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Molecular Details of Membrane Protein Receptor Dynamics

Periodic Reporting for period 1 - GPCR-MS (Molecular Details of Membrane Protein Receptor Dynamics)

Reporting period: 2019-08-01 to 2021-07-31

G protein-coupled receptors (GPCRs) are the proteins responsible for transducing “outside” signals into an intracellular response and their dysfunction is implicated in many diseases. As a result, they are the target of ~40% of drugs on the market. However, many GPCR drugs elicit off-target effects and can produce unwanted effects such as receptor desensitization and/or drug dependence. Currently, the mechanism of action of many GPCR drug targets is poorly understood.

GPCRs don’t act in isolation; proteins, metabolites, and lipids, and other molecules all interact synergistically to generate distinct signaling outputs. However, conventional ‘omics’ approaches will never be able to connect the status of critical proteins with metabolites or other effectors since the links between them are broken during sample preparation. It is therefore imperative to maintain molecular interactions to understand the molecular network that contributes to a biological signaling output.

This project successfully developed state-of-the-art methods in native mass spectrometry (MS) to interrogate membrane proteins in complex with lipids, ligands, and other molecular interactors intact in the mass spectrometer. By maintaining complexes from the cell to the mass spectrometer, and by dissecting protein-effector interactions in a controlled manner, we are now better equipped to understand mechanisms of membrane protein signal transduction. Specific knowledge of how protein receptors translate “outside” signals into a biological response will change how we approach the treatment of human disease, pain, and other conditions.
The project required interdisciplinary training in molecular biology and physical analytical chemistry. Mammalian expression systems were used to produce and isolate membrane protein receptors which provided several advantages toward achieving the goals of the project such as (i) realistic native environments with proper lipid environment, (ii) proper post-translational modifications, and were (iii) easily manipulated by drugs or other stimulants in vitro to induce a desired pathophysiological response (e.g. specific signalling outputs). In addition, novel mass spectrometry workflows were developed to interrogate membrane proteins and their molecular interactors. The use of novel mass spectrometry workflows on proteins involved in human health and disease resulted in three peer-reviewed publications, two conference presentations, and were highlighted in three workshop presentations.
This project successfully developed state-of-the-art methods in native mass spectrometry (MS) to interrogate membrane proteins in complex with lipids, ligands, and other molecular interactors intact in the mass spectrometer. By maintaining complexes from the cell to the MS, and by dissecting protein-effector interactions in a controlled manner, we are now better equipped to understand mechanisms of membrane protein signal transduction. Specific knowledge of how protein receptors translate “outside” signals into a biological response will change how we approach the treatment of human disease, pain, and other conditions.
The fellow using the newest state-of-the-art mass spectrometer to look at membrane protein complexes