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Activation of the mu opioid receptor involves conformational rearrangements of multiple transmembrane domains

Wei Xu1, Arantxa Sanz, Leonardo Pardo

  • 1Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

Biochemistry
|September 10, 2008
PubMed

Insights

Mutations in the mu opioid receptor (MOPR) cause constitutive activation. Identifying accessible cysteines using methanethiosulfonate ethylammonium (MTSEA) reveals distinct conformational changes in activated MOPR variants.

Area of Science:

  • Pharmacology
  • Molecular Biology
  • Structural Biology

Background:

  • The mu opioid receptor (MOPR) plays a crucial role in pain perception and reward pathways.
  • Mutations such as D3.49(164)Y and T6.34(279)K have been shown to cause agonist-independent activation of MOPR.
  • Understanding the conformational changes associated with MOPR activation is essential for drug development.

Purpose of the Study:

  • To identify cysteine residues in the transmembrane domains of the D3.49(164)Y MOPR mutant that become accessible in the binding site.
  • To infer conformational changes related to MOPR activation by probing cysteine accessibility with methanethiosulfonate ethylammonium (MTSEA).
  • To compare the conformational properties of different constitutively active MOPR mutants.

Main Methods:

  • Site-directed mutagenesis of rat MOPR, including single and triple mutants.
  • Cysteine accessibility assays using MTSEA to probe the binding-site crevice.
  • Radioligand binding assays with [3H]diprenorphine and DAMGO to assess receptor affinity.
  • Analysis of MTSEA reaction kinetics to quantify cysteine accessibility.
  • Molecular modeling of MOPR monomer and homodimer structures.

Main Results:

  • The D3.49(164)Y mutant exhibits accessible cysteines in the binding-site crevice, with C7.38(321) being inaccessible.
  • Mutations at C3.44(159)S, C4.48(190)S, C5.41(235)S, and C7.47(330)S significantly reduced MTSEA sensitivity in the D3.49(164)Y/C7.38(321)S background.
  • Most MOPR mutants retained high affinity for [3H]diprenorphine and DAMGO, except for the C6.47(292)S triple mutant.
  • The D3.49(164)Y and T6.34(279)K mutants, even when C7.38(321) was mutated, showed differential sensitivity to MTSEA, suggesting distinct active conformations.

Conclusions:

  • Specific cysteines within the MOPR transmembrane domains become accessible during constitutive activation, indicating conformational rearrangements.
  • The identified accessible cysteines (C3.44, C4.48, C5.41, C7.47) likely undergo rotation or tilting to become solvent-exposed.
  • Constitutively active MOPR mutants (D3.49Y and T6.34K) adopt different conformational states and dynamic properties.

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