Dopamine D2 receptor signaling dynamics of dopamine D2-neurotensin 1 receptor heteromers Dasiel O. Borroto-Escuela a , Annalisa Ravani b , Alexander O. Tarakanov c , Ismel Brito a,d , Manuel Narvaez e , Wilber Romero-Fernandez a , Fidel Corrales f , Luigi F. Agnati g , Sergio Tanganelli b , Luca Ferraro b , Kjell Fuxe a,⇑ a Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden b Department of Clinical and Experimental Medicine, Pharmacology Section and LTTA Centre, University of Ferrara, Ferrara, Italy c Russian Academy of Sciences, St. Petersburg Institute for Informatics and Automation, Saint Petersburg, Russia d III-A, CSIC, Barcelona, Spain e Department of Physiology, School of Medicine, University of Málaga, Spain f Centro Nacional de Neurociencias, La Habana, Cuba g IRCCS Lido Venice, Italy article info Article history: Received 15 April 2013 Available online 25 April 2013 Keywords: Dopamine D 2 R receptor Neurotensin 1 receptor Heteromerization G protein-coupled receptors Allosteric modulation Protein–protein interactions abstract Biochemical, histochemical and coimmunoprecipitation experiments have indicated the existence of antagonistic dopamine D2 (D 2 R) and neurotensin 1 (NTS1R) receptor–receptor interactions in the dorsal and ventral striatum indicating a potential role of these receptor–receptor interactions in Parkinson’s dis- ease and schizophrenia. By means of Bioluminiscence Resonance energy transfer (BRET 2 ) evidence has for the first time been obtained in the current study for the existence of both D 2L R/NTS1R and D 2S R/NTS1R het- eromers in living HEK293T cells. Through confocal laser microscopy the NTS1R GFP2 and D2R YFP were also shown to be colocated in the plasma membrane of these cells. A bioinformatic analysis suggests the exis- tence of a basic set of three homology protriplets (TVM, DLL and/or LRA) in the two participating receptors which may contribute to the formation of the D 2 R/NTS1R heteromers by participating in guide–clasp inter- actions in the receptor interface. The CREB reporter gene assay indicated that the neurotensin receptor ago- nist JMV 449 markedly reduced the potency of the D 2 R like agonist quinpirole to inhibit the forskolin induced increase of the CREB signal. In contrast, the neurotensin agonist was found to markedly increase the quinpirole potency to activate the MAPK pathway as also studied with luciferase reporter gene assay measuring the degree of SRE activity as well as with ERK1/2 phosphorylation assays. These dynamic changes in D 2 R signaling produced by the neurotensin receptor agonist may involve antagonistic allosteric receptor–receptor interactions in the D 2L R–NTS1R heteromers at the plasma membrane level (CREB path- way) and synergistic interactions in PKC activation at the cytoplasmatic level (MAPK pathway). Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Neurotensin (NT), a tridecapeptide originally identified in ex- tracts of bovine hypothalamus by Carraway and Leeman [1], is widely distributed in the central nervous system (CNS) and in the periphery acting as a neurotransmitter [1,2]. In mammalian brain NT and its receptors are widely distributed especially in dopamine (DA)-enriched regions and several studies demonstrate the existence of a close functional relationship between NT and DA neurons [3,4]. The interaction between NT and DA has been implicated in the pathogenesis and treatment of schizophrenia [3–5]. The molecular mechanism underlying the NT-induced mod- ulation of dopamine transmission, especially in the nigrostriatal, mesolimbic and meso-cortical dopamine pathways, has been pro- posed to be a direct antagonistic interaction between the NTS1R and the dopamine D2 receptor (D 2 R), including the D2 autorecep- tor [2,5–7]. The NT-induced reduction in D 2 R agonist affinity is found both in striatal sections and in striatal membrane prepara- tions [7–10], and may reflect direct allosteric NTS1R/D 2 R interac- tions antagonizing the DA agonist affinity at the D 2 R [5]. More recently, biochemical, neurochemical and coimmunoprecipitation experiments have supported the existence of direct NTS1R–D2R interactions [11]. Given the importance of D 2 Rs in the pathophysiology of schizo- phrenia and Parkinson’s disease and their treatments, it is of 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.04.058 ⇑ Corresponding author. Address: Retzius väg 8, 17177 Stockholm, Sweden. Fax: +46 8 315721. E-mail addresses: Dasiel.Borroto-Escuela@ki.se (D.O. Borroto-Escuela), tar@ iias.spb.s (A.O. Tarakanov), ismel@iiia.es (I. Brito), mnarvaez@uma.es (M. Narvaez), Wilber.Romero.Fernandez@ki.se (W. Romero-Fernandez), fidel@cneuro.edu.cu (F. Corrales), luigiagnati@tin.it (L.F. Agnati), kjell.Fuxe@ki.se (K. Fuxe). Biochemical and Biophysical Research Communications 435 (2013) 140–146 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc