Investigating the effects of palmitoylation on the dopamine 1 receptor (D1)
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The dopamine D1 receptor (D1) is a G protein-coupled receptor (GPCR) which regulates various key brain functions like attention, movement, reward, and memory. Understanding D1 signalling may open the horizon for novel treatments for neurological disorders. Upon agonist activation, the heterotrimeric G proteins Gαs activate adenylyl cyclase to increase cAMP/PKA signalling. D1 also engages β-arrestin proteins leading to β-arrestin dependent signalling. The D1 has two palmitoylation sites on cysteines 347&351 in its C-tail domain. However, the distinct roles and implications of palmitoylation on the D1 signalling, trafficking and β-arrestins recruitment are still largely unexplored. A palmitoylation D1 mutant was generated and luminescent based techniques such as BRET and split-Nanoluc complementation assay were employed, to delineate D1 palmitoylation effects on its pharmacology and signalling. The D1 agonists induced 50% less cAMP production in the mutant compared to wildtype (WT) and WT showed a more efficient dissociation of its Gαs. Moreover, the mutant receptor failed to recruit β-arrestin1&2, induced less ERK1/2 activation and internalises in an agonist-independent process while showing an altered intracellular Golgi trafficking. Also, in β-arrestin 1&2 KO HEK 293 cells similar cAMP production levels were reported for D1 WT and palmitoylation mutant. β-arrestin 1&2 KO blocked agonist-induced WT D1 plasma membrane trafficking, indicating that these β-arrestins are driving the differences between WT and the palmitoylation mutant D1. Taken together, our studies indicate that Gαs is the main transducer for D1 cAMP and ERK1/2 signalling and that palmitoylation is essential for its β-arrestin 1&2 interactions and modulating D1 signalling cascades in a drug-dependant process.
Authors
Chalhoub, GCollections
- Theses [4125]