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.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

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."]
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: 15843396External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin Inference Prediction 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Inference Prediction 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Yeast two-hybrid Human 1-215 Human Heart cDNA library 15843396
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Fusion protein-pull down assay Human 1-215 E. coli Chicken Not specified Not specified 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Fusion protein-pull down assay Human 821-968 E. coli Chicken Not specified Not specified 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Fusion protein-pull down assay Human 1-215 E. coli Chicken Not specified Not specified 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Fusion protein-pull down assay Human 821-968 E. coli Chicken Not specified Not specified 15843396
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  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 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunoprecipitation HEK293 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunoprecipitation MDCK 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunoprecipitation Rat kidney tissue 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunofluorescence staining NIH-3T3 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunofluorescence staining MDCK 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunofluorescence staining Mouse inner medullary collecting duct cell 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Co-immunoprecipitation Rat heart tissue 15843396
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Fusion protein-pull down assay Human 821-878 Chicken Not determined 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Yeast two-hybrid Human 821-878 Human 334-892 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Fusion protein-pull down assay Human 821-878 Chicken Not determined 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Yeast two-hybrid Human 821-878 Human 341-894 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Yeast two-hybrid Human 1-215 Human 341-894 15843396
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-2 Yeast two-hybrid Human 1-215 Human 596-728 15843396
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin Patch clamp Activation 15843396
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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