Guan Jiazhen, Luo Yuan, Denker Bradley M
Renal Division, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Biochem J. 2005 Dec 1;392(Pt 2):389-97. doi: 10.1042/BJ20042102.
Purkinje cell protein-2 (Pcp2 or L7) is highly expressed in cerebellar Purkinje cells and retinal bipolar neurons and interacts with the Galpha(i/o) family of G-proteins. Although the expression pattern of Pcp2 in the developing central nervous system suggests a role in differentiation, its function remains unknown. We established Tet-off inducible expression of Pcp2 in PC12 cells (rat pheochromocytoma cells) to determine whether Pcp2 regulates neuronal differentiation. Utilizing a polyclonal antibody, Pcp2 was localized in the cell body and throughout neurites of differentiated PC12 cells, similar to its localization in cerebellar Purkinje cells. Pcp2 expression in PC12 cells stimulated process formation (5-fold) and NGF (nerve growth factor)-stimulated neurite length (2-fold). Under basal conditions, Pcp2-PC12 cells demonstrated a 5-fold increase in Ras activation relative to non-induced PC12 cells and there was no change in extracellular-signal-regulated kinase 1/2 activity with Pcp2 expression. However, Pcp2 induction led to a >3-fold increase in basal p38 MAPK (mitogen-activated protein kinase) activity and the addition of NGF significantly stimulated both Ras and p38 MAPK in Pcp2-PC12 cells relative to the controls. Pretreatment of Pcp2-PC12 cells with the p38-specific inhibitor SB203580 blocked both the increased neurite formation and NGF-stimulated neurite growth. Pertussis toxin treatment had no effect on neurite growth in control cells, but completely blocked Pcp2-mediated increased neurite growth. Transient transfection of the beta-adrenergic receptor kinase C-terminus to prevent signalling through Gbetagamma in Pcp2-PC12 cells also inhibited the Pcp2-induced phenotype and reduced the Pcp2-stimulated Ras activation. Taken together, these findings demonstrate that Pcp2 induces differentiation in PC12 cells, in part through Gbetagamma-mediated Ras and p38 MAPK activation and suggest the potential for similar signalling mechanisms in Purkinje cells.
浦肯野细胞蛋白2（Pcp2或L7）在小脑浦肯野细胞和视网膜双极神经元中高度表达，并与G蛋白的Gα（i/o）家族相互作用。尽管Pcp2在发育中的中枢神经系统中的表达模式表明其在分化中起作用，但其功能仍然未知。我们在PC12细胞（大鼠嗜铬细胞瘤细胞）中建立了Pcp2的Tet-off诱导表达，以确定Pcp2是否调节神经元分化。利用多克隆抗体，Pcp2定位于分化的PC12细胞的细胞体和整个神经突中，类似于其在小脑浦肯野细胞中的定位。PC12细胞中Pcp2的表达刺激了突起形成（5倍）和神经生长因子（NGF）刺激的神经突长度（2倍）。在基础条件下，相对于未诱导的PC12细胞，Pcp2-PC12细胞的Ras激活增加了5倍，并且Pcp2表达后细胞外信号调节激酶1/2活性没有变化。然而，Pcp2的诱导导致基础p38丝裂原活化蛋白激酶（MAPK）活性增加>3倍，并且相对于对照，添加NGF显著刺激了Pcp2-PC12细胞中的Ras和p38 MAPK。用p38特异性抑制剂SB203580预处理Pcp2-PC12细胞可阻断神经突形成增加和NGF刺激的神经突生长。百日咳毒素处理对对照细胞的神经突生长没有影响，但完全阻断了Pcp2介导的神经突生长增加。在Pcp2-PC12细胞中瞬时转染β-肾上腺素能受体激酶C末端以防止通过Gβγ信号传导也抑制了Pcp2诱导的表型并降低了Pcp2刺激的Ras激活。综上所述，这些发现表明Pcp2部分通过Gβγ介导的Ras和p38 MAPK激活诱导PC12细胞分化，并提示浦肯野细胞中存在类似信号机制的可能性。
