Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) to generate the second messengers inositol trisphosphate (IP₃) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein–coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation–dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P₂-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P₂ from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P₂. Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P₂ pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

磷脂酶C（PLC）酶水解质膜（PM）脂质磷脂酰肌醇4,5-双磷酸酯（PI4,5P ₂）生成第二个信使肌醇三磷酸酯（IP ₃）和二酰基甘油（DAG）响应几乎所有哺乳动物细胞中的受体激活。我们先前发现，心脏细胞中G蛋白偶联受体（GPCR）的刺激导致高尔基体PLC依赖的磷脂酰肌醇4-磷酸（PI4P）水解，这是激活核蛋白激酶D（PKD）所需的过程在心脏肥大期间。我们假设GPCR刺激的PLC活化导致直接PI4P水解可能是DAG生产的一般机制。我们使用基于GFP的PI4P检测，测量了各种细胞中PM和高尔基PI4P库中GPCR活化依赖性的变化。用各种激动剂刺激在高尔基体和PM处引起PI4P相关的荧光随时间的减少，但与PI4,5P ₂相关的荧光未随时间的减少。有针对性地消耗PI4,5PGPCR刺激之前的PM中的₂对PM或高尔基体PI4P的耗竭，总肌醇磷酸（IP）产生或PKD激活没有影响。相反，PI4P的急性耗竭特别是在PM处完全阻断了GPCR依赖性IP的产生和PKD的激活，但并未改变PI4,5P ₂的丰度。高尔基体PI4P的急性耗竭对这些过程没有影响。这些数据表明，大多数PM PI4,5P ₂库不参与GPCR刺激的磷酸肌醇水解，而PM上的PI4P负责受体刺激的磷酸肌醇水解和DAG产生。