Biochim Biophys Acta. 2008 Apr;1778(4):974-82. doi: 10.1016/j.bbamem.2008.01.011. Epub 2008 Jan 18.
Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface.
Hannan, M. A., Kabbani, N., Paspalas, C. D., Levenson, R.,
["Department of Pharmacology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA."]
["Department of Pharmacology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA."]
Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.
PMID: 18261457

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPC1 |
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D2R | Yeast two-hybrid | Not used as a bait | Human | Fetal brain | cDNA library | 18261457 | ||
TRPC1 |
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NCS-1 | Inference | Prediction | 18261457 | |||||
TRPC4 |
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D2R | Inference | Prediction | 18261457 | |||||
TRPC5 |
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D2R | Inference | Prediction | 18261457 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPC1 |
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D2R | Fusion protein-pull down assay | Human | 638-759 | E. coli | Human | 131-151 | E. coli | 18261457 |
TRPC1 |
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NCS-1 | Fusion protein-pull down assay | Human | 639-759 | E. coli | Human | Full-length | E. coli | 18261457 |
TRPC4 |
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D2R | Fusion protein-pull down assay | Human | 621-893 | E. coli | Human | 131-151 | E. coli | 18261457 |
TRPC5 |
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D2R | Fusion protein-pull down assay | Human | 619-973 | E. coli | Human | 131-151 | E. coli | 18261457 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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Assay with endogenous proteins | Assay with overexpressed proteins | Reference | ||||||||
Cell or tissue | Cell or tissue | TRP channel construct | Interactor construct | |||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC1 |
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D2R | Co-immunoprecipitation | Rat striatal tissue | 18261457 | |||||
TRPC1 |
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D2R | Co-immunoprecipitation | Rat cortex tissue | 18261457 | |||||
TRPC1 |
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D2R | Co-immunofluorescence staining | Monkey prefrontal cortex | 18261457 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC1 |
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D2R | Yeast two-hybrid | Human | 726-745 | Human | 131-151 | 18261457 | ||
TRPC4 |
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D2R | Yeast two-hybrid | Human | 712-731 | Human | 131-151 | 18261457 | ||
TRPC5 |
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D2R | Yeast two-hybrid | Human | 719-738 | Human | 131-151 | 18261457 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Functional consequence
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TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPC1 |
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D2R | Cell surface biotinylation | Increase in plasma membrane level | 18261457 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
