title: Receptor for activated C kinase 1 (RACK1) inhibits function of transient receptor potential (TRP)-type channel Pkd2L1 through physical interaction.
abstract: Pkd2L1 (also called TRPP3) is a non-selective cation channel permeable to Ca(2+), Na(+), and K(+) and is activated by Ca(2+). It is also part of an acid-triggered off-response cation channel complex. We previously reported roles of the Pkd2L1 C-terminal fragments in its channel function, but the role of the N terminus remains unclear. Using a yeast two-hybrid screening, we found that the Pkd2L1 N terminus interacts with the receptor for activated C kinase 1 (RACK1), a scaffolding/anchoring protein implicated in various cellular functions. This interaction requires the last two Trp-Asp (WD) motifs of RACK1 and fragment Ala(19)-Pro(45) of Pkd2L1. The interaction was confirmed by GST pulldown, blot overlay, and co-immunoprecipitation assays. By (45)Ca tracer uptake and two-microelectrode voltage clamp electrophysiology, we found that in Xenopus oocytes with RACK1 overexpression Pkd2L1 channel activity is abolished or substantially reduced. Combining with oocyte surface biotinylation experiments, we demonstrated that RACK1 inhibits the function of Pkd2L1 channel on the plasma membrane in addition to reducing its total and plasma membrane expression. Overexpressing Pkd2L1 N- or C-terminal fragments as potential blocking peptides for the Pkd2L1-RACK1 interaction, we found that Pkd2L1 N-terminal fragment Met(1)-Pro(45), but not Ile(40)-Ile(97) or C-terminal fragments, abolishes the inhibition of Pkd2L1 channel by overexpressed and oocyte-native RACK1 likely through disrupting the Pkd2L1-RACK1 association. Taken together, our study demonstrated that RACK1 inhibits Pkd2L1 channel function through binding to domain Met(1)-Pro(45) of Pkd2L1. Thus, Pkd2L1 is a novel target channel whose function is regulated by the versatile scaffolding protein RACK1.
authors.affiliation: Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Jungwoo
authors.initials: J
authors.lastname: Yang
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Wang
authors.initials: W
authors.lastname: Zheng
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Tuli
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Wu
authors.forename: Shaimaa
authors.initials: S
authors.lastname: Hussein
authors.forename: Xiao-Qing
authors.initials: XQ
authors.lastname: Dai
authors.forename: Shiva
authors.initials: S
authors.lastname: Shafiei
authors.forename: Xiao-Gai
authors.initials: XG
authors.lastname: Li
authors.forename: Patrick Y
authors.initials: PY
authors.lastname: Shen
authors.forename: Jian-Cheng
authors.initials: JC
authors.lastname: Tu
authors.forename: Xing-Zhen
authors.initials: XZ
authors.lastname: Chen
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis / Q000502:physiology; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012333:RNA, Messenger / Q000494:pharmacology; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D014981:Xenopus
chemical_list: D015220:Calcium Channels; D009363:Neoplasm Proteins; C117185:PKD2L1 protein, human; C419368:RACK1 protein, human; D012333:RNA, Messenger; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins; D002118:Calcium