PKD2 Functions as an Epidermal Growth Factor-Activated Plasma Membrane Channel

Abstract

PKD2, or polycystin 2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, belongs to the transient receptor potential channel superfamily and has been shown to function as a nonselective cation channel in the plasma membrane. However, the mechanism of PKD2 activation remains elusive. We show that PKD2 overexpression increases epidermal growth factor (EGF)-induced inward currents in LLC-PK₁ kidney epithelial cells, while the knockdown of endogenous PKD2 by RNA interference or the expression of a pathogenic missense variant, PKD2-D511V, blunts the EGF-induced response. Pharmacological experiments indicate that the EGF-induced activation of PKD2 occurs independently of store depletion but requires the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP₂) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP₃) does not have any effect on this conductance. Overexpression of type Iα phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kα], which catalyzes the formation of PIP₂, suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-γ2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP₂ in the primary cilium of LLC-PK₁ cells. We propose that plasma membrane PKD2 is under negative regulation by PIP₂. EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP₂-mediated inhibition.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant DK59599 from NIH/NIDDK and the John S. Gammill Endowed Chair in Polycystic Kidney Disease to L.T. and grants from the Polycystic Kidney Disease Foundation to D.R. and R.M.

The first two authors contributed equally to this work.

We thank Brian Ceresa, George Dale, Eric Howard, and Mark Coggeshall for critical reading of the manuscript; Scott Plafker for pUB/RNAi/Ubc-H6; and Chris Carpenter for PIP(5)Kα.