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.

G protein‐coupled receptor kinase‐2 confers isoform‐specific calcium sensitivity to dopamine D 2 receptor desensitization

“…While GRK2 co‐expression clearly increased the rates of β‐arr2‐mediated desensitization of both D 2S R and D 2L R, Cmpd101 had no significant effect on desensitization rate in D 2L R‐expressing cells, suggesting that the enhancement of the rate of D 2L R desensitization observed upon co‐expression of GRK2 with β‐arr2, as compared to β‐arr2 alone, may not be dependent on GRK2 kinase activity. This is in agreement with observations from mammalian cells suggesting that GRK2‐mediated enhancement of D 2L R β‐arr2 recruitment does not require receptor phosphorylation, 36,37 although other recent studies have come to different conclusions 38 . In oocytes expressing D 2S R and WT GRK2, but lacking β‐arr2, desensitization of DA‐evoked Kir3 currents was also unaffected by Cmpd101, again in agreement with earlier reports that the rapid desensitization of Kir3 currents mediated by GRK2 is independent of its kinase activity but rather due to Gβγ sequestration 27 …”

“…This is in agreement with observations from mammalian cells suggesting that GRK2-mediated enhancement of D 2L R β-arr2 recruitment does not require receptor phosphorylation, 36,37 although other recent studies have come to different conclusions. 38 In oocytes expressing D 2S R and WT GRK2, but lacking β-arr2, desensitization of DA-evoked Kir3 currents was also unaffected by Cmpd101, again in agreement with earlier reports that the rapid desensitization of Kir3 currents mediated by GRK2 is independent of its kinase activity but rather due to Gβγ sequestration. 27 The phosphorylation site targeted by GRK2 to mediate the Ca 2+ -sensitive desensitization of D 2S R remains unknown.…”

In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

“…Antibodies that specifically recognize the GPCR phosphorylation state have been available in some cases over the last 20 years. Indeed, phosphosite-specific GPCR antibodies have proven useful to detect receptor activation and to profile the pharmacological properties of new ligands [12][13][14][15] . To date, however, their utility has been largely limited to immunoblotting approaches using heterologous receptor expression systems, while detection of phosphorylated GPCRs from tissue lysates has been mostly unsuccessful, due to their low abundance in vivo.…”

“…A widely used approach to analyze GPCR phosphorylation is the use of phosphosite-specific antibodies [6][7][8][9] . When available, such antibodies are valuable tools to elucidate the temporal dynamics of receptor phosphorylation, identify relevant kinases and phosphatases, and detect receptor activation using immunoblotting techniques 7,[10][11][12][13][14][15] .…”

Behavioral and Neuroanatomical Consequences of Cell-Type Specific Loss of Dopamine D2 Receptors in the Mouse Cerebral Cortex

“…Expression analyses and/or electrophysiological characterization of NMDA receptors in cortical interneurons vs. pyramidal cells will also be important. D2R signaling and function are linked to its phosphorylation by G protein receptor kinases and interactions with arrestins, and new reagents are available to probe these differences (Mann et al, 2021 ). D2Rs expressed by excitatory and inhibitory neurons may signal through distinct protein interactions and/or modifications.…”