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

ABSTRACT: The 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.

SUBMITTER: Mann A

PROVIDER: S-EPMC8050214 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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

Mann Anika A Keen Alastair C AC Mark Hanka H Dasgupta Pooja P Javitch Jonathan A JA Canals Meritxell M Schulz Stefan S Robert Lane J J

Scientific reports 20210415 1

The 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 D<su ...[more]

PMID: 33859231

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Project description:Background and purposeMorphine activates the µ-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [d-Ala(2) -MePhe(4) -Gly-ol]enkephalin (DAMGO) or etonitazene stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood.Experimental approachHere, we have generated phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363-, Thr370- or Ser375-phosphorylated form of the receptor.Key resultsWe showed that agonist-induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that µ receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation.Conclusions and implicationsTogether, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit µ-opioid receptor sequestration.Linked articleThis article is commented on by Kelly, pp. 294-297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x.

Project description:We developed a unified model of the GRK-mediated beta2 adrenergic receptor (beta2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the beta2AR; (3) beta2AR internalization; (4) recycling of the beta2AR post isoproterenol treatment; (5) beta2AR desensitization; and (6) beta2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation "seen" by the beta2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the beta2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the beta2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the beta2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.

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

Publications

New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D<sub>2</sub> receptor regulation and signaling.

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