New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D2 receptor regulation and signaling

AbstractThe dopamine D2 receptor (D2R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.

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The dynamic role of regulator of G-protein signalling (RGS) proteins in partial agonism

The Journal of Physiology ◽

10.1113/jphysiol.2014.279562 ◽

2014 ◽

Vol 592 (17) ◽

pp. 3701-3702

Author(s):

Joobin Sattar ◽

Kevin P. Grace ◽

Guillaume Bastin

Keyword(s):

G Protein ◽

Rgs Proteins ◽

Partial Agonism ◽

G Protein Signalling

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Role of dopamine D2 receptor in hypothalamic regulation of energy homeostasis

Frontiers in Neuroendocrinology ◽

10.1016/j.yfrne.2006.03.192 ◽

2006 ◽

Vol 27 (1) ◽

pp. 88

Author(s):

Sa Yong Kim ◽

Kyu seok Kim ◽

Seung Woo Shin ◽

Min Sun Kim ◽

Ja Hyun Baik

Keyword(s):

Energy Homeostasis ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Dopamine D2 ◽

Hypothalamic Regulation

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Computational design of orthogonal membrane receptor-effector switches for rewiring signaling pathways

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718489115 ◽

2018 ◽

Vol 115 (27) ◽

pp. 7051-7056 ◽

Cited By ~ 6

Author(s):

M. Young ◽

T. Dahoun ◽

B. Sokrat ◽

C. Arber ◽

K. M. Chen ◽

...

Keyword(s):

Signaling Pathways ◽

G Protein ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

High Specificity ◽

Computational Design ◽

Membrane Receptor ◽

Membrane Receptors ◽

Cellular Functions ◽

Hierarchical Code

Membrane receptors regulate numerous intracellular functions. However, the molecular underpinnings remain poorly understood because most receptors initiate multiple signaling pathways through distinct interaction interfaces that are structurally uncharacterized. We present an integrated computational and experimental approach to model and rationally engineer membrane receptor-intracellular protein systems signaling with novel pathway selectivity. We targeted the dopamine D2 receptor (D2), a G-protein–coupled receptor (GPCR), which primarily signals through Gi, but triggers also the Gq and beta-arrestin pathways. Using this approach, we designed orthogonal D2–Gi complexes, which coupled with high specificity and triggered exclusively the Gi-dependent signaling pathway. We also engineered an orthogonal chimeric D2–Gs/i complex that rewired D2 signaling from a Gi-mediated inhibitory into a Gs-dependent activating pathway. Reinterpreting the evolutionary history of GPCRs in light of the designed proteins, we uncovered an unforeseen hierarchical code of GPCR–G-protein coupling selectivity determinants. The results demonstrate that membrane receptor–cytosolic protein systems can be rationally engineered to regulate mammalian cellular functions. The method should prove useful for creating orthogonal molecular switches that redirect signals at the cell surface for cell-engineering applications.

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Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Science Advances ◽

10.1126/sciadv.abb8368 ◽

2020 ◽

Vol 6 (37) ◽

pp. eabb8368 ◽

Cited By ~ 1

Author(s):

Hemlata Dwivedi-Agnihotri ◽

Madhu Chaturvedi ◽

Mithu Baidya ◽

Tomasz Maciej Stepniewski ◽

Shubhi Pandey ◽

...

Keyword(s):

G Protein ◽

Phosphorylation Site ◽

Dynamics Simulation ◽

Phosphorylation Sites ◽

Functional Responses ◽

Biased Signaling ◽

The Stability ◽

G Protein Coupled ◽

Structural Aspects

Agonist-induced phosphorylation of G protein–coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

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Preferential Coupling of Dopamine D2S and D2L Receptor Isoforms with Gi1 and Gi2 Proteins—In Silico Study

International Journal of Molecular Sciences ◽

10.3390/ijms21020436 ◽

2020 ◽

Vol 21 (2) ◽

pp. 436 ◽

Cited By ~ 4

Author(s):

Justyna Żuk ◽

Damian Bartuzi ◽

Dariusz Matosiuk ◽

Agnieszka A. Kaczor

Keyword(s):

Dopamine D2 Receptor ◽

D2 Receptor ◽

Antipsychotic Agents ◽

Intracellular Loop ◽

Dopamine D2 ◽

Receptor Isoforms ◽

In Silico Study ◽

Third Intracellular Loop ◽

G Protein Coupled ◽

Insight Into

The dopamine D2 receptor belongs to rhodopsin-like G protein-coupled receptors (GPCRs) and it is an important molecular target for the treatment of many disorders, including schizophrenia and Parkinson’s disease. Here, computational methods were used to construct the full models of the dopamine D2 receptor short (D2S) and long (D2L) isoforms (differing with 29 amino acids insertion in the third intracellular loop, ICL3) and to study their coupling with Gi1 and Gi2 proteins. It was found that the D2L isoform preferentially couples with the Gi2 protein and D2S isoform with the Gi1 protein, which is in accordance with experimental data. Our findings give mechanistic insight into the interplay between isoforms of dopamine D2 receptors and Gi proteins subtypes, which is important to understand signaling by these receptors and their mediation by pharmaceuticals, in particular psychotic and antipsychotic agents.

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Publisher Correction: Role of Dopamine D2 Receptor in Stress-Induced Myelin Loss

Scientific Reports ◽

10.1038/s41598-018-22954-x ◽

2018 ◽

Vol 8 (1) ◽

Author(s):

Mi-Hyun Choi ◽

Ji Eun Na ◽

Ye Ran Yoon ◽

Hyo Jin Lee ◽

Sehyoun Yoon ◽

...

Keyword(s):

Dopamine D2 Receptor ◽

D2 Receptor ◽

Dopamine D2

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Dopamine D2R is Required for Hippocampal-dependent Memory and Plasticity at the CA3-CA1 Synapse

Cerebral Cortex ◽

10.1093/cercor/bhaa354 ◽

2020 ◽

Author(s):

Isabel Espadas ◽

Oscar Ortiz ◽

Patricia García-Sanz ◽

Adrián Sanz-Magro ◽

Samuel Alberquilla ◽

...

Keyword(s):

Synaptic Plasticity ◽

Associative Learning ◽

Dopamine Receptors ◽

Learning And Memory ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Long Term Potentiation ◽

D1 Receptor ◽

Term Potentiation

Abstract Dopamine receptors play an important role in motivational, emotional, and motor responses. In addition, growing evidence suggests a key role of hippocampal dopamine receptors in learning and memory. It is well known that associative learning and synaptic plasticity of CA3-CA1 requires the dopamine D1 receptor (D1R). However, the specific role of the dopamine D2 receptor (D2R) on memory-related neuroplasticity processes is still undefined. Here, by using two models of D2R loss, D2R knockout mice (Drd2−/−) and mice with intrahippocampal injections of Drd2-small interfering RNA (Drd2-siRNA), we aimed to investigate how D2R is involved in learning and memory as well as in long-term potentiation of the hippocampus. Our studies revealed that the genetic inactivation of D2R impaired the spatial memory, associative learning, and the classical conditioning of eyelid responses. Similarly, deletion of D2R reduced the activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. Our results demonstrate the first direct evidence that D2R is essential in behaving mice for trace eye blink conditioning and associated changes in hippocampal synaptic strength. Taken together, these results indicate a key role of D2R in regulating hippocampal plasticity changes and, in consequence, acquisition and consolidation of spatial and associative forms of memory.

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