Cheng J, Baldassare J J, Raben D M
Department of Physiology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Biochem J. 1999 Jan 1;337 ( Pt 1)(Pt 1):97-104.
Addition of alpha-thrombin to quiescent IIC9 cells results in the activation of lipid-metabolizing enzymes associated with signal-transduction cascades. These enzymes include phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), phosphatidylcholine (PC)-specific phospholipases C and D and phospholipase A2 (PLA2). Whereas the alpha-thrombin receptor has been shown to couple with PI-PLCs, it is not clear whether this receptor, or a putative second receptor, couples to one or more of the other phospholipases. In this report we determine whether the cloned receptor couples to all or a subset of these enzymes. We show that (i) an alpha-thrombin-receptor-activating peptide also elicits the above responses and (ii) addition of enterokinase to IIC9 cells, stably transfected with an alpha-thrombin receptor (enterokinase- responsive alpha-thrombin receptor, EKTR) containing an enterokinase cleavage site in place of an alpha-thrombin cleavage site, stimulates both PI and PC hydrolysis, including PLA2. Enterokinase also induces mitogenesis in the IIC9s transfected with EKTR. These results indicate that, in addition to initiating a mitogenic signalling cascade, the cloned alpha-thrombin receptor couples to enzymes involved in generating PC-derived, as well as PI-derived, second-messenger molecules in IIC9s. Additionally, using the cells transfected with EKTR, we further demonstrate that only activated, i. e. cleaved, receptors are desensitized.
向静止的IIC9细胞中添加α-凝血酶会导致与信号转导级联相关的脂质代谢酶被激活。这些酶包括磷脂酰肌醇（PI）特异性磷脂酶C（PI-PLC）、磷脂酰胆碱（PC）特异性磷脂酶C和D以及磷脂酶A2（PLA2）。虽然已证明α-凝血酶受体与PI-PLCs偶联，但尚不清楚该受体或假定的第二受体是否与一种或多种其他磷脂酶偶联。在本报告中，我们确定克隆的受体是否与这些酶的全部或一部分偶联。我们发现：（i）一种α-凝血酶受体激活肽也能引发上述反应；（ii）向稳定转染了含肠激酶切割位点而非α-凝血酶切割位点的α-凝血酶受体（肠激酶反应性α-凝血酶受体，EKTR）的IIC9细胞中添加肠激酶，会刺激PI和PC的水解，包括PLA2。肠激酶还能诱导转染了EKTR的IIC9细胞发生有丝分裂。这些结果表明，除了启动有丝分裂信号级联反应外，克隆的α-凝血酶受体还与参与在IIC9细胞中生成源自PC以及源自PI的第二信使分子的酶偶联。此外，利用转染了EKTR的细胞，我们进一步证明只有活化的，即被切割的受体才会脱敏。
