Related Experiment Videos
The splice variant D3nf reduces ligand binding to the D3 dopamine receptor: evidence for heterooligomerization
J L Elmhurst1, Z Xie, B F O'Dowd
1Department of Pharmacology, University of Toronto, Ontario, Canada.
Abstract:
The D3 dopamine receptor belongs to the D2-like family of dopamine receptors. As with other members of this group, the D3 dopamine receptor gene contains introns which allow for alternative splicing of gene products. The best characterized of the human D3 dopamine receptor mRNA splice variants encodes a truncated protein called D3nf. The D3 dopamine receptor and D3nf were epitope-tagged and expressed in Sf9 insect cells by recombinant baculovirus infection. The D3 dopamine receptor showed saturable, high affinity binding of agonists and antagonists, consistent with reported D3 dopamine receptor pharmacology. When the D3 dopamine receptor and D3nf were co-expressed, the apparent density of D3 dopamine receptor expression, as determined by radioligand binding, was significantly lowered compared to D3 dopamine receptor expressed alone. This effect of D3nf was specific for the D3 dopamine receptor, since co-expression with the D2 dopamine receptor or beta2-adrenoceptor had no effect on binding. Confocal immunofluorescence studies were used to confirm that both D3 dopamine receptor and D3nf were well expressed on the cell surface and densitometric analysis of cell surface membrane protein confirmed that D3nf did not significantly alter the amount of D3 dopamine receptor expressed. Photoaffinity labelling with [125I]azidonemonapride showed that the amount of ligand bound by membranes co-expressing D3 dopamine receptor and D3nf was significantly less than that bound by membranes expressing D3 dopamine receptor alone. The greatest decrease in binding was observed in the D3 dopamine receptor oligomeric forms. Ligand binding to dimers and tetramers was reduced by 69 and 46%, respectively, indicating effects of a protein-protein interaction. Co-immunoprecipitation confirmed that the D3DR and D3nf interact with each other. These data indicate that D3nf heterodimerizes with the D3 dopamine receptor and decreases the capacity of D3 dopamine receptor to bind ligand.
Insights
The D3 dopamine receptor
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- The D3 dopamine receptor is part of the D2-like family.
- Its gene allows for alternative splicing, producing variants like D3nf.
- Dopamine receptor signaling is crucial in various brain functions.
Purpose of the Study:
- To investigate the functional consequences of D3 dopamine receptor splice variants.
- To determine if D3nf interacts with the D3 dopamine receptor and affects its ligand binding.
- To explore the potential for protein-protein interactions in dopamine receptor regulation.
Main Methods:
- Expression of epitope-tagged D3 dopamine receptor and D3nf in Sf9 insect cells using recombinant baculovirus.
- Radioligand binding assays to assess receptor density and affinity.
- Confocal immunofluorescence microscopy to confirm cell surface expression.
- Photoaffinity labeling and co-immunoprecipitation to study protein interactions.
Main Results:
- D3 dopamine receptor exhibited high-affinity binding, consistent with known pharmacology.
- Co-expression of D3 dopamine receptor and D3nf significantly reduced radioligand binding compared to D3 dopamine receptor alone.
- D3nf specifically affected D3 dopamine receptor binding, not D2 dopamine receptor or beta2-adrenoceptor.
- Photoaffinity labeling revealed reduced ligand binding, particularly in D3 dopamine receptor oligomers (dimers and tetramers).
- Co-immunoprecipitation confirmed a direct interaction between D3 dopamine receptor and D3nf.
Conclusions:
- The D3nf splice variant heterodimerizes with the D3 dopamine receptor.
- This interaction significantly decreases the ligand-binding capacity of the D3 dopamine receptor.
- Protein-protein interactions involving splice variants play a role in regulating dopamine receptor function.