Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms

Sharon Anavi-Goffer, Daniel Fleischer, Dow P. Hurst, Diane L. Lynch, Judy Barnett-Norris, Shanping Shi, Deborah L. Lewis, Somnath Mukhopadhyay, Allyn C. Howlett, Patricia H. Reggio, Mary E. Abood* (Corresponding Author)

*Corresponding author for this work

Research output: Contribution to journalArticle

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Abstract

The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.
Original languageEnglish
Pages (from-to)25100-25113
Number of pages14
JournalThe Journal of Biological Chemistry
Volume282
Issue number34
Early online date26 Jun 2007
DOIs
Publication statusPublished - 24 Aug 2007

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Cannabinoid Receptor CB1
Signal transduction
GTP-Binding Proteins
Signal Transduction
rimonabant
Guanosine 5'-O-(3-Thiotriphosphate)
Somatostatin Receptors
Mutation
Pertussis Toxin
Molecular Dynamics Simulation
Hydrogen Bonding
Elbow
G-Protein-Coupled Receptors
Immunoprecipitation
Molecular dynamics
Assays
Hydrogen bonds
Geometry
Computer simulation
Experiments

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Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms. / Anavi-Goffer, Sharon; Fleischer, Daniel; Hurst, Dow P.; Lynch, Diane L.; Barnett-Norris, Judy; Shi, Shanping; Lewis, Deborah L.; Mukhopadhyay, Somnath; Howlett, Allyn C.; Reggio, Patricia H.; Abood, Mary E. (Corresponding Author).

In: The Journal of Biological Chemistry, Vol. 282, No. 34, 24.08.2007, p. 25100-25113.

Research output: Contribution to journalArticle

Anavi-Goffer, S, Fleischer, D, Hurst, DP, Lynch, DL, Barnett-Norris, J, Shi, S, Lewis, DL, Mukhopadhyay, S, Howlett, AC, Reggio, PH & Abood, ME 2007, 'Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms', The Journal of Biological Chemistry, vol. 282, no. 34, pp. 25100-25113. https://doi.org/10.1074/jbc.M703388200
Anavi-Goffer, Sharon ; Fleischer, Daniel ; Hurst, Dow P. ; Lynch, Diane L. ; Barnett-Norris, Judy ; Shi, Shanping ; Lewis, Deborah L. ; Mukhopadhyay, Somnath ; Howlett, Allyn C. ; Reggio, Patricia H. ; Abood, Mary E. / Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms. In: The Journal of Biological Chemistry. 2007 ; Vol. 282, No. 34. pp. 25100-25113.
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title = "Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms",
abstract = "The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.",
author = "Sharon Anavi-Goffer and Daniel Fleischer and Hurst, {Dow P.} and Lynch, {Diane L.} and Judy Barnett-Norris and Shanping Shi and Lewis, {Deborah L.} and Somnath Mukhopadhyay and Howlett, {Allyn C.} and Reggio, {Patricia H.} and Abood, {Mary E.}",
note = "This work was supported by National Institute on Drug Abuse Grants DA09978, DA05274 (to M. E. A.), DA00489, DA039434 (to P. H. R.), DA03690, and DA12385 (to A. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.",
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T1 - Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms

AU - Anavi-Goffer, Sharon

AU - Fleischer, Daniel

AU - Hurst, Dow P.

AU - Lynch, Diane L.

AU - Barnett-Norris, Judy

AU - Shi, Shanping

AU - Lewis, Deborah L.

AU - Mukhopadhyay, Somnath

AU - Howlett, Allyn C.

AU - Reggio, Patricia H.

AU - Abood, Mary E.

N1 - This work was supported by National Institute on Drug Abuse Grants DA09978, DA05274 (to M. E. A.), DA00489, DA039434 (to P. H. R.), DA03690, and DA12385 (to A. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

PY - 2007/8/24

Y1 - 2007/8/24

N2 - The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.

AB - The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.

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DO - 10.1074/jbc.M703388200

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