Characterizing phosphorylation-dependent regulation of metabotropic glutamate receptors by middle-down mass spectrometry

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ABSTRACT G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in eukaryotes and mediate a wide range of physiological processes. Extensive evidence has shown that GPCRs have complex signaling repertoires, and signal through multiple effector molecules beyond the canonical heterotrimeric G protein. It is believed that constitutive and ligand-induced GPCR phosphorylation is a key regulator in their complex signaling and modulation, and aberrations in receptor phosphorylation have been linked to the manifestation and persistence of disease states. Biochemical evidence suggests that these phosphorylation states are likely combinatorial in nature and highly heterogenous; however, mapping GPCR phosphorylation states remains out of reach given the limitations of common proteomics techniques which are low-throughput or blind to combinatorial information. Herein, we propose an innovative strategy to use intact protein mass spectrometry to characterize and quantify phosphorylation states of metabotropic glutamate receptor 2 which will allow for direct visualization of stoichiometric and positional phosphorylation heterogeneity that has previously been inaccessible. The central hypothesis of this proposal is that mGluR2 is subject to constitutive and ligand-induced phosphorylation that is highly heterogenous and these phosphorylations mediate several key aspects of mGluR2 function including mGluR2-mediated G protein signaling and receptor internalization. To test this hypothesis, we will use a combination of intact protein mass spectrometry and bottom-up proteomics to characterize phosphorylation states of mGluR2 in response to various ligands in a heterologous expression system (Aim 1). Based on the phosphorylation states discovered in Aim 1, we will then perform site-directed mutagenesis of mGluR2 to determine the impact of these phosphorylation states upon downstream G protein signaling and receptor internalization (Aim 2). To our knowledge, this study will be the first available demonstration of intact protein mass spectrometry to determine the identity and function of intact GPCR phosphorylation states; this will have broader implications as the approaches are generalizable to any receptor. The proposed work will be completed in collaboration between the Kelleher and Vafabakhsh groups and provide comprehensive training in cutting edge mass spectrometry-based proteomics, confocal microscopy, and traditional biochemical techniques. Both labs are respective experts in the fields of mass spectrometry and microscopy of GPCRs and are equipped with the instrumentation and expertise to support my training plan. I have constructed the training plan in close collaboration with Drs. Kelleher and Vafabakhsh to develop skills in targeted proteomics, advanced data analysis, live cell imaging via confocal microscopy, traditional biochemical techniques, scientific writing, and scientific presentation. These skills will afford me the opportunity to become an independent researcher and conduct hypothesis driven explorations of proteins and their modification states in my future career.