictor estimated the aa as ‘disordered’) and 1 (all the predictors estimated the aa as ‘disordered’). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4.
Fig. 4.
Functional consequences of D2LR–D4.xR heteromerization on ERK1/2 phosphorylation. (A) An in cell western assay was used to measure ERK1/2 phosphorylation in HEK293T cells co-expressing D4.xR and D2R receptors and treated in presence or absence of D2R and D4R receptor selective agonists (quinerolane, 50 nM and PD168077 (PD), 50 nM) in the time frame of 0–45 min. Similar experiments were carried out without stimulation (vehicle). The data represent the mean ± SEM; n = 3 in quadruplicate. Combined quinerolane and PD168077 D2LR–D4.2R is significantly different compared to quinerolane D2LR–D4.2R in the range of 5–25 min (+++: P < 0.001). Combined quinerolane and PD168077 D2LR–D4.4R is significantly different compared to quinerolane D2LR–D4.4R in the range of 5–20 min (***: P < 0.001) and 20–25 min (**: P < 0.01), by two-way analysis of variance (ANOVA). (B) Concentration-response curve of quinerolane induced ERK1/2 phosphorylation. Cells co-expressing D2R and D4.xR were stimulated in presence or absence of the D4R selective agonist PD168077 (100 nM) during 10 min. PD168077 at 100 nM shifts the concentration-response curve of quinerolane on ERK1/2 phosphorylation to the left in cells co-expressing only D2LR–D4.2R and D2LR–D4.4R. Data represent the mean ± SEM; n = 3 in triplicate. Qlane: quinerolane.
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