Molecular Psychiatry (2012) 17, 650–662 & 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12 www.nature.com/mp ORIGINAL ARTICLE Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain S Gonza´lez1, C Rangel-Barajas2, M Peper3, R Lorenzo3, E Moreno1, F Ciruela4, J Borycz5, J Ortiz6, C Lluı´s1, R Franco7, PJ McCormick1, ND Volkow8, M Rubinstein3, B Floran2 and S Ferre´ 5 1Department of Biochemistry and Molecular Biology, Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas, University of Barcelona, Barcelona, Spain; 2Departamento de Fisiologı´a, Biofı´sica y Neurociencias, Centro de Investigacio´n y de Estudios Avanzados del Instituto Polite´cnico Nacional, Me´xico D.F., Me´xico; 3Departamento de Fisiologı´a y Biologı´a Molecular y Celular, Instituto de Investigaciones en Ingenierı´a Gene´tica y Biologı´a Molecular, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina; 4Unitat de Farmacologia, Departament Patologia i Terape`utica Experimental, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain; 5CNS Receptor-Receptor Interactions Unit, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD, USA; 6Department of Biochemistry and Molecular Biology, Neuroscience Institute, Universitat Auto´noma de Barcelona, Bellaterra, Spain; 7Departamento de Neurociencias, Centro de Investigacio´nMe´dica Aplicada, Universidad de Navarra, Pamplona, Spain and 8National Institute on Drug Abuse, National Institutes of Health, Bethesda MD, USA Polymorphic variants of the dopamine D4 receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D4.4) and the 2-repeat (D4.2) variants form functional heteromers with the short isoform of the dopamine D2 receptor (D2S), the 7-repeat risk allele (D4.7) does not. D2 receptor activation in the D2S–D4 receptor heteromer potentiates D4 receptor- mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D4.7 or in the striatum of knockin mutant mice carrying the 7 repeats of the human D4.7 in the third intracellular loop of the D4 receptor. In the striatum, D4 receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D2S receptors. This interaction shows the same qualitative characteristics than the D2S–D4 receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D2S receptor activation potentiates D4 receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D2S–D4.7 heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD. Molecular Psychiatry (2012) 17, 650–662; doi:10.1038/mp.2011.93; published online 16 August 2011 Keywords: dopamine receptors; receptor heteromers; ADHD; striatum; glutamate Introduction human D4 receptor contains a large number of polymorphisms in its coding sequence.4 The most Dopamine D4 receptors are expressed in the prefrontal extensive polymorphism is found in exon 3, a region cortex, in GABAergic interneurons and in gluta- that codes for the third intracellular loop (3IL) of the matergic pyramidal neurons, including their striatal receptor. This polymorphism consists of a variable 1–3 projections. D4 receptors have been implicated in number of tandem repeats in which a 48-bp sequence attention-deficit hyperactivity disorder (ADHD).1,4–6 exists as 2- to 11-fold repeats.7 The three most In fact, the prefrontal cortex and associated fronto- common variants contain 2, 4 and 7 repeats (D4.2, striatal circuits are critical for executive function and D4.4 and D4.7, respectively). D4.4 constitutes the most are involved in ADHD.5 The gene encoding the frequent variant, with a global frequency of 64%, 8 followed by D4.7 (21%) and D4.2 (8%). Importantly, a high prevalence of the D4.7 variant has been demon- Correspondence: Dr S Ferre´, National Institute on Drug Abuse, strated in children diagnosed with ADHD.5 Though Intramural Research Program, National Institutes of Health, 251 stimulation of the D4,7 variant has been reported to be Bayview Boulevard, Baltimore, MD 21224, USA. 9 E-mail: [email protected] less potent at inhibiting cAMP than D4.2 or D4.4, the Received 25 March 2011; revised 27 June 2011; accepted 7 July functional significance of these variants are poorly 2011; published online 16 August 2011 understood. Dopamine D2–D4 heteromers S Gonza´lez et al 651 Receptor heteromers are becoming the focus of fragment of 1500 bp. Equimolar quantities of both extensive research in the field of G-protein-coupled fragments were used to produce a third product 10 receptors. A receptor heteromer is currently defined corresponding to the myc-D4.2, myc-D4.4 or myc-D4.7- as a macromolecular complex composed of at least tagged gene using the sense primer (50-GTGCTCGAG two (functional) receptor units with biochemical CACCATGGGTAACCGAAGCACAG-30) and antisense properties that are demonstrably different from those primer without its stop codon (50-GCGAATTCTCAG of its individual components.10 In some cases, CAGCAAGCACGTAGAGCCTTACG-30), harboring un- receptor heteromers provide a framework in which ique XhoI and EcoRI restriction sites, respectively. to understand the role of receptors with no clear The fragments were then subcloned in-frame into functional significance, and example being the D3 XhoI/EcoRI sites of the pcDNA3.1 vector (Invitrogen, receptor, which forms heteromers with the D1 recep- Paisley, Scotland, UK). Next, the human cDNAs 11 tor and modifies its function. A recent study for the adenosine A1 receptor and dopamine D4.2, showed that in mammalian transfected cells, the long D4.4,D4.7 and D2S receptors, cloned in pcDNA3.1 isoform of the D2 receptor (D2L) heteromerizes with were amplified without their stop codons using sense the three main D4 receptor variants, D4.2,D4.4 and and antisense primers harboring unique XhoI and 12 D4.7. Interestingly, results from the same study EcoRI sites to clone A1,D4.2,D4.4 and D4.7 receptors in suggested that D4.7 was less effective in forming the RLuc and the yellow fluorescent protein (YFP) 12 heteromers with D2L receptors. In view of the corresponding vectors, and HindIII and BamHI to reported evidence of predominant co-localization of clone D2S in the RLuc and the YFP corresponding D4 receptors with the short isoform of the D2 receptor vectors. The mouse cDNAs for the D4 and D2S 2,3,13 (D2S) in corticostriatal glutamatergic terminals, we receptors, cloned in pCMV-SPORT6 (American Type first investigated if any of the three main human Culture Collection, Manassas, USA) and pReceiver- variants of the D4 receptor could interact both M16 vectors, respectively (GeneCopoeia, Rockville, physically and functionally with D2S. By using the MD, USA), were amplified without their stop codons Bioluminescence Resonance Energy Transfer (BRET) using sense and antisense primers harboring unique technique, here we show evidence for the formation XhoI and EcoRV sites to clone D4 receptor in the RLuc of heteromers between D2S and D4.2 and D4.4 variants corresponding vector, and XhoI and KpnI to clone D2S of the D4 receptor. In contrast, the D4.7 variant failed to receptor in the RLuc and the YFP corresponding form heteromers with the D2S receptor. In transfected vectors. The amplified fragments were subcloned to cells, we found a biochemical property of the D2S–D4 be in-frame into restriction sites of the multiple receptor heteromer, which consists of the ability of cloning sites of EYFP-N3 vector (enhanced yellow D2S receptor activation to potentiate D4 receptor- variant of YFP; Clontech, Heidelberg, Germany) or the mediated mitogen-activated protein kinase (MAPK) mammalian humanized pRluc-N1 vectors (Perkin- signaling. A similar result was observed in striata Elmer, Waltham, MA, USA) to give the plasmids that from wild-type (WT) mice, a species that expresses D4 express the receptors fused to either Rluc or YFP on receptors with a short 3IL comparable to human D4.2. the C-terminal end of the receptor (D4.2-RLuc, D4.4- In contrast, potentiation of D4 receptor-mediated RLuc, D4.7-Rluc, D2S-RLuc and A1-RLuc or D2S-YFP, MAPK signaling was not observed in transfected D4.7-YFP and D1-YFP, respectively). All constructs cells expressing D4.7 or in striata taken from knockin were verified by nucleotide sequencing and the mice carrying a humanized 7-repeat intracellular fusion proteins are functional and expressed at the loop identical to that found in human D4.7. Finally, membrane level (see Results). analyzing neurotransmitter release in striatal slices and with in vivo microdialysis in rats, evidence Cell culture and transient transfection was obtained for a key role of D2–D4 receptor inter- HEK (human embryonic kidney)-293T cells were action in the modulation of striatal glutamatergic grown in DMEM (Dulbecco’s modified Eagle’s neurotransmission. medium) (Gibco Paisley, Scotland, UK) supplemented with 2 mML-glutamine, 100 U mlÀ1 penicillin/strepto- mycin and 5% (v/v) heat-inactivated fetal bovine Materials and methods serum (all supplements were from Invitrogen). Fusion proteins and expression vectors CHO cell lines were maintained in a´-MEM The synthetic cDNAs for the human D4.2,D4.4 and, D4.7 medium without nucleosides, containing 10% fetal receptor gene (kindly provided by TP Sakmar, Rocke- calf serum, 50 mgmlÀ1 penicillin, 50 mgmlÀ1 strepto- feller University, USA) were amplified using sense mycin and 2 mML-glutamine (300 mgmlÀ1). Cells oligonucleotide primer (50-TCAACGGGACTTTCCA were maintained at 371C in an atmosphere of 5% 0 0 AAATGT-3 ) and antisense primer (5 -CTCCGAGAT CO2, and were passaged when they were 80–90% CAACTTCTGCTCGCTTCGGTTACCC-30), resulting in confluent, twice a week.