D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson’s disease
Irene Sebastianutto, … , M. Angela Cenci, Julie Perroy
Irene Sebastianutto, … , M. Angela Cenci, Julie Perroy
Published February 10, 2020
Citation Information: J Clin Invest. 2020;130(3):1168-1184. https://doi.org/10.1172/JCI126361.
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D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson’s disease

Abstract

Dopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson’s disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine. In rodent models of Parkinson’s disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5–dependent PLC signaling was causally linked with excessive activation of extracellular signal–regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson’s disease.

Authors

Irene Sebastianutto, Elise Goyet, Laura Andreoli, Joan Font-Ingles, David Moreno-Delgado, Nathalie Bouquier, Céline Jahannault-Talignani, Enora Moutin, Luisa Di Menna, Natallia Maslava, Jean-Philippe Pin, Laurent Fagni, Ferdinando Nicoletti, Fabrice Ango, M. Angela Cenci, Julie Perroy

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Figure 1

Heteromerization between mGlu5 and D1 receptors in living cells.

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Heteromerization between mGlu5 and D1 receptors in living cells. (A–C) B...
(A–C) BRET titration curves measured on HEK293 cells cotransfected with N- (A) or C-tagged (B) receptors. BRET signals were measured for increasing expression levels of acceptor (Venus-D1, A; or D1-Venus, B) at constant levels of donor expression (Rluc8-mGlu5, A; or mGlu5-Nluc, B). Results were analyzed by nonlinear regression on a pooled data set from 3 independent experiments, assuming a model with 1-site binding (GraphPad Prism 7). (A) BRET signals measured between Venus-D1 receptor and Rluc8-mGlu5 (purple curve) or Rluc8-mGlu5 deleted from its C-tail (Rluc8-mGlu5-DelCtail, black curve); b.u., BRET unit. (B) BRET signals measured between D1-Venus and mGlu5-Nluc, with (black curve) or without (purple curve) coexpression of mGlu5-Ctail fused to the CD4 membrane domain (CD4-CtailmGlu5). (C) Decrease of net BRET signal between D1-Venus and mGlu5-Nluc in cells coexpressing CD4-CtailmGlu5 (black) for identical D1-Venus/mGlu5-Nluc expression ratios. P = 0.0571, Mann-Whitney U test. (D–F) BiFC measured on transiently transfected HEK293 cells. (D) Schematic representation of BiFC principle. Nonfluorescent fragments from the Venus fluorescent protein (V1 and V2) are fused to putative interaction partners. Physical association triggers bimolecular fluorescent Venus complex. (E) BiFC images of receptors fused to nonfluorescent monomeric Venus split V1 or V2. Specificity was controlled in cells expressing D1-V1 or 5a-V1 together with CD8-V2 (last 2 rows). The green channel illustrates the expression of Venus complementation (V1 + V2), whereas DAPI and pmRFP fluorescences stain the nucleus and plasma membrane, respectively. Scale bar: 10 μm. (F) Quantification of complemented Venus fluorescence intensity at the membrane (colocalized with pmRFP) expressed as a percentage of whole-cell Venus fluorescence. Box and whiskers plots of 21 to 54 measurements. *P < 0.05; ***P < 0.001; ****P < 0.0001, Kruskal-Wallis test. Box and whiskers plots: in this and the following figures: line, median; bounds: 25th to 75th percentiles; whiskers, minimum to maximum.