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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

Functional competition between ORG27569 and cholesterol.(a) Rat brain membranes were left untreated (blue bar) or were cholesterol depleted by treatement with MβC (10 mM, 15 minutes) to be then replenished (red bar) or not (green bar) with soluble cholesterol (1 mM, 15 minutes). In each bar is indicated the amount cholesterol measured in the membranes after each treatment (expressed as % of the amount present in untreated samples, see methods for details). T1117 binding measurement performed as described in Fig.1b. Specific binding is indicated (data depict the mean +/− s.e.m. and are representative of 4 independent experiments. P < 0.05. One-way ANOVA-test was employed). (b) Rat brain membranes were treated with MβC and then replenished or not with cholesterol as described above. Membranes were incubated with the indicated amount of ORG27569. T1117 (2.5 μM) was then added and specific binding measured as described in Fig. 1b. Data were fitted with a dose response curve as described in the Method Sections. (data depict the mean +/− s.e.m. and are representative of three or more independent experiments. One-way ANOVA was employed. P < 0.05).
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f3: Functional competition between ORG27569 and cholesterol.(a) Rat brain membranes were left untreated (blue bar) or were cholesterol depleted by treatement with MβC (10 mM, 15 minutes) to be then replenished (red bar) or not (green bar) with soluble cholesterol (1 mM, 15 minutes). In each bar is indicated the amount cholesterol measured in the membranes after each treatment (expressed as % of the amount present in untreated samples, see methods for details). T1117 binding measurement performed as described in Fig.1b. Specific binding is indicated (data depict the mean +/− s.e.m. and are representative of 4 independent experiments. P < 0.05. One-way ANOVA-test was employed). (b) Rat brain membranes were treated with MβC and then replenished or not with cholesterol as described above. Membranes were incubated with the indicated amount of ORG27569. T1117 (2.5 μM) was then added and specific binding measured as described in Fig. 1b. Data were fitted with a dose response curve as described in the Method Sections. (data depict the mean +/− s.e.m. and are representative of three or more independent experiments. One-way ANOVA was employed. P < 0.05).

Mentions: Cholesterol has been shown to affect GPCRs either directly, by binding to them and affecting their conformation, or indirectly, by influencing the membranous environment in which they are embedded. Effect of cholesterol and its precursor pregnenolone on CB1 binding was already demonstrated, with the lipids increasing the affinity of the receptor for inverse agonists2122. We started proving that depletion of cholesterol, similarly to ORG27569, reduces CB1 affinity for T1117 (Fig. 3a,b). Rat brain membranes were treated or not with methyl-β-cyclodextrin (MβD) to selectively extract cholesterol and thus T1117 binding was measured. Treatment with MβD (98–99% of total cholesterol extracted) drastically reduced T1117 binding. The loss of affinity for T1117 is indeed due to cholesterol withdrawal since the exogenous replenishment of cholesterol (50–70% of total cholesterol re-uptake) recovered the ability to bind the inverse agonist (Fig. 3a). This suggests that, in absence of cholesterol, the conformation of CB1 is less prone to bind the inverse agonist T1117.


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)

Functional competition between ORG27569 and cholesterol.(a) Rat brain membranes were left untreated (blue bar) or were cholesterol depleted by treatement with MβC (10 mM, 15 minutes) to be then replenished (red bar) or not (green bar) with soluble cholesterol (1 mM, 15 minutes). In each bar is indicated the amount cholesterol measured in the membranes after each treatment (expressed as % of the amount present in untreated samples, see methods for details). T1117 binding measurement performed as described in Fig.1b. Specific binding is indicated (data depict the mean +/− s.e.m. and are representative of 4 independent experiments. P < 0.05. One-way ANOVA-test was employed). (b) Rat brain membranes were treated with MβC and then replenished or not with cholesterol as described above. Membranes were incubated with the indicated amount of ORG27569. T1117 (2.5 μM) was then added and specific binding measured as described in Fig. 1b. Data were fitted with a dose response curve as described in the Method Sections. (data depict the mean +/− s.e.m. and are representative of three or more independent experiments. One-way ANOVA was employed. P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Functional competition between ORG27569 and cholesterol.(a) Rat brain membranes were left untreated (blue bar) or were cholesterol depleted by treatement with MβC (10 mM, 15 minutes) to be then replenished (red bar) or not (green bar) with soluble cholesterol (1 mM, 15 minutes). In each bar is indicated the amount cholesterol measured in the membranes after each treatment (expressed as % of the amount present in untreated samples, see methods for details). T1117 binding measurement performed as described in Fig.1b. Specific binding is indicated (data depict the mean +/− s.e.m. and are representative of 4 independent experiments. P < 0.05. One-way ANOVA-test was employed). (b) Rat brain membranes were treated with MβC and then replenished or not with cholesterol as described above. Membranes were incubated with the indicated amount of ORG27569. T1117 (2.5 μM) was then added and specific binding measured as described in Fig. 1b. Data were fitted with a dose response curve as described in the Method Sections. (data depict the mean +/− s.e.m. and are representative of three or more independent experiments. One-way ANOVA was employed. P < 0.05).
Mentions: Cholesterol has been shown to affect GPCRs either directly, by binding to them and affecting their conformation, or indirectly, by influencing the membranous environment in which they are embedded. Effect of cholesterol and its precursor pregnenolone on CB1 binding was already demonstrated, with the lipids increasing the affinity of the receptor for inverse agonists2122. We started proving that depletion of cholesterol, similarly to ORG27569, reduces CB1 affinity for T1117 (Fig. 3a,b). Rat brain membranes were treated or not with methyl-β-cyclodextrin (MβD) to selectively extract cholesterol and thus T1117 binding was measured. Treatment with MβD (98–99% of total cholesterol extracted) drastically reduced T1117 binding. The loss of affinity for T1117 is indeed due to cholesterol withdrawal since the exogenous replenishment of cholesterol (50–70% of total cholesterol re-uptake) recovered the ability to bind the inverse agonist (Fig. 3a). This suggests that, in absence of cholesterol, the conformation of CB1 is less prone to bind the inverse agonist T1117.

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