Stimulation by parathyroid hormone of a NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cβ, and actin increases intracellular calcium in opossum kidney cells
Stimulation by parathyroid hormone of a NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cβ, and actin increases intracellular calcium in opossum kidney cells
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DOI：
10.1074/jbc.m313229200
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发表时间：
2004-05-28
影响因子：
4.8
通讯作者：
Segre, GV
中科院分区：
文献类型：
--
作者：
Mahon, MJ;Segre, GV
甲状旁腺激素(PTH)通过腺苷环化酶和磷脂酶C(PLC)结合其同源G蛋白偶联受体(PTH1R)和信号。PTH1R的C末端决定簇通过与两个PDZ(PSD95，Diss-Large，ZO-1)结构域中的第一个结构域结合，与Na+/H+交换调节因子1(NHERF-1)相互作用。与野生型负鼠肾(OK)细胞相比，OKH细胞不表现为PTH介导的[Ca~(2+)](I)升高，NHERF-1的表达水平明显低于野生型。NHERF-1在OKH亲本(OKH-N1)中的稳定表达恢复了PTH介导的细胞外钙内流引起的[Ca~(2+)](I)升高，这种升高既是PLC依赖的，也是百日咳毒素敏感的。从形态学的角度来看，NHERF-1和PTH1R共同定位于OKH-N1细胞的顶端斑块，这一表达模式在OKH-N1细胞中是不存在的，并且依赖于OKH-N1细胞中NHERF-1和PTH1R的直接相互作用。肌动蛋白、PLCbeta1和-beta3与NHERF-1和PTH1R共同定位于OKH-N1细胞的顶端斑块。肌动蛋白也是NHERF-1组装复合体的组成部分，因为细胞松弛素D扰乱了NHERF-1和PTH1R的顶端定位，并抑制了PTH介导的[Ca~(2+)](I)升高。NHERF-1的第一个PDZ结构域的表达通过阻断PTH1R的顶端定位和抑制PTH诱导的[Ca~(2+)](I)升高而起到显性-负性相互作用。因此，NHERF-1在OK细胞的顶端区域组装了一个包含PTH1R、PLCβ和肌动蛋白细胞骨架的信号复合体。该复合体的破坏可阻断甲状旁腺素介导的细胞内钙的增加。
Parathyroid hormone (PTH) binds its cognate G-protein-coupled receptor (PTH1R) and signals through both adenylyl cyclase and phospholipase C ( PLC). C-terminal determinants of the PTH1R interact with the Na+/H+ exchanger regulatory factor 1 (NHERF-1) by binding the first of two PDZ (psd95, discs-large, ZO-1) domains. Compared with wild-type opossum kidney (OK) cells, OKH cells, a sub-clone, do not display PTH-mediated increases of [Ca2+](i) and express NHERF-1 at markedly lower levels. Stable expression of NHERF-1 in the OKH parent (OKH-N1) restores the PTH-mediated increase of [Ca2+](i) that arises from an influx of extracellular calcium and is both PLC-dependent and pertussis toxin-sensitive. From a morphological perspective, NHERF-1 and the PTH1R co-localize to apical patches of OKH-N1 cells, an expression pattern that is absent in OKH cells and depends on a direct NHERF-1-PTH1R interaction in OKH-N1 cells. Actin and PLCbeta1 and -beta3 co-localize with NHERF-1 and the PTH1R in OKH-N1 cell apical patches. Actin is also an integral component of the NHERF-1-assembled complex because cytochalasin D disrupts apical localization of both NHERF-1 and the PTH1R and inhibits the PTH-mediated increase of [Ca2+](i). Expression of the first PDZ domain of NHERF-1 acts as a dominant-negative interactor by blocking apical localization of the PTH1R and inhibiting PTH-elicited increases of [Ca2+](i). Thus, NHERF-1 assembles a signaling complex in the apical domains of OK cells that contains the PTH1R, PLCbeta, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium.