Cytoplasmic tail of D1 dopaminergic receptor differentially regulates desensitization and phosphorylation by G protein-coupled receptor kinase 2 and 3
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Keyvan Sedaghat1, Mario Tiberi
1Ottawa Hospital Research Institute, Neuroscience Program and Departments of Medicine/Cellular and Molecular Medicine/Psychiatry, University of Ottawa, Ottawa, ON, Canada.
Abstract:
Herein, we investigate the differential D1 dopaminergic receptor (D1R) regulation by G protein-coupled receptor kinase (GRK) 2 and 3 using two truncated receptors lacking the distal (Δ425) and distal-central (Δ379) cytoplasmic tail (CT) regions. We first show the association between D1R and GRKs in co-transfected cells and rat striatum. Our studies further indicate that deletion of distal CT region of D1R does not alter the association between receptor and GRK2. Meanwhile, removal of both distal and central CT regions culminates in a drastic increase in the basal association between Δ379 and GRK2 relative to D1R and Δ425. Interestingly, CT truncations have no effect on the basal and DA-induced association of receptors with GRK3. Furthermore, we demonstrate that desensitization of D1R is considerably more robust in cells expressing GRK3. Notably, the robust GRK3-induced D1R desensitization is not attenuated by CT deletions. However, GRK2-induced Δ425 desensitization is not detectable whereas we unexpectedly find that Δ379 desensitization is similar to GRK2-induced D1R desensitization. GRK2 and GRK3-dependent desensitization of wild type D1R is not linked to differences in the extent of DA-induced receptor phosphorylation. Moreover, our studies show that GRK2-induced D1R phosphorylation is only modulated by deletion of distal CT region while distal and central CT regions control GRK3-induced D1R phosphorylation. Intriguingly, dopamine-induced Δ379 phosphorylation by GRK3 was significantly lower than receptor phosphorylation in cells harboring Δ379 alone or Δ379 and GRK2. Overall, our study suggests an intricate interplay between CT regions of D1R in differentially regulating receptor responsiveness by GRK2 and GRK3.
Insights
The study reveals how specific parts of the D1 dopamine receptor (D1R) affect its interaction with G protein-coupled receptor kinases (GRKs). Findings show distinct roles for receptor regions in GRK2 and GRK3 regulation of D1R signaling.
Area of Science:
- Neuroscience
- Molecular Pharmacology
- Cell Biology
Background:
- G protein-coupled receptor kinases (GRKs) play a crucial role in regulating G protein-coupled receptor (GPCR) function, including the D1 dopamine receptor (D1R).
- The cytoplasmic tail (CT) of GPCRs is known to be critical for GRK-mediated regulation, but the specific contributions of different CT regions to D1R desensitization and phosphorylation by GRKs remain incompletely understood.
Purpose of the Study:
- To investigate the differential regulation of the D1 dopamine receptor (D1R) by G protein-coupled receptor kinase (GRK) 2 and GRK3.
- To elucidate the role of specific cytoplasmic tail (CT) regions of D1R in mediating interactions with GRK2 and GRK3, and in subsequent receptor desensitization and phosphorylation.
Main Methods:
- Co-transfection of cells with wild-type and truncated D1R constructs (Δ425 and Δ379) and GRK2 or GRK3.
- Assessment of basal and dopamine-induced receptor-GRK association using co-immunoprecipitation.
- Measurement of D1R desensitization and receptor phosphorylation in response to GRK2 and GRK3 activity.
Main Results:
- Deletion of the distal CT region (Δ425) did not affect D1R association with GRK2, while removal of both distal and central CT regions (Δ379) significantly increased basal GRK2 association.
- CT truncations had no impact on basal or dopamine-induced association of D1R with GRK3.
- GRK3 induced more robust D1R desensitization than GRK2, and this effect was not altered by CT deletions.
- GRK2-induced desensitization was abolished in Δ425 but surprisingly maintained in Δ379, while GRK2 and GRK3 differentially modulated D1R phosphorylation based on CT deletions.
Conclusions:
- The cytoplasmic tail of D1R plays a complex, region-specific role in its differential regulation by GRK2 and GRK3.
- Distinct CT regions dictate the extent of D1R association with GRK2 and influence GRK-mediated desensitization and phosphorylation.
- These findings highlight an intricate mechanism by which D1R signaling is fine-tuned by specific interactions with GRKs, mediated by its cytoplasmic tail.
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