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J. Biol. Chem., Vol. 282, Issue 34, 25100-25113, August 24, 2007

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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^||1, Patricia H. Reggio, and Mary E. Abood²

From the California Pacific Medical Center Research Institute, San Francisco, California 94107, Center for Drug Discovery, Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, North Carolina 27402, the ^¶Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, and the ^||Neuroscience of Drug Abuse Research Program, North Carolina Central University, Durham, North Carolina 27707

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 G_i3 but not G_0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by G_i3. Furthermore, G_i3 but not G_0A enhanced basal facilitation ratio, suggesting that G_i3 is responsible for CB₁ tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with G_i1 or G_i2 but not with G_i3. 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.

Received for publication, April 24, 2007 , and in revised form, June 19, 2007.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 10 and 11.

¹ Present address: Dept. of Physiology and Pharmacology, Wake Forest University, Winston-Salem, NC 27157.

² To whom correspondence should be addressed: CA Pacific Medical Center Research Institute, 475 Brannan St., Ste. 220, San Francisco, CA 94107. Tel.: 415-600-3607; Fax: 415-600-1725; E-mail: aboodm{at}cpmcri.org.

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