Fischer G A, Clementi E, Raichman M, Südhof T, Ullrich A, Meldolesi J
Department of Molecular Biology, Max-Planck-Institut fur Biochemie, Martinsried, Federal Republic of Germany.
J Biol Chem. 1994 Jul 29;269(30):19216-24.
Stable clones of NIH 3T3 fibroblasts transfected with the cDNA of either the wild-type or deletion forms of the rat type I (or cerebellar) inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) were investigated. The delta form, missing the NH2-terminal sequence that includes the IP3-binding site, is expected to be still assembled with wild-type subunits to yield a tetrameric Ca2+ channel across the endoplasmic reticulum membrane; the s form, missing the membrane-spanning sequences, is expected to remain as a soluble monomer in the cytosol. With respect to control clones transfected with the vector only, the synthesis fo IP3Rs was markedly stimulated in the receptor-transfected clones. The mass accumulation, however, was increased only moderately (deletion forms = 15-30% of the endogenous IP3R), apparently because of a compensatory increase in receptor turnover. Coordinate changes in IP3 generation and Ca2+ release were revealed in the delta clones by experiments in both intact and permeabilized cells. In these clones, the IP3R was more sensitive to IP3, and IP3 generation at the ATP P2u surface receptor was decreased. This latter effect was due neither to a defect in G protein coupling nor to changes in phospholipase C expression, but to down-regulation of the P2u receptor. In the cells expressing the s- and delta-IP3R subunits, no differences with respect to the controls were observed in epidermal growth factor-induced DNA synthesis, whereas long-term growth stimulated by serum was reduced. Even more marked, especially in the delta clones (-90%), was the inhibition of cell transformation induced by autocrine stimulation with transforming growth factor alpha of the overexpressed epidermal growth factor receptors or by other growth factor receptors and oncogenes (platelet-derived growth factor/platelet-derived growth factor receptor beta, HER2/neu, and v-erbB). These effects appear not to be connected to the signaling processes mediated by tyrosine phosphorylation since the latter was unchanged in the delta clones. These results demonstrate for the first time (a) that the changes in cellular homeostasis directly induced by deleted IP3R subunits (increased receptor synthesis and increased IP3R sensitivity) are largely compensated by indirect coordinate changes apparently aimed to keep near normal the signaling properties of the cells; (b) that modulation of intracellular Ca2+ channels induces profound consequences that differentially affect growth and oncogenesis; and (c) that IP3Rs and the Ca2+ stores are important cross-roads of intracellular signaling pathways.
对用大鼠I型（或小脑）肌醇1,4,5 -三磷酸（IP3）受体的野生型或缺失形式的cDNA转染的NIH 3T3成纤维细胞的稳定克隆进行了研究。δ形式缺失包含IP3结合位点的NH2末端序列，预计仍会与野生型亚基组装形成跨内质网膜的四聚体Ca2+通道；s形式缺失跨膜序列，预计会以可溶性单体形式保留在细胞质中。与仅用载体转染的对照克隆相比，在受体转染的克隆中IP3Rs的合成受到显著刺激。然而，其质量积累仅适度增加（缺失形式为内源性IP3R的15 - 30%），这显然是由于受体周转的代偿性增加。在完整细胞和透化细胞中进行的实验均揭示了δ克隆中IP3生成和Ca2+释放的协同变化。在这些克隆中，IP3R对IP3更敏感，并且ATP P2u表面受体处的IP生产生减少。后一种效应既不是由于G蛋白偶联缺陷，也不是由于磷脂酶C表达的变化，而是由于P2u受体的下调。在表达s -和δ - IP3R亚基的细胞中观察到，在表皮生长因子诱导的DNA合成方面与对照没有差异，而血清刺激的长期生长则减少。更显著的是，特别是在δ克隆中（-90%），由过表达的表皮生长因子受体的自分泌刺激或其他生长因子受体和癌基因（血小板衍生生长因子/血小板衍生生长因子受体β、HER2/neu和v - erbB）诱导的细胞转化受到抑制。这些效应似乎与酪氨酸磷酸化介导的信号传导过程无关，因为在δ克隆中酪氨酸磷酸化没有变化。这些结果首次证明：（a）缺失的IP亚基直接诱导的细胞内稳态变化（受体合成增加和IP3R敏感性增加）在很大程度上被间接的协同变化所补偿，这些协同变化显然旨在使细胞的信号传导特性保持接近正常；（b）细胞内Ca2+通道的调节会产生深远影响，对生长和肿瘤发生有不同影响；（c）IP3Rs和Ca2+储存是细胞内信号传导途径的重要交叉点。
