Cyclic AMP‐dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase‐α and synaptic 2‐arachidonoyl glycerol signaling

• Published in: Journal of neurochemistry Vol. 153; no. 3; pp. 334 - 345
• Main Authors: Shonesy, Brian C., Stephenson, Jason R., Marks, Christian R., Colbran, Roger J.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.05.2020
• Subjects: Animals; Antibodies; Arachidonic Acids - metabolism; Brain; Brain - drug effects; Brain - metabolism; Calcium; Calcium influx; Cannabinoid receptors; Cannabinoids; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases - metabolism; Cyclic AMP-Dependent Protein Kinases - pharmacology; D1 dopamine receptor; Depolarization; diacylglycerol lipase; Diglycerides; Dopamine; Dopamine Agonists - pharmacology; Dopamine Antagonists - pharmacology; Dopamine D1 receptors; Dose-Response Relationship, Drug; endocannabinoid; Endocannabinoids - metabolism; Excitation; Glutamatergic transmission; Glutamic acid receptors (metabotropic); Glycerides - metabolism; Glycerol; HEK293 Cells; Humans; Incubation; Kinases; Lipase; Lipoprotein lipase; Lipoprotein Lipase - metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neostriatum; Neurons; Neurotransmitters; Organ Culture Techniques; Phosphorylation; PKA; Protein kinase A; protein phosphorylation; Proteins; Receptors; Receptors, Dopamine D1 - agonists; Receptors, Dopamine D1 - antagonists & inhibitors; Receptors, Dopamine D1 - metabolism; Signal transduction; Signal Transduction - drug effects; Signal Transduction - physiology; Signaling; Spiny neurons; Synaptic depression; Synthesis; γ-Aminobutyric acid; endocannabinoid; D1 dopamine receptor; protein phosphorylation; diacylglycerol lipase; PKA
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.14972

Brain endocannabinoids serve as retrograde neurotransmitters, being synthesized in post‐synaptic neurons “on demand” and released to bind pre‐synaptic cannabinoid receptors and suppress glutamatergic or GABAergic transmission. The most abundant brain endocannabinoid, 2 arachidonoyl glycerol (2‐AG), is primarily synthesized by diacylglycerol lipase‐α (DGLα), which is activated by poorly understood mechanisms in response to calcium influx following post‐synaptic depolarization and/or the activation of Gq‐coupled group 1 metabotropic glutamate receptors. However, the impact of other neurotransmitters and their downstream signaling pathways on synaptic 2‐AG signaling has not been intensively studied. Here, we found that DGLα activity in membrane fractions from transfected HEK293T cells was significantly increased by in vitro phosphorylation using cyclic AMP‐dependent protein kinase (PKA). Moreover, PKA directly phosphorylated DGLα at Ser798 in vitro. Elevation of cAMP levels in HEK293 cells expressing DGLα increased Ser798 phosphorylation, as detected using a phospho‐Ser798‐specific antibody, and enhanced DGLα activity; this in situ enhancement of DGLα activity was prevented by mutation of Ser798 to Ala. We investigated the impact of PKA on synaptic 2‐AG mobilization in mouse striatal slices by manipulating D1‐dopamine receptor (D1R) signaling and assessing depolarization‐induced suppression of excitation, a DGLα‐ and 2‐AG‐dependent form of short‐term synaptic depression. The magnitude of depolarization‐enhanced suppression of excitation in direct pathway medium spiny neurons was increased by pre‐incubation with a D1R agonist, and this enhancement was blocked by post‐synaptic inhibition of PKA. Taken together, these findings provide new molecular insights into the complex mechanisms regulating synaptic endocannabinoid signaling. Post‐synaptic synthesis of a major brain endocannabinoid, 2‐arachidonoyl glycerol (2‐AG), from diacylglycerol (DAG) by diacylglycerol lipase‐α (DGLα) is stimulated by L‐type voltage‐gated calcium channels (LTCC) and/or metabotropic glutamate receptors (mGluR1/5). Shonesy et al show that cyclic AMP‐dependent protein kinase (PKA) phosphorylates Ser798 in DGLα to increase activity. Their data indicate that D1‐dopamine receptors (D1R) stimulate adenylyl cyclase (Ca2+‐AC) and PKA to enhance synaptic 2‐AG production by DGLα and short‐term depression of glutamatergic transmission, which depends on pre‐synaptic endocannabinoid 1 receptors (CB1R). Ca2+/calmodulin‐dependent protein kinase II (CaMKII) was previously shown to phosphorylate distinct sites in DGLα to restrain synaptic 2‐AG synthesis. Read the Editorial Highlight for this article on https://doi.org/10.1111/jnc.14977