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 CB(1) cannabinoid receptor deviates from the highly conserved NPXXY(X)(5,6)F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB(1) with those of an L7.60F mutation and an L7.60I mutation that mimics the CB(2) sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [(35)S]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 Ca(2+) 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 Galpha(i3) but not Galpha(0A) in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Galpha(i3). Furthermore, Galpha(i3) but not Galpha(0A) enhanced basal facilitation ratio, suggesting that Galpha(i3) is responsible for CB(1) tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Galpha(i1) or Galpha(i2) but not with Galpha(i3). Molecular dynamics simulations of WT CB(1) 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)(5,6)L contributes to maximal activity of the CB(1) receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.
authors.affiliation: California Pacific Medical Center Research Institute, San Francisco, California 94107, USA.
authors.forename: Sharon
authors.initials: S
authors.lastname: Anavi-Goffer
authors.forename: Daniel
authors.initials: D
authors.lastname: Fleischer
authors.forename: Dow P
authors.initials: DP
authors.lastname: Hurst
authors.forename: Diane L
authors.initials: DL
authors.lastname: Lynch
authors.forename: Judy
authors.initials: J
authors.lastname: Barnett-Norris
authors.forename: Shanping
authors.initials: S
authors.lastname: Shi
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Lewis
authors.forename: Somnath
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: Allyn C
authors.initials: AC
authors.lastname: Howlett
authors.forename: Patricia H
authors.initials: PH
authors.lastname: Reggio
authors.forename: Mary E
authors.initials: ME
authors.lastname: Abood
mesh_terms: D000595:Amino Acid Sequence; D000700:Analgesics / Q000494:pharmacology; D048588:Benzoxazines / Q000494:pharmacology; D003511:Cyclohexanols / Q000494:pharmacology; D013759:Dronabinol / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D016296:Mutagenesis; D009281:Naphthalenes / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry*; D015398:Signal Transduction
chemical_list: D000700:Analgesics; D048588:Benzoxazines; D003511:Cyclohexanols; D008051:Lipid Bilayers; D009025:Morpholines; D009281:Naphthalenes; D043884:Receptor, Cannabinoid, CB1; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D013759:Dronabinol; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D037342:Pertussis Toxin; D007930:Leucine; C062018:HU 211