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Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A2A receptor.

ABSTRACT: Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, indirectly via modifications to the membrane, or via both mechanisms is not well understood. High-resolution crystal structures of GPCRs have identified bound cholesterols; based on a ?2-adrenergic receptor (?2AR) structure bound to cholesterol and the presence of conserved amino acids in class A receptors, the cholesterol consensus motif (CCM) was identified. Here in mammalian cells expressing the adenosine A2A receptor (A2AR), ligand dependent production of cAMP is reduced following membrane cholesterol depletion with methyl-beta-cyclodextrin (M?CD), indicating that A2AR signaling is dependent on cholesterol. In contrast, ligand binding is not dependent on cholesterol depletion. All-atom molecular simulations suggest that cholesterol interacts specifically with the CCM when the receptor is in an active state, but not when in an inactive state. Taken together, the data support a model of receptor state-dependent binding between cholesterol and the CCM, which could facilitate both G-protein coupling and downstream signaling of A2AR.

SUBMITTER: McGraw C

PROVIDER: S-EPMC6383678 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A2A receptor.

McGraw Claire C Yang Lewen L Levental Ilya I Lyman Edward E Robinson Anne Skaja AS

Biochimica et biophysica acta. Biomembranes 20190108 4

Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, indirectly via modifications to the membrane, or via both mechanisms is not well understood. High-resolution crystal structures of GPCRs have identified bound cholesterols; based on a β2-adrenergic receptor (β2AR) structure bound to cholesterol and the presence of conserved amino acids in class A receptors, the chol ...[more]

PMID: 30629951

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Project description:Autophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

Dataset Information

Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A2A receptor.

Publications

Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A<sub>2A</sub> receptor.

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