Omoto Yujiro, Higa-Nakamine Sayomi, Higa Airi, Yamamoto Hideyuki
Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
Eur J Pharmacol. 2017 Mar 15;799:171-179. doi: 10.1016/j.ejphar.2017.02.006. Epub 2017 Feb 4.
The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G-protein-coupled receptors, and its stimulation activates extracellular signal-regulated protein kinase (ERK). In the present study, we first examined the actions of GnRH on the ErbB family using two types of cultured gonadotroph cells. As reported previously, AG1478, an inhibitor of the ErbB family tyrosine kinase, inhibited GnRH-induced ERK activation in undifferentiated gonadotroph αT3-1 cells. However, AG1478 did not inhibit ERK activation in differentiated gonadotroph LβT2 cells, suggesting that transactivation of the ErbB family was not necessary for ERK activation in LβT2 cells. We found that ErbB4 was expressed in αT3-1 cells but not in LβT2 cells. GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment in αT3-1 cells. Pharmacological experiments suggested that G and tumor necrosis factor-α-converting enzyme (TACE) were essential for GnRH-induced ErbB4 cleavage. GnRH increased the phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), indicating that GnRH activated protein kinase C (PKC). Down-regulation of PKC and bisindolylmaleimide I, a PKC inhibitor, strongly inhibited the GnRH-induced cleavage of ErbB4. It was surprising that GnRH treatment of LβT2 cells after overexpression of ErbB4 induced ErbB4 cleavage in a TACE-dependent manner. ErbB4 cleavage was induced also by treatment of αT3-1 cells, ErbB4-overexpressing LβT2 cells, and immortalized GnRH neurons (GT1-7 cells) with leuprorelin acetate. These results may suggest that the pharmacological effects of leuprorelin acetate are conducted through TACE-mediated proteolysis of membrane proteins, including ErbB4, in gonadotroph cells and GnRH neurons.
促性腺激素释放激素（GnRH）受体属于G蛋白偶联受体，其刺激可激活细胞外信号调节蛋白激酶（ERK）。在本研究中，我们首先使用两种类型的培养促性腺激素细胞研究了GnRH对表皮生长因子受体（ErbB）家族的作用。如先前报道，ErbB家族酪氨酸激酶抑制剂AG1478可抑制未分化促性腺激素αT3-1细胞中GnRH诱导的ERK激活。然而，AG1478并未抑制分化的促性腺激素LβT2细胞中的ERK激活，这表明ErbB家族的反式激活对于LβT2细胞中的ERK激活并非必需。我们发现ErbB4在αT3-1细胞中表达，但在LβT2细胞中不表达。GnRH诱导αT3-1细胞中ErbB4的裂解和一个80 kDa片段的积累。药理学实验表明，G蛋白和肿瘤坏死因子-α转换酶（TACE）对于GnRH诱导的ErbB4裂解至关重要。GnRH增加了豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）的磷酸化，表明GnRH激活了蛋白激酶C（PKC）。PKC的下调和PKC抑制剂双吲哚马来酰亚胺I强烈抑制了GnRH诱导的ErbB4裂解。令人惊讶的是，在过表达ErbB4后用GnRH处理LβT2细胞会以TACE依赖的方式诱导ErbB4裂解。用醋酸亮丙瑞林处理αT3-1细胞、过表达ErbB4的LβT2细胞和永生化GnRH神经元（GT1-7细胞）也可诱导ErbB4裂解。这些结果可能表明，醋酸亮丙瑞林的药理作用是通过TACE介导的促性腺激素细胞和GnRH神经元中包括ErbB4在内的膜蛋白的蛋白水解来实现的。
