QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: jpet.107.133256v1 325/1/341    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kapur, A. Articles by Abood, M. E. Search for Related Content PubMed PubMed Citation Articles by Kapur, A. Articles by Abood, M. E.

CELLULAR AND MOLECULAR

Mapping the Structural Requirements in the CB₁ Cannabinoid Receptor Transmembrane Helix II for Signal Transduction

California Pacific Medical Center Research Institute, San Francisco, California (A.K., P.S., M.E.A.); and Center for Drug Discovery, Northeastern University, Boston, Massachusetts (G.A.T., A.M.)

Amino acid residues in the transmembrane domains of the CB₁ receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB₁) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB₁ receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(–)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (–)-11β-hydroxy-3-(1',1'-dimethylheptyl) hexahydrocannabinol (AM4056), and (–)-11-hydroxyldimethylheptyl-⁸-tetrahydrocannabinol (HU210) for the stimulation of GTPS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by 50-fold) in the EC₅₀ for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.

Address correspondence to: Dr. Mary E. Abood. California Pacific Medical Center Research Institute, 475 Brannan St., San Francisco, CA 94107. E-mail: aboodm{at}cpmcri.org

This article has been cited by other articles:

				A. Kapur, P. Zhao, H. Sharir, Y. Bai, M. G. Caron, L. S. Barak, and M. E. Abood Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands J. Biol. Chem., October 23, 2009; 284(43): 29817 - 29827. [Abstract] [Full Text] [PDF]