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J. Biol. Chem., Vol. 279, Issue 46, 48024-48037, November 12, 2004

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Structural Mimicry in Class A G Protein-coupled Receptor Rotamer Toggle Switches

THE IMPORTANCE OF THE F3.36(201)/W6.48(357) INTERACTION IN CANNABINOID CB₁ RECEPTOR ACTIVATION^*

Sean D. McAllister, Dow P. Hurst||, Judy Barnett-Norris||, Diane Lynch||, Patricia H. Reggio||, and Mary E. Abood¶

From the California Pacific Medical Center Research Institute, San Francisco, California 94115 and Kennesaw State University, Kennesaw, Georgia 30144

In this study, we tested the hypothesis that a CB₁ TMH3-4-5-6 aromatic microdomain, which includes F3.25(190), F3.36(201), W5.43(280), and W6.48(357), is centrally involved in CB₁ receptor activation, with the F3.36(201)/W6.48(357) interaction key to the maintenance of the CB₁-inactive state. We have shown previously that when F3.36(201), W5.43(280), and W6.48(357) are individually mutated to alanine, a significant reduction in ligand binding affinity is observed in the presence of WIN 55,212-2 and SR141716A but not CP55,940 and anandamide. In the work presented here, we report a detailed functional analysis of the F3.36(201)A, F3.25(190)A, W5.43(280)A, and W6.48(357)A mutant receptors in stable cell lines created in HEK cells for agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPS) binding and GIRK1/4 channel current effects in Xenopus oocytes where the mutant proteins were expressed transiently. The F3.36(201)A mutation showed statistically significant increases in ligand-independent stimulation of GTPS binding versus wild type CB₁, although basal levels for the W6.48(357)A mutant were not statistically different from wild type CB₁. F3.36(201)A demonstrated a limited activation profile in the presence of multiple agonists. In contrast, enhanced agonist activation was produced by W6.48(357)A. These results suggest that a F3.36(201)/W6.48(357)-specific contact is an important constraint for the CB₁-inactive state that may need to break during activation. Modeling studies suggest that the F3.36(201)/W6.48(357) contact can exist in the inactive state of CB₁ and be broken in the activated state via a ₁ rotamer switch (F3.36(201) trans, W6.48(357) g+) (F3.36(201) g+, W6.48(357) trans). The F3.36(201)/W6.48(357) interaction therefore may represent a "toggle switch" for activation of CB₁.

Received for publication, June 15, 2004 , and in revised form, August 19, 2004.

^* This work has been supported by National Institutes on Drug Abuse Grants DA09978, DA05274 (to M. E. A.), DA00489, and DA03934 (to P. H. R.). 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.

^|| Current address: University of North Carolina, Greensboro, NC 27402.

^¶ To whom correspondence should be addressed: Forbes Norris ALS/MDA Research Center, 2351 Clay St, Suite 416, California Pacific Medical Center Research Institute, San Francisco, CA 94115. Tel.: 415-600-3607; Fax: 415-563-7325; E-mail: mabood{at}cooper.cpmc.org.

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