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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 pocket identification.(a) The 5 putative allosteric pockets mapped onto the CB1 homology model. Probes identifying each site are represented by differently colored surfaces. The three mutated residues for each site are highlighted in colored sticks. P1 is defined by TM1-TM7 and H8 domains, P2 by TM1-4, P3 by the same TM domains of P2 but towards the extracellular region, P4 is defined by residues on TM3 and TM7, finally P5 is defined by TM3-5. (b) Human CB1wt receptor and the indicated CB1 mutants were transiently expressed in HEK293 cells. Membrane homogenates were obtained and T1117 binding measurement performed as described in the On line Method Sections. Specific binding correlates with the fold change increase of T1117 fluorescence in presence of AM251. (c) Membrane homogenates were obtained from cells expressing CB1wt receptor or the indicated CB1 mutants. Samples were incubated with ORG27569 (3 μM) for 30 minutes. T1117 specific binding measurement was performed as described above. Effect of ORG27569 treatment is expressed as change in T1117 specific binding upon ORG27569 treatment (for panels b and c the data depict the mean +/− s.e.m. and are representative of three or more independent experiments. P < 0.05. ANOVA-test was employed). (d) Peptides identified by LC/MS analysis and presenting ORG27569alk3 covalently linked to S2.45 or to S3.42 of the P2 pocket. Peptide abundance is plotted as a function of mass/charge (m/z). Amino acids that could present ORG27569alk3 covalently bound are shown in red. The inset shows the region addressed by the probe (red, surface) superimposed with the P2 binding pocket (cyan, surface); (e) rat CB1wt-GFP and CB1(H2.41L)-GFP constructs were transiently expressed in HuH7 (upper panels) and SHSY-5Y (lower panels) that were treated (+ORG27569) or not (ctrl) with ORG27569 (3 μM) for 4 hours (see also Supplementary Fig. S6).
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f1: ORG27569 pocket identification.(a) The 5 putative allosteric pockets mapped onto the CB1 homology model. Probes identifying each site are represented by differently colored surfaces. The three mutated residues for each site are highlighted in colored sticks. P1 is defined by TM1-TM7 and H8 domains, P2 by TM1-4, P3 by the same TM domains of P2 but towards the extracellular region, P4 is defined by residues on TM3 and TM7, finally P5 is defined by TM3-5. (b) Human CB1wt receptor and the indicated CB1 mutants were transiently expressed in HEK293 cells. Membrane homogenates were obtained and T1117 binding measurement performed as described in the On line Method Sections. Specific binding correlates with the fold change increase of T1117 fluorescence in presence of AM251. (c) Membrane homogenates were obtained from cells expressing CB1wt receptor or the indicated CB1 mutants. Samples were incubated with ORG27569 (3 μM) for 30 minutes. T1117 specific binding measurement was performed as described above. Effect of ORG27569 treatment is expressed as change in T1117 specific binding upon ORG27569 treatment (for panels b and c the data depict the mean +/− s.e.m. and are representative of three or more independent experiments. P < 0.05. ANOVA-test was employed). (d) Peptides identified by LC/MS analysis and presenting ORG27569alk3 covalently linked to S2.45 or to S3.42 of the P2 pocket. Peptide abundance is plotted as a function of mass/charge (m/z). Amino acids that could present ORG27569alk3 covalently bound are shown in red. The inset shows the region addressed by the probe (red, surface) superimposed with the P2 binding pocket (cyan, surface); (e) rat CB1wt-GFP and CB1(H2.41L)-GFP constructs were transiently expressed in HuH7 (upper panels) and SHSY-5Y (lower panels) that were treated (+ORG27569) or not (ctrl) with ORG27569 (3 μM) for 4 hours (see also Supplementary Fig. S6).

Mentions: Consensus pocket prediction on the entire CB1 receptor was performed to identify ORG27569 candidate binding sites. Beside the canonical orthosteric pocket, nine potential allosteric sites were identified (See Computational Protocol and Supplementary Fig. S1–3). Since ORG27569 selectively binds CB1 over CB22324, we only selected pockets presenting at least one aminoacidic difference between CB1 and CB2. Thus, only five potential binding sites (P1-5) for ORG27569 were further considered (Fig. 1a). With the exception of pocket 4 (P4), which partially overlaps with the orthosteric pocket, the other sites are all lipid exposed (Fig. 1a). Noteworthy, P1, P2, and P4 were previously reported as putative allosteric pocket for other GPCRs283031. For each candidate site only 3 residues (not conserved in CB2) were considered for site-directed mutagenesis (Table 1). These were mutated in the corresponding CB2 residues rather than in Alanines, to avoid non-functional mutant receptors (see Supplementary Fig. S4 for details).


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 pocket identification.(a) The 5 putative allosteric pockets mapped onto the CB1 homology model. Probes identifying each site are represented by differently colored surfaces. The three mutated residues for each site are highlighted in colored sticks. P1 is defined by TM1-TM7 and H8 domains, P2 by TM1-4, P3 by the same TM domains of P2 but towards the extracellular region, P4 is defined by residues on TM3 and TM7, finally P5 is defined by TM3-5. (b) Human CB1wt receptor and the indicated CB1 mutants were transiently expressed in HEK293 cells. Membrane homogenates were obtained and T1117 binding measurement performed as described in the On line Method Sections. Specific binding correlates with the fold change increase of T1117 fluorescence in presence of AM251. (c) Membrane homogenates were obtained from cells expressing CB1wt receptor or the indicated CB1 mutants. Samples were incubated with ORG27569 (3 μM) for 30 minutes. T1117 specific binding measurement was performed as described above. Effect of ORG27569 treatment is expressed as change in T1117 specific binding upon ORG27569 treatment (for panels b and c the data depict the mean +/− s.e.m. and are representative of three or more independent experiments. P < 0.05. ANOVA-test was employed). (d) Peptides identified by LC/MS analysis and presenting ORG27569alk3 covalently linked to S2.45 or to S3.42 of the P2 pocket. Peptide abundance is plotted as a function of mass/charge (m/z). Amino acids that could present ORG27569alk3 covalently bound are shown in red. The inset shows the region addressed by the probe (red, surface) superimposed with the P2 binding pocket (cyan, surface); (e) rat CB1wt-GFP and CB1(H2.41L)-GFP constructs were transiently expressed in HuH7 (upper panels) and SHSY-5Y (lower panels) that were treated (+ORG27569) or not (ctrl) with ORG27569 (3 μM) for 4 hours (see also Supplementary Fig. S6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: ORG27569 pocket identification.(a) The 5 putative allosteric pockets mapped onto the CB1 homology model. Probes identifying each site are represented by differently colored surfaces. The three mutated residues for each site are highlighted in colored sticks. P1 is defined by TM1-TM7 and H8 domains, P2 by TM1-4, P3 by the same TM domains of P2 but towards the extracellular region, P4 is defined by residues on TM3 and TM7, finally P5 is defined by TM3-5. (b) Human CB1wt receptor and the indicated CB1 mutants were transiently expressed in HEK293 cells. Membrane homogenates were obtained and T1117 binding measurement performed as described in the On line Method Sections. Specific binding correlates with the fold change increase of T1117 fluorescence in presence of AM251. (c) Membrane homogenates were obtained from cells expressing CB1wt receptor or the indicated CB1 mutants. Samples were incubated with ORG27569 (3 μM) for 30 minutes. T1117 specific binding measurement was performed as described above. Effect of ORG27569 treatment is expressed as change in T1117 specific binding upon ORG27569 treatment (for panels b and c the data depict the mean +/− s.e.m. and are representative of three or more independent experiments. P < 0.05. ANOVA-test was employed). (d) Peptides identified by LC/MS analysis and presenting ORG27569alk3 covalently linked to S2.45 or to S3.42 of the P2 pocket. Peptide abundance is plotted as a function of mass/charge (m/z). Amino acids that could present ORG27569alk3 covalently bound are shown in red. The inset shows the region addressed by the probe (red, surface) superimposed with the P2 binding pocket (cyan, surface); (e) rat CB1wt-GFP and CB1(H2.41L)-GFP constructs were transiently expressed in HuH7 (upper panels) and SHSY-5Y (lower panels) that were treated (+ORG27569) or not (ctrl) with ORG27569 (3 μM) for 4 hours (see also Supplementary Fig. S6).
Mentions: Consensus pocket prediction on the entire CB1 receptor was performed to identify ORG27569 candidate binding sites. Beside the canonical orthosteric pocket, nine potential allosteric sites were identified (See Computational Protocol and Supplementary Fig. S1–3). Since ORG27569 selectively binds CB1 over CB22324, we only selected pockets presenting at least one aminoacidic difference between CB1 and CB2. Thus, only five potential binding sites (P1-5) for ORG27569 were further considered (Fig. 1a). With the exception of pocket 4 (P4), which partially overlaps with the orthosteric pocket, the other sites are all lipid exposed (Fig. 1a). Noteworthy, P1, P2, and P4 were previously reported as putative allosteric pocket for other GPCRs283031. For each candidate site only 3 residues (not conserved in CB2) were considered for site-directed mutagenesis (Table 1). These were mutated in the corresponding CB2 residues rather than in Alanines, to avoid non-functional mutant receptors (see Supplementary Fig. S4 for details).

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