PKD2 functions as an epidermal growth factor-activated plasma membrane channel
PKD2 functions as an epidermal growth factor-activated plasma membrane channel
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DOI：
10.1128/mcb.25.18.8285-8298.2005
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发表时间：
2005-09-01
影响因子：
5.3
通讯作者：
Tsiokas, L
中科院分区：
文献类型：
--
作者：
Ma, R;Li, WP;Tsiokas, L
PKD2或Pollycystin 2是该基因在2型常染色体显性遗传性多囊肾病中突变的产物，属于瞬时受体电位通道超家族，是细胞膜上的一种非选择性阳离子通道。然而，PKD2的激活机制仍然不清楚。我们发现，PKD2过表达增加了表皮生长因子(EGF)诱导的LLC-PK1肾上皮细胞的内向电流，而内源性PKD2被RNA干扰或致病错义变异体PKD2-D511V的表达下调，钝化了EGF诱导的反应。药理实验表明，EGF诱导的PKD2的激活不依赖于储备物的消耗，但需要磷脂酶C(PLC)和磷脂酰肌醇3-激酶(PI3K)的活性。吸管注入纯化的磷脂酰肌醇-4，5-二磷酸(PIP2)可抑制PKD2介导的EGF诱导的电导效应，而吸管注入磷脂酰肌醇-3，4，5-三磷酸(PIP3)对该电导无影响。过表达I型磷脂酰肌醇-4-磷酸5-激酶[PIP(5)Kα]可催化PIP_1的形成，抑制EGF诱导的电流。生化实验表明，在HEK293T细胞中，PKD2与PLC-γ2和EGF受体(EGFR)发生物理相互作用，并与EGFR和PIP共存于LLC-PK细胞的原代纤毛中。我们认为质膜PKD2受到PIP2的负调控。EGF可能通过释放PIP2介导的抑制来降低机械刺激和其他刺激对PKD2激活的阈值。
PKD2, or pollycystin 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 I alpha phosphatidylinositol-4-phosphate 5-kinase [PIP(5)K alpha], which catalyzes the formation of PIP,, suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-gamma 2 and EGF receptor (EGFR) in transfected HEK293T cells and collocalizes with EGFR and PIP, in the primary cilium of LLC-PK, 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.