ORCID

http://orcid.org/0000-0002-9044-8313

Date of Award

Winter 12-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

G protein coupled receptors transduce a truly staggering number of diverse extracellular signals including chemical messengers, physical force, and even photons into specific cellular responses through their coupling to heterotrimeric G proteins. G proteins amplify the originating signal through their binding to downstream effectors, activating a complex network of overlapping responses that allow the cell to respond perfectly to that specific stimulus. It is critical to the cell that this process is carried out faithfully in order to respond to the myriad environmental cues and avoid injury, exhaustion, and death for the individual cell or the development of pathology if part of a multicellular organism. Unfortunately, there are many common scenarios that result in exactly this kind of signaling dysregulation. Some of these can be abrogated by targeting the receptors, for which we have developed a veritable suite of agonists and antagonists to tune receptor activity. However, there exist pathologies, such as uveal melanoma which is driven by a mutant G protein or asthma which is the result of pathological overstimulation by overlapping G protein coupled receptors, where this approach is rendered completely ineffective and a method to target the G protein responsible for propagating the aberrant signal is necessary. Additionally, there are many questions in both pathological and normal signaling biology that are difficult or even unanswerable without a way to specifically remove a signaling branch in order to tease out subtle effects. Two such inhibitors currently exist, the natural product depsipeptides YM-254890 and FR900359, representing an entirely new class of potential therapeutic and research tools that are already in use to discover novel signaling intricacies and as potential therapeutics. In order to improve upon these natural inhibitors and further develop the class, the mechanism of action must be more completely understood as well as the determinants that render such exquisite specificity for the Gαq/11 family of G proteins in order to design new inhibitors with the same degree of specificity for the other G protein families. Chapter 2 of this dissertation aims to answer fundamental questions about the mechanism of action of these inhibitors on the disease relevant constitutively active mutant responsible for uveal melanoma through the use of biochemical and cellular assays and establishes the first G protein outside of the Gαq/11 family with engineered sensitivity to these inhibitors, demonstrating for the first time that this inhibitory mechanism can be utilized beyond the Gαq/11 family. This work is expanded upon in Chapter 3 to render a member of the last remaining inhibitor insensitive G protein family with the furthest diverged binding site sequence sensitive, finally demonstrating that this is a universal mechanism of G protein inhibition which can be exploited in all G protein families. Finally, Chapter 3 directly interrogates the hypothesis that these inhibitors stabilize interdomain closure to prevent G protein activation through the use of single molecule Förster’s Resonance Energy Transfer and demonstrates that this is indeed the inhibitory mechanism of action. The research contained in this dissertation will aid in the further development of novel inhibitors and pave the way for exciting new discoveries in G protein signaling biology.

Language

English (en)

Chair and Committee

Kendall J. Blumer

Committee Members

John A. Cooper, Joseph M. Jez, Linda J. Pike, Andrea Soranno,

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