J Biol Chem. 2012 Feb 24;287(9):6551-61. doi: 10.1074/jbc.M111.305854. Epub 2011 Dec 15.
Receptor for activated C kinase 1 (RACK1) inhibits function of transient receptor potential (TRP)-type channel Pkd2L1 through physical interaction.
Yang, J., Wang, Q., Zheng, W., Tuli, J., Li, Q., Wu, Y., Hussein, S., Dai, X. Q., Shafiei, S., Li, X. G., Shen, P. Y., Tu, J. C., Chen, X. Z.,
["Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada."]
["Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada."]
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.
PMID: 22174419

![]() ![]() ![]() ![]() ![]() |
![]() |
Screening
![]() |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|
Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPP3 |
![]() |
RACK1 | Yeast two-hybrid | Human | 1-291 | Human | Kidney | cDNA library | 22174419 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

![]() ![]() ![]() ![]() ![]() |
![]() |
Validation: In vitro validation
![]() |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|
Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPP3 |
![]() |
RACK1 | Fusion protein-pull down assay | Human | 1-291 | HEK293 lysates | Human | Full-length | E. coli | 22174419 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

![]() ![]() ![]() ![]() ![]() |
![]() |
Validation: In vivo validation
![]() |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|
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 | ||||
TRPP3 |
![]() |
RACK1 | Co-immunoprecipitation | Xenopus oocyte | Human | Full-length | Human | Full-length | 22174419 | |
TRPP3 |
![]() |
RACK1 | Co-immunoprecipitation | HEK293 | Human | Full-length | Human | Full-length | 22174419 | |
TRPP3 |
![]() |
RACK1 | Co-immunoprecipitation | mIMCD-3 | Human | Full-length | Human | Full-length | 22174419 | |
TRPP3 |
![]() |
RACK1 | Co-immunoprecipitation | Mouse kidney lysates | 22174419 | |||||
TRPP3 |
![]() |
RACK1 | Co-immunoprecipitation | Mouse heart tissue lysates | 22174419 | |||||
TRPP3 |
![]() |
RACK1 | Co-immunoprecipitation | Mouse brain tissue lysates | 22174419 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

![]() ![]() ![]() ![]() ![]() |
![]() |
Characterization
![]() |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|
Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPP3 |
![]() |
RACK1 | Yeast two-hybrid | Human | 1-291 | Human | 415-669 | 22174419 | ||
TRPP3 |
![]() |
RACK1 | Yeast two-hybrid | Human | 1-291 | Human | 649-951 | 22174419 | ||
TRPP3 |
![]() |
RACK1 | Yeast two-hybrid | Human | 55-291 | Human | 415-951 | 22174419 | ||
TRPP3 |
![]() |
RACK1 | Yeast two-hybrid | Human | 1696-2415 | Human | Not specified | 22174419 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

![]() ![]() ![]() ![]() ![]() |
![]() |
Functional consequence
![]() |
||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPP3 |
![]() |
RACK1 | Calcium measurement | Inhibition | 22174419 | |||||||
TRPP3 |
![]() |
RACK1 | Cell surface biotinylation | Decrease in plasma membrane level | 22174419 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
