Insight into Mechanisms of Multiplicity of GPCR-G protein Signaling
Malik, Rabia
2017
Abstract
G protein coupled receptors (GPCR) are sensory proteins that transduce a wide range of extracellular stimuli including drugs, neurotransmitters, and hormones into diverse physiological responses by activating intracellular effectors (G proteins or arrestins). Dysfunction in GPCR signaling is implicated in many disease states including cardiomyopathies, neuropathies, and cancer. Multiple factors complicate dissection of GPCR-G protein signaling in live cells. First, there is an imbalance between the number of distinct GPCRs and G proteins. For example, in the human genome, there are over 700 GPCRs but only 16 G proteins. Next, GPCRs are known to adopt multiple structural conformations and differentially activate functionally distinct G proteins through mechanisms that are poorly understood. Additional complexity arises from differences in cellular GPCR and G protein concentrations, localization of GPCR and G protein to micro-domains, and regulation by regulatory elements (RGS’s, GRKs, and arrestins). How the cell prevents misfiring and interprets cross talk between different GPCR-G protein interactions remains an ongoing area of research. Studies presented herein aim to understand the mechanisms of specificity and multiplicity of GPCR signaling in live cells. The first set of studies explores the mechanisms by which GPCRs selectively activate one or more G proteins. Sensors were designed based on the well-established interaction between GPCRs and peptides derived from the Gα C-terminal α5 helix, a known determinant of the specificity of GPCR-G protein interaction. Proof-of-concept studies demonstrate that the Gα C-terminus probes for a G protein-selective conformation of GPCRs in live cells. Following ligand stimulation, G protein-activating GPCR conformations interact specifically with the corresponding Gα C-terminus. Additional collaborative efforts using molecular dynamic simulation (MD), revealed hot spot residues on the Gαs and Gαq C-termini that confer specificity of GPCR-G protein interactions. A second set of MD and functional studies identified the role of conserved Gαi residues, L32 and D333, in G protein activation. A parallel set of studies examines the influence of GPCR and G protein concentration and interaction strengths on downstream second messenger response. Therein, the well-characterized ER/K linker was leveraged to tether GPCRs to G proteins. The ER/K linker sets the effective concentration of the tethered GPCR-G protein and thus enabled a pairwise comparison across fusions. Linker length systematically modulated association between cognate and non-cognate G proteins; however, it only tuned cognate second messenger response downstream of the Gs, Gi, and Gq coupled receptors. Agonist stimulation provides changes in FRET (ΔFRET) comparable to those previously reported using co-expressed FRET/BRET pairs. Hence, the SPASM sensors serve the dual purpose of probing agonist-stimulated changes in GPCR-G protein interaction while examining the downstream response using the same construct. These two parallel approaches seek to better understand the mechanism regulating multiplicity in the GPCR-G protein signaling pathway. Findings shed light into the structural basis of GPCR activation and provide a mechanism for ligand-dependent GPCR-G protein selection. Initial studies provide proof of concept for the use of fusions to probe the multiplicity of GPCR-G protein signaling in live cells, however, additional research is needed to explore the functional consequence of these findings in a physiologically relevant context.Subjects
GPCR FRET sensors G protein FRET membrane protein signaling
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.