Honors Theses
Date of Award
Spring 5-9-2026
Document Type
Undergraduate Thesis
Department
Biomolecular Sciences
First Advisor
Brenton Laing
Second Advisor
Gregg Roman
Relational Format
Dissertation/Thesis
Abstract
G protein-coupled receptors (GPCRs) represent one of the most versatile and widely studied families of signaling proteins, yet current experimental approaches often struggle to resolve pathway-specific activation across the three distinct G protein subclasses. This study aimed to develop complementary methods for the interrogation of GPCR signaling, with a focus on the Gq- and Gi-mediated pathways.
Components of the TRUPATH assay were established to enable future investigation of Gi-coupled receptor signaling. Fluorescent imaging and RT-PCR results validated successful transcription of the TRUPATH constructs, with transfected samples showing vivid fluorescence under imaging and elevated expression of target genes relative to the control groups.
A TRH-sniffer cell system was engineered to detect Gq-mediated calcium signaling through the expression of a calcium-sensitive fluorescent reporter in response to the activation of the thyrotropin-releasing hormone receptor (TRHR). Functional analysis demonstrated that cells in the ligand-receptor (LR) group, defined as cells co-expressing TRHR and a calcium sensitive reporter GCaMP while receiving TRH stimulation, exhibited a significant increase in calcium-dependent fluorescence compared to the receptor-only (R) control group, which expressed TRHR without ligand stimulation, and the ligand-only (L), which received TRH stimulation without TRHR expression. Quantification using area under the curve analysis confirmed that responses were both ligand-dependent and receptor-dependent.
Together, these findings demonstrate the successful development of complementary experimental platforms for the analysis of GPCR signaling. The interrogation of these approaches improves the resolution of GPCR signaling measurements and provides a framework for future studies investigating ligand-specific signaling dynamics.
Recommended Citation
Roberts, Ethan J., "Development of Methods for GPCR Function Interrogation" (2026). Honors Theses. 3564.
https://egrove.olemiss.edu/hon_thesis/3564