title: Polycystin-2 associates with tropomyosin-1, an actin microfilament component.
abstract: Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel. PC2 has been shown to connect indirectly with the actin microfilament. Here, we report a direct association between PC2 and the actin microfilament. Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex. Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells. We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2. In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms. Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a. Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells. Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs. Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney. Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered.
authors.affiliation: Membrane Protein Research Group, Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, T6G 2H7, Edmonton, AB, Canada.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Yue
authors.initials: Y
authors.lastname: Dai
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Chunhai
authors.initials: C
authors.lastname: Hao
authors.forename: Guanqing
authors.initials: G
authors.lastname: Wu
authors.forename: Nuria
authors.initials: N
authors.lastname: Basora
authors.forename: Marek
authors.initials: M
authors.lastname: Michalak
authors.forename: Xing Zhen
authors.initials: XZ
authors.lastname: Chen
mesh_terms: D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000737:chemistry; D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000378:metabolism; D029721:Drosophila Proteins; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D016891:Polycystic Kidney, Autosomal Dominant / Q000378:metabolism; D011233:Precipitin Tests; D021381:Protein Transport; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D050396:TRPP Cation Channels; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D000199:Actins; D015220:Calcium Channels; D029721:Drosophila Proteins; D005982:Glutathione Transferase; D006639:Histidine; D008565:Membrane Proteins; D050396:TRPP Cation Channels; D014335:Tropomyosin; C490775:TPM1 protein, human; C097163:Tm2 protein, Drosophila; C490774:Tpm1 protein, mouse