Na Wu1, Agnieszka M. Olechwier2,3, Cyrill Brunner1, Patricia C. Edwards4, Ching-Ju Tsai2, Christopher G. Tate4, Gebhard F. X. Schertler2, Gisbert Schneider1, Xavier Deupi2, Renato Zenobi1, and Pikyee Ma2
- Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland
- Laboratory of Biomolecular Research, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland
- Department of Biology, ETH Zürich, CH-8093 Zürich, Switzerland
- Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
- Condensed Matter Theory Group, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland
G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR–G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand–GPCR–partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαiβγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gαiβγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.
CovalX Technology Used
Many important categories of drugs are made to target G protein-coupled receptors (GPCRs). These drug targets are widely used, but their signaling patterns are not fully understood. Specifically, the mechanism of how GPCR uses ligand-induced coupling selectivity. In order to gain more knowledge of this specific mechanism, a full examination of the network of protein interactions between GPCRs and their cytoplasmic transducers (G proteins, kinases, and arrestins) is required. Traditional methods of measuring such protein interactions (ex. SPR, fluorescence resonance energy transfer, and isothermal titration calorimetry) were difficult to apply due to the lack of buffer conditions compatible with functional membrane proteins. CovalX was able to offer an alternative. Through the development of a quantitative high-mass matrix-assisted laser desorption/ionization mass spectrometry strategy, combining cross-linking and quantification, an assay was able to measure the interplay between receptors, ligands, and interacting proteins. This method had a higher tolerance to buffers, salts, detergents, or lipids compared to ESI-MS. This protocol allowed for visualization of coupling activity for over 70 ligand–GPCR–partner protein combinations, using as little as 1.25 pmol protein per sample. The high mass MALDI mass spectrometry strategy produced the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). Through these profiles, it was discovered that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. The quantification method was represented using β1AR which demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. The complex formation with wild-type heterotrimeric Gαiβγ and β-arrestin-1 was quantified, showing that carvedilol induces an increase in the coupling of β-arrestin-1 and Gαto β1AR. A normalization strategy allowed for quantitative measurement of the binding affinities of GPCRs to partner proteins. This methodology using the CovalX detection system provided clear results and will be useful in the future for the discovery and development phases of GPCR-targeting drugs.