title: Activation of the mu 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.
authors.affiliation: Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Arantxa
authors.initials: A
authors.lastname: Sanz
authors.forename: Leonardo
authors.initials: L
authors.lastname: Pardo
authors.forename: Lee-Yuan
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004174:Diprenorphine / Q000378:metabolism / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004174:Diprenorphine