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Date of Award

Summer 8-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

R7-RGS proteins (consisting of RGS6, RGS7, RGS9, and RGS11) potently regulate GPCR signaling by functioning as GAPs (GTPase accelerating proteins) for Gαi/o subunits in neurons. Interestingly, several findings suggest that R7-RGS proteins have functions beyond serving as GAPs. For example, they are structurally more complex than other RGS proteins that are GAPs for Gαi/o because they form obligate heterotrimers (R7-RGS heterotrimers) with Gβ5, a diverged Gβ subunit, and R7BP, a dynamically palmitoylated membrane anchor. The interaction between an R7-RGS protein and Gβ5 structurally resembles classic Gβγ dimers, suggesting that R7-RGS heterotrimers could activate effector proteins similarly to Gβγ. Additionally, R7-RGS heterotrimers could have subcellular location-specific functions because the palmitoylation state of R7BP determines if R7-RGS heterotrimers are located at the plasma membrane, on endomembranes, or in the nucleus. Furthermore, R7-RGS heterotrimers are critical for neuronal function because mice lacking in these proteins have deficiencies in an array of biological processes including viability, weight gain, photoresponses of the retina, neurobehavioral development, motor coordination, cerebellar and hippocampal development, and analgesic responses to opioids. These phenotypes are more diverse than the phenotypes that occur when GAP activity for Gαi/o is blocked, suggesting that R7-RGS heterotrimers have other roles that are important for neuronal function. The goal of this dissertation is to identify and characterize novel functions of R7-RGS heterotrimers.

This dissertation is divided into two parts: i) a proteomics study to identify novel binding partners of R7-RGS heterotrimers in brain, and ii) a microarray study to test if nuclear R7-RGS heterotrimers regulate gene transcription. In part i), we generated a transgenic mouse expressing tandem affinity-tagged R7BP (SF-R7BP) in neurons as a tool to isolate novel binding partners of R7-RGS heterotrimers. Gα13 co-purified with SF-R7BP from brain homogenates and was identified by mass spectrometry. Gα13 interacts with R7-RGS heterotrimers containing any R7-RGS family member and R7BP is required for the interaction to occur in cells. Intriguingly, R7-RGS heterotrimers enhance basal Gα13 signaling in differentiated mouse neuroblastoma cells, resulting in cells bearing fewer projections. In part ii), we used a palmitoylation-deficient R7BP mutant (R7BP SS) to drive endogenous R7-RGS and Gβ5 to the nucleus of neuroblastoma cells in order to test if nuclear R7-RGS heterotrimers regulate gene transcription. Microarray analysis showed that there was no dramatic difference in the mRNA expression profiles of cells stably expressing R7BP SS, R7BP WT, or no R7BP, supporting a model in which nuclear R7-RGS heterotrimers are not transcriptional regulators. These results taken together represent a significant advancement of our knowledge of R7-RGS heterotrimers as multi-functional protein complexes that are critical for neuronal function.

Language

English (en)

Chair and Committee

Kendall J. Blumer

Committee Members

Kendall J. Blumer, Thomas J. Baranski, Michael R. Bruchas, Karen L. O'Malley, Linda J. Pike

Comments

Permanent URL: https://doi.org/doi:10.7936/K7JH3JKR

Available for download on Saturday, August 15, 2116

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