Abstract
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).
Highlights
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
P6.50 of the highly conserved CWXP motif in TMH6 of G protein-coupled receptors (GPCRs) may act as a flexible hinge, permitting TMH6 to straighten upon activation, moving its intracellular end away from TMH3 and upwards toward the lipid bilayer (12)
Mutation, and functional studies undertaken to test the importance of the TMH3-4-5-6 aromatic microdomain in ligand recognition and in the conformational changes that accompany activation of CB1 suggest that a F3.36(201)/W6.48(357)specific contact is an important constraint for the CB1inactive state
Summary
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. There is a growing body of evidence in the literature that activation of GPCRs is accompanied by rigid domain motions and rotations of transmembrane helices (TMHs) 3 and 6 (6 – 8) At their intracellular ends, TMHs 3 and 6 in Rho are constrained by an E3.49(134)/R3.50(135)/E6.30 (247) salt bridge that limits the relative mobility of the cytoplasmic ends of TMH3 and TMH6 in the inactive state (3) and acts like an “ionic lock” (10, 11). Light activation of Rho does not require the breaking and forming of thousands of specific contacts within nanoseconds, rather only a few specific contacts restricting the inactive state, including indole side chain contacts of tryptophan residues, need to break on activation These changes can be transmitted through the entire membrane protein because of its dynamic plasticity. This movement releases the constraint on W6.48(265), making it possible for W6.48(265)
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