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.

In this study, we tested the hypothesis that a CB(1) 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 CB(1) receptor activation, with the F3.36(201)/W6.48(357) interaction key to the maintenance of the CB(1)-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 (GTPgammaS) 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 GTPgammaS binding versus wild type CB(1), although basal levels for the W6.48(357)A mutant were not statistically different from wild type CB(1). 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 CB(1)-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 CB(1) and be broken in the activated state via a chi(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 CB(1).