Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

Abstract

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P) to generate the second messengers inositol trisphosphate (IP) 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 PI4,5P-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P 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 PI4,5P Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P 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.

Gq-coupled muscarinic receptor enhancement of KCNQ2/3 channels and activation of TRPC channels in multimodal control of excitability in dentate gyrus granule cells

“…As above, neurons were studied under whole-cell brain-slice voltage-clamp conditions. From the robust literature documenting M 1 R-mediated depression of I M in multiple types of peripheral neurons and the known increase in neuronal excitability in hippocampus and other brain regions resulting from mAChR stimulation (Bymaster et al, 2003;Shen et al, 2005;Sheffler et al, 2009;Chiang et al, 2010), we expected parallel results from DGGCs and CA1 neurons. However, this was not the case.…”

“…In a variety of tissues, stimulation of G q/11 -coupled receptors stimulates PIP 2 synthesis concurrently with PLC hydrolysis, driven by rises in [Ca 2ϩ ] i (Lassing and Lindberg, 1990; Racaud-Sultan et al, 1993; Loew, 2007). Both physiological data in cerebellar neurons and biophysical model-ing (Finch and Augustine, 1998;Takechi et al, 1998;Brown et al, 2008) indicate that stimulation of G q/11 -coupled receptors must increase PIP 2 abundance in dendrite spine membrane by 2.5-fold to account for accumulation of local intracellular IP 3 . In sympathetic neurons, subcellular microdomain organization precludes M 1 R stimulation from inducing IP 3 -mediated [Ca 2ϩ ] i rises, allowing substantial PIP 2 depletion, whereas bradykinin B 2 R stimulation, for example, provokes sufficient rises in [Ca 2ϩ ] i and stimulation of PIP 2 synthesis to compensate for any decrease in PIP 2 abundance by PLC hydrolysis (Falkenburger et al, 2010).…”

“…Voltage-gated "M-type" (KCNQ, Kv7) K ϩ channels are ubiquitously expressed in the nervous system, where they regulate neuronal resting membrane potential, spike-frequency adaptation (SFA), bursting, and hyperexcitatory states (Cooper and Jan, 2003;Brown and Passmore, 2009;Gamper and Shapiro, 2015;Greene and Hoshi, 2017). M-channel function is modulated by metabotropic G-protein-coupled receptors, providing a powerful mechanism by which neuronal networks alter signaling functions.…”

Uveal Melanoma OncogeneCYSLTR2Encodes a Constitutively Active GPCR Highly Biased Toward Gq Signaling

“…It has a high catalytic activity to cleave the substrate phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). It has been suggested that PLC-βdependent IP1 accumulation also includes contributions from the IP2 formed by direct hydrolysis of PI4P, instead of IP2 formed by the dephosphorylation of IP3 formed by hydrolysis of PIP2 45 . Since this novel pathway does not depend on PIP2, which is predominantly found at the plasma membrane, the PI4P-dependent IP1 accumulation could have a substantial contribution to PLC-β signaling from the endosomal compartment.…”

Functional and structural characterization of allosteric activation of Phospholipase Cε by Rap1A

“…These proteins hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) at the plasma membrane, producing the second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which increase Ca 2+ in the cytoplasm and activate protein kinase C (PKC), respectively. However, some PLC subfamilies, such as PLCe, also hydrolyze other phosphatidylinositol phosphate (PIP) species at internal membranes (3)(4)(5)(6). Thus, PLC enzymes regulate multiple pathways from different subcellular locations (1,2).…”