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Probing conformation of opioid receptor by in-cell chemical cross-linking and mass spectrometry

Wednesday, October 10, 2012 — Poster Session II

Noon – 2:00 p.m

Natcher Conference Center, Building 45




  • M Akbar
  • H-Y Kim


Opioid receptors mediate the actions of opioids on physiological processes such as analgesia and mood changes. Despite recent breakthrough in crystallographic studies of ligand-bound opioid receptors, detailed molecular mechanisms for both the extracellular binding of the receptors to various drugs and subsequent downstream signaling within the cells are not fully understood. In this study, we probe the conformation of μ-opioid receptor (μ-OR) by in-cell chemical cross-linking and mass spectrometry. Neuro-2A cells overexpressing Flag-tagged human μ-OR were stimulated with DAMGO and incubated with various cross-linkers. The immunopurified μ-OR was subjected to SDS-PAGE, chymotryptic digestion and nanoLC-ESI-MS/MS analysis. The MS-based approach identified three cross-linked lysine pairs (K211-K235, K235-K305, and K211-K305) in the extracellular loops 2 and 3 of μ-OR. The data suggest that the spatial distance among these lysine residues is within 24 Å defined by the cross-linkers, consistent with crystallographic data. Moreover, μ-OR phosphorylation was inhibited when cells were treated with long (24 Å) but not short (19 Å) cross-linkers prior to DAMGO stimulation, suggesting that μ-OR activation depended on its conformational profile which can be probed with different cross-linkers. The conformation-based approach should be useful in providing insight into activation processes of opioid receptors in response to various drug molecules.

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