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Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments.

Stornaiuolo M, Bruno A, Botta L, La Regina G, Cosconati S, Silvestri R, Marinelli L, Novellino E - Sci Rep (2015)

Bottom Line: This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs.ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands.Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples "Federico II", via D. Montesano 49, 80131 Naples, Italy.

ABSTRACT
A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

No MeSH data available.


Related in: MedlinePlus

ORG27569 and cholesterol dependent shuttling of CB1 among axons and soma of the neurons.SHSY-5Y were treated with MβC and replenished or not with cholesterol. After cholesterol manipulation cells were treated or not with ORG27569 (3 μM) for the indicated time. After being fixed and permeabilized, cells were processed for immunfluorescence to detect endogenous CB1 receptor (panel (a)), endogenous CB2 (panel (b)) or transiently expressed CB1-H2.41L-GFP (panel (b)). White arrows and red arrows indicate axonal region and soma of the cell, respectively.
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f4: ORG27569 and cholesterol dependent shuttling of CB1 among axons and soma of the neurons.SHSY-5Y were treated with MβC and replenished or not with cholesterol. After cholesterol manipulation cells were treated or not with ORG27569 (3 μM) for the indicated time. After being fixed and permeabilized, cells were processed for immunfluorescence to detect endogenous CB1 receptor (panel (a)), endogenous CB2 (panel (b)) or transiently expressed CB1-H2.41L-GFP (panel (b)). White arrows and red arrows indicate axonal region and soma of the cell, respectively.

Mentions: The effects of competition between ORG27569 and cholesterol were analyzed in cultured cells by following CB1 intracellular localization (Fig. 4). As already shown, upon agonist treatment, CB1 rapidly moves from the axons/dendrites to the neuron soma42 where endocytosis via chlatrin coated vesicles and receptor recycling occur. CB1 diffusion between axons/dendrites and soma was shown to be essential for its function43. We thus decided to follow change in CB1 localization in neurons upon ORG27569 treatment. After short treatment (30 minutes) with the allosteric molecule, the endogenous CB1 moved from axons to the cell soma (Fig. 4a), similarly to what has been reported after agonist treatment42. Noteworthy, longer treatment with ORG27569 (4 hours) induced the internalization of CB1. The effect of treatment with ORG27569 was specific for CB1 since neither CB1(H2.41L)-GFP or CB2 changed their localization after treatment with the allosteric molecule (Fig. 4b).


Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments.

Stornaiuolo M, Bruno A, Botta L, La Regina G, Cosconati S, Silvestri R, Marinelli L, Novellino E - Sci Rep (2015)

ORG27569 and cholesterol dependent shuttling of CB1 among axons and soma of the neurons.SHSY-5Y were treated with MβC and replenished or not with cholesterol. After cholesterol manipulation cells were treated or not with ORG27569 (3 μM) for the indicated time. After being fixed and permeabilized, cells were processed for immunfluorescence to detect endogenous CB1 receptor (panel (a)), endogenous CB2 (panel (b)) or transiently expressed CB1-H2.41L-GFP (panel (b)). White arrows and red arrows indicate axonal region and soma of the cell, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612305&req=5

f4: ORG27569 and cholesterol dependent shuttling of CB1 among axons and soma of the neurons.SHSY-5Y were treated with MβC and replenished or not with cholesterol. After cholesterol manipulation cells were treated or not with ORG27569 (3 μM) for the indicated time. After being fixed and permeabilized, cells were processed for immunfluorescence to detect endogenous CB1 receptor (panel (a)), endogenous CB2 (panel (b)) or transiently expressed CB1-H2.41L-GFP (panel (b)). White arrows and red arrows indicate axonal region and soma of the cell, respectively.
Mentions: The effects of competition between ORG27569 and cholesterol were analyzed in cultured cells by following CB1 intracellular localization (Fig. 4). As already shown, upon agonist treatment, CB1 rapidly moves from the axons/dendrites to the neuron soma42 where endocytosis via chlatrin coated vesicles and receptor recycling occur. CB1 diffusion between axons/dendrites and soma was shown to be essential for its function43. We thus decided to follow change in CB1 localization in neurons upon ORG27569 treatment. After short treatment (30 minutes) with the allosteric molecule, the endogenous CB1 moved from axons to the cell soma (Fig. 4a), similarly to what has been reported after agonist treatment42. Noteworthy, longer treatment with ORG27569 (4 hours) induced the internalization of CB1. The effect of treatment with ORG27569 was specific for CB1 since neither CB1(H2.41L)-GFP or CB2 changed their localization after treatment with the allosteric molecule (Fig. 4b).

Bottom Line: This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs.ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands.Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples "Federico II", via D. Montesano 49, 80131 Naples, Italy.

ABSTRACT
A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

No MeSH data available.


Related in: MedlinePlus