Title

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

Presenter Information

Hannah Frye

Department

Chemistry

Major

Chemistry, Biochemistry Emphasis

Research Advisor

Aronstam, Robert

Advisor's Department

Biological Sciences

Funding Source

OURE; cDNA Resource Center

Abstract

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.

Biography

Hannah Frye is a senior in Chemistry with a Biochemistry emphasis and a minor in Biological Sciences. She has worked in Dr. Robert Aronstam’s lab for over three years and has presented her work at both the 2013 American Society for Cell Biology Meeting in New Orleans and the 2013 Undergraduate Research Conference, at which she won third place in the Sciences Poster Division. Upon graduation she will be continuing her education towards a doctorate in Neuroscience. On the Missouri S&T campus she is involved with the International Genetically Engineered Machines Team, the National Residence Hall Honorary, and Alpha Chi Sigma.

Research Category

Sciences

Presentation Type

Poster Presentation

Document Type

Poster

Location

Upper Atrium/Hall

Presentation Date

15 Apr 2015, 9:00 am - 11:45 am

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Apr 15th, 9:00 AM Apr 15th, 11:45 AM

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

Upper Atrium/Hall

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.