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Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells
Published on: September 16, 2014
Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer
S J Farrar1, P J Whiting, T P Bonnert
1Department of Biochemistry and Molecular Biology, Merck Sharp and Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, United Kingdom.
Insights
Researchers developed a novel method to determine cell surface receptor stoichiometry using fluorescently tagged antibodies. This technique confirms the gamma-aminobutyric acid (GABA) A receptor alpha1beta2gamma2 subtype has two alpha, two beta, and one gamma subunit.
Area of Science:
- Molecular Biology
- Neuroscience
- Biochemistry
Background:
- Oligomeric cell surface receptors play crucial roles in cellular signaling.
- Determining the precise subunit stoichiometry of these receptors is essential for understanding their function.
- The gamma-aminobutyric acid (GABA) A receptor is a key inhibitory neurotransmitter receptor with a complex subunit composition.
Purpose of the Study:
- To develop and validate a novel method for determining the subunit stoichiometry of oligomeric cell surface receptors.
- To elucidate the subunit composition of the gamma-aminobutyric acid (GABA) A receptor alpha1beta2gamma2 subtype.
- To assess the general applicability of the developed method for other multisubunit cell surface proteins.
Main Methods:
- Utilized fluorescently tagged antibodies to label individual subunits of the GABA A receptor in intact cells.
- Employed europium cryptate (EuK) as a fluorophore and XL665 as a fluorescence energy transfer acceptor.
- Measured fluorescence and energy transfer signals to deduce subunit stoichiometry.
Main Results:
- Fluorescence signal intensity suggested a 2:2:1 ratio for alpha, beta, and gamma subunits, respectively.
- Fluorescence energy transfer experiments confirmed the presence of two alpha and two beta subunits.
- Energy transfer data indicated the absence of a second gamma subunit, confirming a 2:2:1 stoichiometry.
Conclusions:
- The gamma-aminobutyric acid (GABA) A receptor alpha1beta2gamma2 subtype is confirmed to have a stoichiometry of (alpha1)2(beta2)2(gamma2)1.
- The developed method using fluorescently tagged antibodies and energy transfer is effective for determining receptor subunit stoichiometry.
- This methodology holds significant potential for characterizing the composition of various multisubunit cell surface proteins.
Abstract:
We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.
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