title: Dopamine D3 and acetylcholine nicotinic receptor heteromerization in midbrain dopamine neurons: Relevance for neuroplasticity.
abstract: Activation of nicotinic acetylcholine receptors (nAChR) promotes the morphological remodeling of cultured dopamine (DA) neurons, an effect requiring functional DA D3 receptors (D3R). The aim of this study was to investigate the mechanisms mediating D3R-nAChR cross-talk in the modulation of DA neuron structural plasticity. By using bioluminescence resonance energy transfer2(BRET2) and proximity ligation assay (PLA), evidence for the existence of D3R-nAChR heteromers has been obtained. In particular, BRET2showed that the D3R directly and specifically interacts with the β2 subunit of the nAChR. The D3R-nAChR complex was also identified in cultured DA neurons and in mouse Substantia Nigra/Ventral Tegmental Area by PLA. Cell permeable interfering peptides, containing highly charged amino acid sequences from the third intracellular loop of D3R (TAT-D3R) or the second intracellular loop of the β2 subunit (TAT-β2), were developed. Both peptides, but not their scrambled counterparts, significantly reduced the BRET2signal generated by D3R-GFP2and β2-Rluc. Similarly, the PLA signal was undetectable in DA neurons exposed to the interfering peptides. Moreover, interfering peptides abolished the neurotrophic effects of nicotine on DA neurons. Taken together these data first demonstrate that a D3R-nAChR heteromer is present in DA neurons and represents the functional unit mediating the neurotrophic effects of nicotine.
authors.affiliation: Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: l.bontempi006@unibs.it.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Bontempi
authors.affiliation: Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Savoia
authors.affiliation: Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Bono
authors.affiliation: Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Fiorentini
authors.affiliation: Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Missale
mesh_terms: D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques; D002478:Cells, Cultured; D059290:Dopaminergic Neurons / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008636:Mesencephalon / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009473:Neuronal Plasticity / Q000187:drug effects / Q000235:genetics*; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D050637:Receptors, Dopamine D3 / Q000172:deficiency* / Q000235:genetics; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins; D009538:Nicotine; D018722:Nicotinic Agonists; D050637:Receptors, Dopamine D3; D011978:Receptors, Nicotinic; D014446:Tyrosine 3-Monooxygenase
keywords: D3 Receptor; Dopamine; Heteromerization; Nicotine; Nicotinic receptor; Plasticity