Alpha-actinin associates with polycystin-2 and regulates its channel activity.
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.
Li
Qiang
Q
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada.
Montalbetti
Nicolás
N
Shen
Patrick Y
PY
Dai
Xiao-Qing
XQ
Cheeseman
Christopher I
CI
Karpinski
Edward
E
Wu
Guanqing
G
Cantiello
Horacio F
HF
Chen
Xing-Zhen
XZ
D000185:Actinin / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016891:Polycystic Kidney, Autosomal Dominant / Q000378:metabolism; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013347:Subcellular Fractions; D050396:TRPP Cation Channels; D020798:Two-Hybrid System Techniques
D000185:Actinin; D000199:Actins; D015220:Calcium Channels; D005982:Glutathione Transferase; D008051:Lipid Bilayers; D008565:Membrane Proteins; D050396:TRPP Cation Channels