Hum Mol Genet. 2005 Jun 15;14(12):1587-603. Epub 2005 Apr 20.
Alpha-actinin associates with polycystin-2 and regulates its channel activity.
Li, Q., Montalbetti, N., Shen, P. Y., Dai, X. Q., Cheeseman, C. I., Karpinski, E., Wu, G., Cantiello, H. F., Chen, X. Z.,
["Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada."]
["Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada."]
Polycystin-2 (PC2) is the product of the PKD2 gene, which is mutated in 10-15% patients of autosomal dominant polycystic kidney disease (ADPKD). PC2 is an integral transmembrane protein and acts as a calcium-permeable cation channel. The functional modulation of this channel by other protein partners remains largely unknown. In the present study, using a yeast two-hybrid approach, we discovered that both intracellular N- and C-termini of PC2 associate with alpha-actinins, actin-binding and actin-bundling proteins important in cytoskeleton organization, cell adhesion, proliferation and migration. The PC2-alpha-actinin association was confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. In addition, the in vivo interaction between endogenous PC2 and alpha-actinins was demonstrated by co-immunoprecipitation in human embryonic kidney 293 and Madin-Darby canine kidney (MDCK) cells, rat kidney and heart tissues and human syncytiotrophoblast (hST) apical membrane vesicles. Immunofluorescence experiments showed that PC2 and alpha-actinin were partially co-localized in epithelial MDCK and inner medullary collecting duct cells, NIH 3T3 fibroblasts and hST vesicles. We studied the functional modulation of PC2 by alpha-actinin in a lipid bilayer electrophysiology system using in vitro translated PC2 and found that alpha-actinin substantially stimulated the channel activity of reconstituted PC2. A similar stimulatory effect of alpha-actinin on PC2 was also observed when hST vesicles were reconstituted in lipid bilayer. Thus, physical and functional interactions between PC2 and alpha-actinin may play an important role in abnormal cell adhesion, proliferation and migration observed in ADPKD.
PMID: 15843396
 Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
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Screening
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| Experimental screening | Non-experimental screening | Reference | ||||||||
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| TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
| TRPP1 |
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ơ-actinin | Inference | Prediction | 15843396 | |||||
| TRPP1 |
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ơ-actinin-1 | Inference | Prediction | 15843396 | |||||
| TRPP1 |
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ơ-actinin-2 | Yeast two-hybrid | Human | 1-215 | Human | Heart | cDNA library | 15843396 | |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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top
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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 | ||
| TRPP1 |
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ơ-actinin-1 | Fusion protein-pull down assay | Human | 1-215 | E. coli | Chicken | Not specified | Not specified | 15843396 |
| TRPP1 |
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ơ-actinin-1 | Fusion protein-pull down assay | Human | 821-968 | E. coli | Chicken | Not specified | Not specified | 15843396 |
| TRPP1 |
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ơ-actinin-2 | Fusion protein-pull down assay | Human | 1-215 | E. coli | Chicken | Not specified | Not specified | 15843396 |
| TRPP1 |
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ơ-actinin-2 | Fusion protein-pull down assay | Human | 821-968 | E. coli | Chicken | Not specified | Not specified | 15843396 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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top
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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 | ||||
| TRPP1 |
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ơ-actinin-1 | Co-immunoprecipitation | HEK293 | 15843396 | |||||
| TRPP1 |
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ơ-actinin-1 | Co-immunoprecipitation | MDCK | 15843396 | |||||
| TRPP1 |
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ơ-actinin-1 | Co-immunoprecipitation | Rat kidney tissue | 15843396 | |||||
| TRPP1 |
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ơ-actinin-1 | Co-immunofluorescence staining | NIH-3T3 | 15843396 | |||||
| TRPP1 |
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ơ-actinin-1 | Co-immunofluorescence staining | MDCK | 15843396 | |||||
| TRPP1 |
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ơ-actinin-1 | Co-immunofluorescence staining | Mouse inner medullary collecting duct cell | 15843396 | |||||
| TRPP1 |
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ơ-actinin-2 | Co-immunoprecipitation | Rat heart tissue | 15843396 | |||||
(:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
|
Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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top
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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 | ||||
| TRPP1 |
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ơ-actinin-1 | Fusion protein-pull down assay | Human | 821-878 | Chicken | Not determined | 15843396 | ||
| TRPP1 |
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ơ-actinin-1 | Yeast two-hybrid | Human | 821-878 | Human | 334-892 | 15843396 | ||
| TRPP1 |
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ơ-actinin-2 | Fusion protein-pull down assay | Human | 821-878 | Chicken | Not determined | 15843396 | ||
| TRPP1 |
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ơ-actinin-2 | Yeast two-hybrid | Human | 821-878 | Human | 341-894 | 15843396 | ||
| TRPP1 |
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ơ-actinin-2 | Yeast two-hybrid | Human | 1-215 | Human | 341-894 | 15843396 | ||
| TRPP1 |
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ơ-actinin-2 | Yeast two-hybrid | Human | 1-215 | Human | 596-728 | 15843396 | ||
(:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
|
Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
|
top
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| Functional consequence
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| TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
| TRPP1 |
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ơ-actinin | Patch clamp | Activation | 15843396 | |||||||
(:
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:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)