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Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor
1Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, USA.
Abstract:
We have found that phosphorylation of a G-protein-coupled receptor by protein kinase C (PKC) disrupts modulation of ion channels by the receptor. In AtT-20 cells transfected with rat cannabinoid receptor (CB1), the activation of an inwardly rectifying potassium current (Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by 100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected, and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop of CB1. Mutating this serine to alanine did not affect the ability of CB1 to modulate currents, but it eliminated disruption by PMA, demonstrating that PKC can disrupt ion channel modulation by receptor phosphorylation.
Insights
Protein kinase C (PKC) phosphorylation of cannabinoid receptors (CB1) disrupts their ability to modulate ion channels. This phosphorylation event prevents CB1 receptors from activating potassium currents and inhibiting calcium channels.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- G-protein-coupled receptors (GPCRs) play crucial roles in cellular signaling.
- GPCRs modulate ion channel activity, influencing neuronal excitability and neurotransmitter release.
- Protein kinase C (PKC) is a key enzyme involved in signal transduction pathways.
Purpose of the Study:
- To investigate the effect of PKC phosphorylation on cannabinoid receptor 1 (CB1) function.
- To determine if PKC-mediated phosphorylation disrupts CB1 receptor modulation of ion channels.
Main Methods:
- Utilized AtT-20 cells expressing rat CB1 receptors.
- Stimulated PKC using phorbol 12-myristate 13-acetate (PMA).
- Measured inwardly rectifying potassium (Kir) currents and P/Q-type calcium channels.
- Performed site-directed mutagenesis on CB1 receptor to identify phosphorylation sites.
Main Results:
- PKC stimulation prevented cannabinoid-induced activation of Kir currents and depression of P/Q-type calcium channels.
- PKC phosphorylation of CB1 receptors occurred at serine 317 (S317).
- Mutating S317 to alanine abolished the disruptive effect of PMA on CB1 receptor function.
Conclusions:
- PKC-mediated phosphorylation of CB1 receptors at S317 disrupts their ability to modulate ion channel activity.
- This mechanism provides insight into the regulation of GPCR signaling and neuronal function.