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).
McAllister
Sean D
SD
California Pacific Medical Center Research Institute, San Francisco, California 94115, USA.
Hurst
Dow P
DP
Barnett-Norris
Judy
J
Lynch
Diane
D
Reggio
Patricia H
PH
Abood
Mary E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D063385:Cannabinoid Receptor Modulators / Q000378:metabolism; D002460:Cell Line; D063388:Endocannabinoids; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D053284:Polyunsaturated Alkamides; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D008024:Ligands; D053284:Polyunsaturated Alkamides; D024661:Potassium Channels, Inwardly Rectifying; D043884:Receptor, Cannabinoid, CB1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C078814:anandamide