Activation of the μ Opioid Receptor Involves Conformational Rearrangements of Multiple Transmembrane Domains

Abstract

We previously demonstrated that D3.49(164)Y or T6.34(279)K mutation in the rat mu opioid receptor (MOPR) resulted in agonist-independent activation. Here, we identified the cysteine(s) within the transmembrane domains (TMs) of the D3.49(164)Y mutant that became accessible in the binding-site crevice by use of methanethiosulfonate ethylammonium (MTSEA) and inferred conformational changes associated with receptor activation. While the C7.38(321)S mutant was insensitive to MTSEA, the D3.49(164)Y/C7.38(321)S mutant showed similar sensitivity as the D3.49(164)Y, suggesting that, in the D3.49(164)Y mutant, C7.38(321) becomes inaccessible while other cysteines are accessible in the binding-site crevice. Each of the other seven cysteines in the TMs was mutated to serine on the background of D3.49(164)Y/C7.38(321)S, and the resulting triple mutants were evaluated for [3H]diprenorphine and [d-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) binding and effect of MTSEA on [3H]diprenorphine binding. The D3.49(164)Y/C7.38(321)S mutant and the triple mutants, except the C6.47(292)S triple mutant, retained similar affinities for [3H]diprenorphine and DAMGO as the D3.49(164)Y mutant. The second-order rate constants for MTSEA reactions showed that C3.44(159)S, C4.48(190)S, C5.41(235)S, and C7.47(330)S significantly reduced sensitivity to MTSEA, compared with the D3.49(164)Y/C7.38(321)S. These results suggest that the four cysteines may be rotated and/or tilted to become accessible. While the D3.49(164)Y/C7.38(321)S was similarly sensitive to MTSEA as the D3.49(164)Y mutant, the T6.34(279)K/C7.38(321)S was much less sensitive to MTSEA than the T6.34(279)K mutant, suggesting that the two constitutively active mutants assume different conformations and/or possess different dynamic properties. Molecular models of the MOPR monomer and homodimer, using the crystal structures of rhodopsin, the beta2-adrenergic receptor, and the ligand-free opsin, which contains several features characteristic of the active state, were employed to analyze these experimental results in a structural context.

Preferences of transmembrane helices for cooperative amplification of Gαs and Gαq signaling of the thyrotropin receptor

“…Surprisingly, in contrast to previous observations for other GPCRs, our approach of double mutant design by combining single CAMs in the helices with each other, reveals that synergistic signaling effects at the TSHR are not restricted to a focused and limited subset of TMHs. In agreement with observations for other GPCRs, like the very recent data about the opioid receptor [58], we conclude that multiple conformational changes in the structural arrangement of the TMHs are necessary for receptor activation and signal transduction. In addition, our results suggest that conformational changes caused at TMH2, 6 and 7 have a higher impact on G a s-mediated signaling, whereas TMH1, 2, 3 and 6 are essential for G a q activation.…”

GPCR Conformations: Implications for Rational Drug Design

“…The difference between cysteine accessibility in WT and CAM backgrounds reflects the conformational change that occurs upon receptor activation. One interesting observation from SCAM studies in WT, partially, and completely activated CAM backgrounds in both the α1B-adrenergic [82] and μ-opioid [83] receptors is that sensitivity of ligand binding to MTS derivative treatment for cysteine mutations at certain sites (3.36 [82] and 7.38 [83]) paralleled the degree of constitutive activation. Additionally, the degree of conformational change upon activation as a function of TM helical segment can be inferred from a series of publications from the Leduc and Guillemette groups that systematically compared SCAM in WT and CAM backgrounds for the angiotensin II type 1 receptor [84-89].…”

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines

“…This includes six out of the seven transmembrane domains which are of special interest because these regions are involved in ligand-binding. 30,31,32 We envisage that these optimized techniques should facilitate a full structural characterization of hMOR binding sites, help in the design of covalent probes and aid in future drug discovery programs which use structure based approaches to find more selective and novel analgesic drugs.…”