Physiological requirement for both SH2 domains for phospholipase C-gamma1 function and interaction with platelet-derived growth factor receptors.
Physiological requirement for both SH2 domains for phospholipase C-gamma1 function and interaction with platelet-derived growth factor receptors.
复制标题
磷脂酶 C-gamma1 功能和与血小板衍生生长因子受体相互作用的两个 SH2 结构域的生理需求。
DOI：
10.1128/mcb.19.7.4961
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发表时间：
1999
影响因子：
5.3
通讯作者：
Carpenter,G
中科院分区：
文献类型：
--
作者：
Ji,QS;Chattopadhyay,A;Vecchi,M;Carpenter,G
已有两种方法用于研究单个SH 2结构域在磷脂酶C-γ1（PLC-γ1）的生长因子活化中的作用。表面等离子体共振分析表明，单个N-SH 2和C-SH 2结构域能够特异性识别对应于血小板衍生生长因子（PDGF）β受体Tyr 1021的含磷酸酪氨酸的肽。为了评估全长PLC-γ1分子以及完整细胞中的SH 2功能，在Plcg 1 −/−成纤维细胞中表达了通过N-SH 2和/或C-SH 2结构域的定点突变而失活的PLC-γ1 SH 2结构域突变体。在平衡孵育条件下（4°C，40 min），N-SH 2结构域（而非C-SH 2结构域）足以介导PLC-γ1与活化的PDGF受体的显著缔合和PLC-γ1酪氨酸磷酸化。当PLC-γ1中的两个SH 2结构域都被禁用时，双突变体不与活化的PDGF受体结合，并且不被酪氨酸磷酸化。然而，没有一个SH 2突变体能够介导生长因子激活Ca 2+动员或肌醇1，4，5-三磷酸（IP 3）的形成。随后的动力学实验表明，每一个单一的SH 2结构域突变体的能力显着受损，介导快速协会与活化的PDGF受体，成为酪氨酸磷酸化。因此，当在实现快速生物反应（Ca 2+动员和IP 3形成）所必需的生理条件下测定时，PLC-γ1的两个SH 2结构域对于生长因子反应性是必需的。
Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-γ1 (PLC-γ1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) β receptor. To assess SH2 function in the context of the full-length PLC-γ1 molecule as well as within the intact cell, PLC-γ1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed inPlcg1−/−fibroblasts. Under equilibrium incubation conditions (4°C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-γ1 association with the activated PDGF receptor and PLC-γ1 tyrosine phosphorylation. When both SH2 domains in PLC-γ1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+mobilization and IP3formation), both SH2 domains of PLC-γ1 are essential to growth factor responsiveness.