Tuesday, May 13, 2025 5/13/2025

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

Award Number: F31MH129114
ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

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


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = -$18,401 )
2024	2023	NORTHWESTERN UNIVERSITY	633 CLARK ST	EVANSTON	IL	60208	COOK	USA	Mental Health Research Grants	000	2	7/16/2024	NON-COMPETING CONTINUATION	-$10,980
2024	2023	NORTHWESTERN UNIVERSITY	633 CLARK ST	EVANSTON	IL	60208	COOK	USA	Mental Health Research Grants	001	2	8/22/2024	NON-COMPETING CONTINUATION	-$7,421
														Subtotal = -$18,401

Issue Date FY: 2023 ( Subtotal = $43,115 )
2023	2023	NORTHWESTERN UNIVERSITY	633 CLARK	EVANSTON	IL	60208	COOK	USA	Mental Health Research Grants	000	2	2/4/2023	NON-COMPETING CONTINUATION	$43,115
														Subtotal = $43,115

Issue Date FY: 2022 ( Subtotal = $42,526 )
2022	2022	NORTHWESTERN UNIVERSITY	633 CLARK	EVANSTON	IL	60208	COOK	USA	Mental Health Research Grants	000	1	12/20/2021	NEW	$41,810
2022	2022	NORTHWESTERN UNIVERSITY	633 CLARK	EVANSTON	IL	60208	COOK	USA	Mental Health Research Grants	002	1	7/5/2022	NEW	$716
2022	2022	NORTHWESTERN UNIVERSITY	633 CLARK	EVANSTON	IL	60208	COOK	USA	Mental Health Research Grants	001	1	12/20/2021	NEW	$0
														Subtotal = $42,526

Grand Total All Awards = $67,240

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

Award Number: F31MH129114

ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

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