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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-PK(1) 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 (PIP(2)) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) does not have any effect on this conductance. Overexpression of type Ialpha phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kalpha], which catalyzes the formation of PIP(2), suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-gamma2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP(2) in the primary cilium of LLC-PK(1) cells. We propose that plasma membrane PKD2 is under negative regulation by PIP(2). EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP(2)-mediated inhibition.

SUBMITTER: Ma R

PROVIDER: S-EPMC1234340 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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PKD2 functions as an epidermal growth factor-activated plasma membrane channel.

Ma Rong R Li Wei-Ping WP Rundle Dana D Kong Jin J Akbarali Hamid I HI Tsiokas Leonidas L

Molecular and cellular biology 20050901 18

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-PK(1) kidney epithelial cells, while the knockdown of endogenous PKD2 by R ...[more]

PMID: 16135816

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Project description:Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP(2)) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P(2) but not PI(4)P(1) or PIP(3), releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP(2) in the plasma membrane and eliminated EGF-induced calpain activation. This PIP(2)-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP(2) in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP(2) concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-gamma hydrolyzes PIP(2) in the protruding lamellipodia.

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PKD2 functions as an epidermal growth factor-activated plasma membrane channel.

Publications

PKD2 functions as an epidermal growth factor-activated plasma membrane channel.

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