An Accessible and Generalizable In Vitro Luminescence Assay for Detecting GPCR Activation
Miller, Ruby
2023
Abstract
G-protein coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. The receptors are responsible for the regulation of signaling passing between different cells and their activation triggers cellular responses in the form of signaling cascades. It is this regulatory role that places GPCRs at the center of many disease mechanisms and creates incentive for better controlling their activation or inhibition. It follows that GPCRs are the targets of a significant percentage of clinically approved drugs. While there are many assays useful for studying the effects of GPCR activation, both live cell and biochemical, most rely on indirect tracking via downstream products or only report whether the drug is bound but not whether structural activation has occurred. None of them provide a direct readout of the activation mechanism or can be performed as a cell free method. This thesis describes the design of a generalizable and accessible In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), having broad and diverse applications. IGNiTR leverages the interaction between a conformation-specific binder and agonist- activated GPCR to reconstitute a split NanoLuciferase (NanoLuc) which can produce a bioluminescent signal in the presence of substrate. I describe the development of protocols for harvesting components expressed in live mammalian cells and for lysing the pellets (stored frozen) before mixing with other components of the assay. This thesis demonstrates IGNiTR with three Gs-coupled GPCRs and a Gi-coupled GPCR with three classes of conformation- specific binders: nanobodies, miniG proteins, and G-protein peptidomimetics. IGNiTR and the lysate conditions also allows the use of a synthetic G-protein peptidomimetics, providing easily standardized reagents for characterizing GPCRs and ligands. Described here is the development of two novel peptidomimetics, a Gs peptidomimetic fused to the SmBiT component of split NanoLuc, as well as a Gi peptidomimetic version. These peptides feature the use of unnatural amino acids that enhance the selectivity of the peptidomimetic for the active conformation of the GPCR. As an in vitro assay, IGNiTR components can be prepared in advance and stored frozen, and mixed, providing ready-to-go reagents and high consistency across reactions. The thesis describes three applications of IGNiTR: 1) a proof of concept high-throughput screening of ligands against DRD1; 2) detection of opioids using less sophisticated imaging instrumentation; and 3) characterizing GPCR functionality during Nanodisc-based reconstitution process. Due to its convenience, accessibility, and consistency, IGNiTR will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.Deep Blue DOI
Subjects
protein engineering chemical biology peptidomimetics G protein coupled receptors
Types
Thesis
Metadata
Show full item recordCollections
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.