Presence of a D2 receptor truncated protein in the brain of the D2 receptor functional knockout mouse, revisiting its molecular and neurochemical features

AbstractNull mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein in the central nervous system. Several lines of D2R knockout mice have been generated, which share some characteristics but differ in others. The D2R functional knockout mouse, first described in 1997, is functionally null for D2R-mediated signaling but the Drd2 gene was interrupted at the most extreme distal end leaving open the question about whether transcript and protein are produced. We decided to determine if there are D2R transcripts, the characteristics of these transcripts and whether they are translated in the brain of D2R functional knockout mice. Sequence analysis of 3’ Rapid Amplification of cDNA Ends showed that D2R functional knockout mice express transcripts that lack only the exon eight. Immunofluorescence showed D2R-like protein in the brain of the knockout mice. As previously reported, D2R functional knockout mice are hypoactive and insensitive to the D2R agonist quinpirole (QNP). However, the heterozygous showed locomotor activity and response to QNP similar to the wild-type mice. Intriguingly, microdialysis experiments showed that heterozygous mice, such as knockouts, have half the normal levels of synaptic dopamine in the striatum. However, heterozygous mice responded similarly to wild-type mice to an acute injection of QNP, showing a 50% decrease in synaptic dopamine. In conclusion, D2R functional knockout mice express transcripts that lead to a truncated D2R protein that lacks from the sixth transmembrane domain to the C-terminal end but retains the third intracellular loop. We discuss the implications of this truncated D2R coexisting with the native D2R that may explain the unexpected outcomes observed in the heterozygous. Finally, we suggest that the D2R functional knockout mouse can be a useful model for studying protein-protein interaction and trafficking of D2R.

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Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice

NeuroSci ◽

10.3390/neurosci2020014 ◽

2021 ◽

Vol 2 (2) ◽

pp. 193-206

Author(s):

Natalia Sánchez ◽

Montserrat Olivares-Costa ◽

Marcela P González ◽

Roberto Munita ◽

Angélica P Escobar ◽

...

Keyword(s):

Golgi Apparatus ◽

Mouse Strain ◽

Knockout Mice ◽

Biochemical Characterization ◽

Transmembrane Domain ◽

Dopamine D2 Receptor ◽

Intracellular Localization ◽

D2 Receptor ◽

Receptor Protein ◽

The Brain

Null mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein. For our research, we obtained the functional D2R knockout mouse strain described initially in 1997. Surprisingly, our biochemical characterization showed that this mouse strain is not a true knockout. We determined by sequence analysis of the rapid 3′ amplification of cDNA ends that functional D2R knockout mice express transcripts that lack only the eighth exon. Furthermore, immunofluorescence assays showed a D2R-like protein in the brain of functional D2R knockout mice. We verified by immunofluorescence that the recombinant truncated D2R is expressed in HEK293T cells, showing intracellular localization, colocalizing in the Golgi apparatus and the endoplasmic reticulum, but with less presence in the Golgi apparatus compared to the native D2R. As previously reported, functional D2R knockout mice are hypoactive and insensitive to the D2R agonist quinpirole. Concordantly, microdialysis studies confirmed that functional D2R knockout mice have lower extracellular dopamine levels in the striatum than the native mice. In conclusion, functional D2R knockout mice express transcripts that lead to a truncated D2R protein lacking from the sixth transmembrane domain to the C-terminus. We share these findings to avoid future confusion and the community considers this mouse strain in D2R traffic and protein–protein interaction studies.

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Dopamine D2 receptor function is compromised in the brain of the methionine sulfoxide reductase A knockout mouse

Journal of Neurochemistry ◽

10.1111/j.1471-4159.2010.06721.x ◽

2010 ◽

pp. no-no ◽

Cited By ~ 4

Author(s):

Derek B. Oien ◽

Andrea N. Ortiz ◽

Alexander G. Rittel ◽

Rick T. Dobrowsky ◽

Michael A. Johnson ◽

...

Keyword(s):

Knockout Mouse ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Receptor Function ◽

Methionine Sulfoxide Reductase A ◽

Dopamine D2 ◽

The Brain

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Ascorbic acid and CoQ10 ameliorate the reproductive ability of superoxide dismutase 1-deficient female mice†

Biology of Reproduction ◽

10.1093/biolre/ioz149 ◽

2019 ◽

Cited By ~ 1

Author(s):

Naoki Ishii ◽

Takujiro Homma ◽

Jaeyong Lee ◽

Hikaru Mitsuhashi ◽

Ken-ichi Yamada ◽

...

Keyword(s):

Ascorbic Acid ◽

Superoxide Dismutase ◽

Coenzyme Q10 ◽

Knockout Mice ◽

Knockout Mouse ◽

Superoxide Dismutase 1 ◽

Wild Type ◽

Positive Effects ◽

Reproductive Ability ◽

Ascorbic Acid Supplementation

Abstract Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q10, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44–75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

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Disruption of the Dopamine D2 Receptor Impairs Insulin Secretion and Causes Glucose Intolerance

Endocrinology ◽

10.1210/en.2009-0996 ◽

2010 ◽

Vol 151 (4) ◽

pp. 1441-1450 ◽

Cited By ~ 67

Author(s):

Isabel García-Tornadú ◽

Ana M. Ornstein ◽

Astrid Chamson-Reig ◽

Michael B. Wheeler ◽

David J. Hill ◽

...

Keyword(s):

Insulin Secretion ◽

Glucose Intolerance ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Insulin Response ◽

Wild Type ◽

Β Cell ◽

Insulin Response To Glucose

The relationship between antidopaminergic drugs and glucose has not been extensively studied, even though chronic neuroleptic treatment causes hyperinsulinemia in normal subjects or is associated with diabetes in psychiatric patients. We sought to evaluate dopamine D2 receptor (D2R) participation in pancreatic function. Glucose homeostasis was studied in D2R knockout mice (Drd2−/−) mice and in isolated islets from wild-type and Drd2−/− mice, using different pharmacological tools. Pancreas immunohistochemistry was performed. Drd2−/− male mice exhibited an impairment of insulin response to glucose and high fasting glucose levels and were glucose intolerant. Glucose intolerance resulted from a blunted insulin secretory response, rather than insulin resistance, as shown by glucose-stimulated insulin secretion tests (GSIS) in vivo and in vitro and by a conserved insulin tolerance test in vivo. On the other hand, short-term treatment with cabergoline, a dopamine agonist, resulted in glucose intolerance and decreased insulin response to glucose in wild-type but not in Drd2−/− mice; this effect was partially prevented by haloperidol, a D2R antagonist. In vitro results indicated that GSIS was impaired in islets from Drd2−/− mice and that only in wild-type islets did dopamine inhibit GSIS, an effect that was blocked by a D2R but not a D1R antagonist. Finally, immunohistochemistry showed a diminished pancreatic β-cell mass in Drd2−/− mice and decreased β-cell replication in 2-month-old Drd2−/− mice. Pancreatic D2Rs inhibit glucose-stimulated insulin release. Lack of dopaminergic inhibition throughout development may exert a gradual deteriorating effect on insulin homeostasis, so that eventually glucose intolerance develops.

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Wnt5a-Dopamine D2 Receptor Interactions Regulate Dopamine Neuron Development via Extracellular Signal-regulated Kinase (ERK) Activation

Journal of Biological Chemistry ◽

10.1074/jbc.m110.188078 ◽

2011 ◽

Vol 286 (18) ◽

pp. 15641-15651 ◽

Cited By ~ 23

Author(s):

Sehyoun Yoon ◽

Mi-hyun Choi ◽

Min Seok Chang ◽

Ja-Hyun Baik

Keyword(s):

Transmembrane Domain ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Dopamine Neuron ◽

Neuronal Cultures ◽

Neuron Development ◽

Erk Activation ◽

Extracellular Signal Regulated Kinase ◽

Dopamine D2 ◽

Extracellular Signal

The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R−/−) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R−/− mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R−/− mice. Co-immunoprecipitation and displacement of [3H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway.

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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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PT545. The Direct Protein-Protein Interaction between Dopamine Transporter and Dopamine D2 Receptor: A Novel Target Site to Modulate Synaptic Dopamine

The International Journal of Neuropsychopharmacology ◽

10.1093/ijnp/pyw044.545 ◽

2016 ◽

Vol 19 (Suppl_1) ◽

pp. 1-2

Keyword(s):

Dopamine Transporter ◽

Protein Interaction ◽

Dopamine D2 Receptor ◽

D2 Receptor ◽

Target Site ◽

Protein Protein Interaction ◽

Dopamine D2 ◽

Novel Target

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Opiate-induced Analgesia Is Increased and Prolonged in Mice Lacking P-glycoprotein

Anesthesiology ◽

10.1097/00000542-200005000-00030 ◽

2000 ◽

Vol 92 (5) ◽

pp. 1392-1399 ◽

Cited By ~ 204

Author(s):

Susan J. Thompson ◽

Kari Koszdin ◽

Christopher M. Bernards

Keyword(s):

Knockout Mice ◽

Transmembrane Protein ◽

Analgesic Efficacy ◽

Cell Types ◽

Acute Administration ◽

Morphine Analgesia ◽

Wild Type ◽

P Glycoprotein ◽

Glycoprotein Inhibitors ◽

The Brain

Background P-glycoprotein is a transmembrane protein expressed by multiple mammalian cell types, including the endothelial cells that comprise the blood-brain-barrier. P-glycoprotein functions to actively pump a diverse array of xenobiotics out of the cells in which it is expressed. The purpose of this study was to determine if P-glycoprotein alters the analgesic efficacy of clinically useful opioids. Methods Using a standard hot-plate method, the magnitude and duration of analgesia from morphine, morphine-6-glucuronide, methadone, meperidine, and fentanyl were assessed in wild-type Friends virus B (FVB) mice and in FVB mice lacking P-glycoprotein [mdr1a/b(-/-)]. Analgesia was expressed as the percent maximal possible effect (%MPE) over time, and these data were used to calculate the area under the analgesia versus time curves (AUC) for all opioids studied. In addition, the effect of a P-glycoprotein inhibitor (cyclosporine, 100 mg/kg) on morphine analgesia in both wild-type and mdr knockout mice was also determined. Results Morphine induced greater analgesia in knockout mice compared with wild-type mice (AUC 6,450 %MPE min vs. 1,610 %MPE min at 3 mg/kg), and morphine brain concentrations were greater in knockout mice. Analgesia was also greater in knockout mice treated with methadone and fentanyl but not meperidine or morphine-6-glucuronide. Cyclosporine pretreatment markedly increased morphine analgesia in wild-type mice but had no effect in knockout mice. Conclusions These results suggest that P-glycoprotein acts to limit the entry of some opiates into the brain and that acute administration of P-glycoprotein inhibitors can increase the sensitivity to these opiates.

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