The Dependence of M2 Muscarinic Receptor Signaling on the Formation of a Disulfide Bridge

Muscarinic receptors are G-protein coupled acetylcholine receptors made up of seven transmembrane loops. On the M2 receptor, the second and the third extracellular domain are linked by two cysteine residues forming an intramolecular disulfide bridge. Little is known of the disulfide bridge’s role in receptor signaling and this project aims to explore how elimination of the disulfide bridge affects M2 receptor signaling. First this was performed through co-transfection of a modified M2 receptor (hM2 aaC96A mutant) where alanine replaces cysteine residues with a chimeric G protein (Gαqi) that binds the M2 receptor but produces a measurable calcium signal (atypical of M2 signaling). Second, cells with a wild-type M2 receptor were dosed with dithiothreitol (DTT) which reduces disulfide bonds. Elimination of the disulfide bridge through point mutation disrupts receptor function by reducing or eliminating receptor signaling, whereas signal was maintained with chemical reduction of the disulfide bridge with DTT