Quantitative properties and receptor reserve of the DAG and PKC branch of Gq-coupled receptor signaling

Björn H. Falkenburger, Eamonn J Dickson, Bertil Hille

Research output: Contribution to journalArticle

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Abstract

Gq protein-coupled receptors (GqPCRs) of the plasma membrane activate the phospholipase C (PLC) signaling cascade. PLC cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers diacylgycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to calcium release, protein kinase C (PKC) activation, and in some cases, PIP2 depletion. We determine the kinetics of each of these downstream endpoints and also ask which is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels in single living tsA-201 cells. We measure DAG production and PKC activity by Förster resonance energy transfer- based sensors, and PIP2 by KCNQ2/3 channels. Fully activating endogenous purinergic receptors by uridine 5triphosphate (UTP) leads to calcium release, DAG production, and PKC activation, but no net PIP2 depletion. Fully activating high-density transfected muscarinic receptors (M1Rs) by oxotremorine-M (Oxo-M) leads to similar calcium, DAG, and PKC signals, but PIP2 is depleted. KCNQ2/3 channels are inhibited by the Oxo-M treatment (85%) and not by UTP (<1%), indicating that depletion of PIP2 isrequired to inhibit KCNQ2/3 in response to receptor activation. Overexpression of A kinase-anchoring protein (AKAP)79 or calmodulin (CaM) does not increase KCNQ2/3 inhibition by UTP.From these results and measurements of IP3 and calcium presented in our companion paper (Dickson et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210886), we extendour kinetic model for signaling from M1Rs to DAG/PKC and IP3/calcium signaling. We conclude that calcium/ CaM and PKC-mediated phosphorylation do not underlie dynamic KCNQ2/3channel inhibition during GqPCR activation in tsA-201 cells. Finally, our experimental data provide indirect evidence for cleavage of PI(4)P by PLC in living cells, and our modeling revisits/explains the concept of receptor reserve with measurements from all steps ofGqPCR signaling.

Original languageEnglish (US)
Pages (from-to)537-555
Number of pages19
JournalJournal of General Physiology
Volume141
Issue number5
DOIs
StatePublished - May 2013
Externally publishedYes

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Protein Kinase C
Calcium
Type C Phospholipases
Gq-G11 GTP-Binding Protein alpha Subunits
Uridine Triphosphate
KCNQ2 Potassium Channel
Purinergic Receptors
Calcium-Calmodulin-Dependent Protein Kinases
Inositol 1,4,5-Trisphosphate
Calcium Signaling
Uridine
Energy Transfer
Second Messenger Systems
Muscarinic Receptors
Calmodulin
Membrane Lipids
Phosphatidylinositols
Protein Kinases
Phosphorylation
Cell Membrane

ASJC Scopus subject areas

  • Physiology

Cite this

Quantitative properties and receptor reserve of the DAG and PKC branch of Gq-coupled receptor signaling. / Falkenburger, Björn H.; Dickson, Eamonn J; Hille, Bertil.

In: Journal of General Physiology, Vol. 141, No. 5, 05.2013, p. 537-555.

Research output: Contribution to journalArticle

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abstract = "Gq protein-coupled receptors (GqPCRs) of the plasma membrane activate the phospholipase C (PLC) signaling cascade. PLC cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers diacylgycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to calcium release, protein kinase C (PKC) activation, and in some cases, PIP2 depletion. We determine the kinetics of each of these downstream endpoints and also ask which is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels in single living tsA-201 cells. We measure DAG production and PKC activity by F{\"o}rster resonance energy transfer- based sensors, and PIP2 by KCNQ2/3 channels. Fully activating endogenous purinergic receptors by uridine 5triphosphate (UTP) leads to calcium release, DAG production, and PKC activation, but no net PIP2 depletion. Fully activating high-density transfected muscarinic receptors (M1Rs) by oxotremorine-M (Oxo-M) leads to similar calcium, DAG, and PKC signals, but PIP2 is depleted. KCNQ2/3 channels are inhibited by the Oxo-M treatment (85{\%}) and not by UTP (<1{\%}), indicating that depletion of PIP2 isrequired to inhibit KCNQ2/3 in response to receptor activation. Overexpression of A kinase-anchoring protein (AKAP)79 or calmodulin (CaM) does not increase KCNQ2/3 inhibition by UTP.From these results and measurements of IP3 and calcium presented in our companion paper (Dickson et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210886), we extendour kinetic model for signaling from M1Rs to DAG/PKC and IP3/calcium signaling. We conclude that calcium/ CaM and PKC-mediated phosphorylation do not underlie dynamic KCNQ2/3channel inhibition during GqPCR activation in tsA-201 cells. Finally, our experimental data provide indirect evidence for cleavage of PI(4)P by PLC in living cells, and our modeling revisits/explains the concept of receptor reserve with measurements from all steps ofGqPCR signaling.",
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N2 - Gq protein-coupled receptors (GqPCRs) of the plasma membrane activate the phospholipase C (PLC) signaling cascade. PLC cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers diacylgycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to calcium release, protein kinase C (PKC) activation, and in some cases, PIP2 depletion. We determine the kinetics of each of these downstream endpoints and also ask which is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels in single living tsA-201 cells. We measure DAG production and PKC activity by Förster resonance energy transfer- based sensors, and PIP2 by KCNQ2/3 channels. Fully activating endogenous purinergic receptors by uridine 5triphosphate (UTP) leads to calcium release, DAG production, and PKC activation, but no net PIP2 depletion. Fully activating high-density transfected muscarinic receptors (M1Rs) by oxotremorine-M (Oxo-M) leads to similar calcium, DAG, and PKC signals, but PIP2 is depleted. KCNQ2/3 channels are inhibited by the Oxo-M treatment (85%) and not by UTP (<1%), indicating that depletion of PIP2 isrequired to inhibit KCNQ2/3 in response to receptor activation. Overexpression of A kinase-anchoring protein (AKAP)79 or calmodulin (CaM) does not increase KCNQ2/3 inhibition by UTP.From these results and measurements of IP3 and calcium presented in our companion paper (Dickson et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210886), we extendour kinetic model for signaling from M1Rs to DAG/PKC and IP3/calcium signaling. We conclude that calcium/ CaM and PKC-mediated phosphorylation do not underlie dynamic KCNQ2/3channel inhibition during GqPCR activation in tsA-201 cells. Finally, our experimental data provide indirect evidence for cleavage of PI(4)P by PLC in living cells, and our modeling revisits/explains the concept of receptor reserve with measurements from all steps ofGqPCR signaling.

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