Chen L, Fitzpatrick V D, Vandlen R L, Tashjian A H
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Jul 25;272(30):18666-72. doi: 10.1074/jbc.272.30.18666.
To elucidate the signaling events mediated by specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and SSTR2 individually in rat pituitary GH12C1 and F4C1 cells, which lack endogenous somatostatin receptors. In transfected GH12C1 cells, both SSTR1 and SSTR2 coupled to inhibition of Ca2+ influx and hyperpolarization of membrane potential via a pertussis toxin (PTx)-sensitive mechanism. These effects reflected modulation of ion channel activities which are important for regulation of hormone secretion. Somatostatin analogs MK678 and CH275 acted as subtype selective agonists as expected. In transfected F4C1 cells, both SSTR1 and SSTR2 mediated somatostatin-induced inhibition of adenylyl cyclase via a PTx-sensitive pathway. In addition, activation of SSTR2 in F4C1 cells, but not SSTR1, stimulated phospholipase C (PLC) activity and an increase in [Ca2+]i due to release of Ca2+ from intracellular stores. Unlike adenylyl cyclase inhibition, the PLC-mediated response was only partially sensitive to PTx. To determine the structural determinants in SSTR2 necessary for activation of PLC, we constructed chimeric receptors in which domains of SSTR2 were introduced into SSTR1. Chimeric receptors containing only the third intracellular loop, or all three intracellular loops from SSTR2, mediated inhibition of adenylyl cyclase, but failed to stimulate PLC activity as did wild-type SSTR2. Furthermore, the C-terminal tail of SSTR2 was not required for coupling to PLC. Thus, by expressing individual somatostatin receptor subtypes in pituitary cells, we have identified both overlapping and distinct signaling pathways for SSTR1 and SSTR2, and have shown that sequences other than simply the intracellular domains are required for SSTR2 to couple to the PLC signaling pathway.
为了阐明特定生长抑素受体（SSTR）亚型介导的信号转导事件，我们分别在缺乏内源性生长抑素受体的大鼠垂体GH12C1和F4C1细胞中表达了SSTR1和SSTR2。在转染的GH12C1细胞中，SSTR1和SSTR2均通过百日咳毒素（PTx）敏感机制与抑制Ca2+内流和膜电位超极化偶联。这些效应反映了对离子通道活性的调节，而离子通道活性对激素分泌的调节很重要。生长抑素类似物MK678和CH275如预期的那样作为亚型选择性激动剂起作用。在转染的F4C1细胞中，SSTR1和SSTR2均通过PTx敏感途径介导生长抑素诱导的腺苷酸环化酶抑制。此外，F4C1细胞中SSTR2的激活而非SSTR1的激活刺激了磷脂酶C（PLC）活性，并由于细胞内储存的Ca2+释放导致[Ca2+]i增加。与腺苷酸环化酶抑制不同，PLC介导的反应仅对PTx部分敏感。为了确定激活PLC所需的SSTR2中的结构决定因素，我们构建了嵌合受体，其中将SSTR2的结构域引入SSTR1。仅包含第三个细胞内环或来自SSTR2的所有三个细胞内环的嵌合受体介导了腺苷酸环化酶的抑制，但未能像野生型SSTR2那样刺激PLC活性。此外，SSTR2的C末端尾巴对于与PLC偶联不是必需的。因此，通过在垂体细胞中表达单个生长抑素受体亚型，我们确定了SSTR1和SSTR2重叠和不同的信号转导途径，并表明SSTR2与PLC信号转导途径偶联所需的序列不仅仅是细胞内结构域。
