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A Dissertation Entitled Interrogation of GPCR-G Protein Signaling Using A Dissertation entitled Interrogation of GPCR-G Protein Signaling using Novel Optogenetic Tools by Kanishka D Senarath Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry ___________________________________________ Dr. Ajith Karunarathne, Committee Chair ___________________________________________ Dr. Donald Ronning, Committee Member ___________________________________________ Dr. Dragan Isailovic, Committee Member ___________________________________________ Dr. Song-Tao Liu, Committee Member ___________________________________________ Dr. Cyndee Gruden, Dean College of Graduate Studies The University of Toledo May 2019 Copyright 2019, Kanishka Senarath This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Interrogation of GPCR-G Protein Signaling using Novel Optogenetic Tools by Kanishka D Senarath Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry The University of Toledo May 2019 G protein coupled receptors (GPCRs) control a vast majority of signaling responses in the body. Therefore, they are at the center of a large number of diseases, ranging from heart diseases to cancer, and have become the primary target of more than one third of pharmaceutical agents currently available in the market. Due to the limitations of current assays to measure activation of GPCRs and G proteins – such as being time-consuming, indirect, and expensive – an efficient method directly measuring the GPCR-G protein activation was in demand. Therefore, a confocal microscopy imaging-based assay was developed to directly measure ligand-induced GPCR and G protein activity in real time in live cells. The number of active GPCRs governs G protein heterotrimer (Gαβγ) dissociation (into Gα and Gβγ), and thereby controls the concentration of free Gβγ subunits. The developed Gγ9 assay measures GPCR activation-induced reversible Gβγ9 subunit exchange between the plasma membrane (PM) and internal membranes (IMs). Since G protein activation and dissociation is the immediate next step of GPCR activation, the live cell imaging-based γ9 assay can be used to directly measure ligand-induced GPCR and G protein activity in real time. The Gγ9 assay quantitatively determines the concentration dependency of ligands on GPCR activation, deactivation, and inhibition. Also, the assay iii demonstrates the high-throughput screening (HTS) adaptability. This assay works effectively for light-sensing GPCRs as well, which enables experimental determination of spatially restricted activation of GPCRs and G proteins not only in single cells but also in subcellular regions of single cells. Overall, the Gγ9 assay provides a robust strategy for quantitative as well as qualitative assessment of GPCR-G protein activation in a single- cell, multicell, and subcellular level, which would be useful in drug discovery efforts. Gβγ is a major signal transducer and controls multiple cellular processes ranging from cell migration to gene transcription. Despite the significant subtype heterogeneity and diverse cell and tissue specific expressions, Gβγ is often treated as a single signaling entity. The Gγ subunit bears the only PM anchoring motif in the Gβγ dimer. Our discoveries demonstrate that the differential PM-affinities differentially modulate Gβγ-effector signaling at the PM and subsequent cellular responses (i.e., cell migration) in a Gγ type specific manner. Also, we show that the overall PM-affinity of the Gβγ-pool of a cell type is a strong predictor of its Gβγ signaling activation efficacy at the PM. Overall, our data discloses crucial aspects of Gβγ signaling and cell behavior regulation by Gγ-type specific differential PM-affinities of Gβγ. Gβγ interacts with the PM through a prenyl group at the carboxy terminus (CT) of Gγ, which strengthens the PM localization of Gαβγ heterotrimer as well. However, it was not clear how Gβγ possesses this unique and G-type dependent range of PM affinities by using only two types of lipid anchors (either farnesyl or geranylgeranyl) on Gγ. Therefore, we explored the sequence properties of the carboxy terminal region Gγ. Results identified a key existence of hydrophobic residues in the pre-prenylation region of several Gγ types that provided the highest PM-affinity for G. Further, we recognized the crucial iv contribution of pre-prenylation residues acting as “on-off’’ switches for Gβγ signaling, regardless of the type of prenylation on Gγ. Also, we show that these pre-prenylation regions of Gγ are evolved to be significantly shorter than other prenylated proteins while still maintaining the G protein heterotrimer formation, GPCR-G protein interaction, and G protein signaling ability. Overall, we showed a simple and unique design of pre-prenylation regions of G that allow Gβγ to act as a regulator of GPCR pathways and as a molecular switch for Gβγ signaling. To optogenetically control GPCR-G protein pathways in vivo, light sensing GPCRs should be able to activate G protein heterotrimers with a comparable efficiency to ligand inducible GPCRs of interest. Over the last two decades, rhodopsin-based chimeric GPCRs have been employed in cultured cells and in vivo to control multiple G protein pathways. However, these chimeric GPCRs in our studies showed severely defective trafficking to the PM as well as extremely poor signaling activation, both of which could go unnoticed in studies in vitro with purified proteins or in in vivo animal studies. Therefore, we examined the feasibility of chimeric receptor engineering by replacing intracellular loops (ILs) of human color opsin and found that even a minor change to IL1 and IL2 either drastically reduces or completely abolishes receptor trafficking to the PM. IL3 exhibited more tolerance for mutagenesis, and together with the CT, it allowed switching opsin signaling towards the signaling of the chemokine GPCR of interest. Compared to rhodopsin-based chimeras, these cone opsin-based chimeric GPCRs exhibited several fold higher G protein signaling that can be measured using fluorescence based assays. They also exhibit uninterrupted trafficking. However, in order to reach near similar signaling efficacies in chemical sensing GPCRs, our chimeric receptors require further engineering. v Overall, our work not only resulted in superior light-activatable GPCRs for in vivo optogenetics, but it also serves as a guide for future chimeric GPCR engineering. Overall, the studies presented establish the significance of the CT of Gγ in determining the PM-affinity of Gβγ and Gβγ mediated signaling activation at the PM. This Gγ-type dependent PM-affinity is achieved by fine-tuning the residues in the relatively short PM-interacting pre-CaaX regions (compared to other prenylated proteins). For instance, hydrophobic residues (i.e., phe) in the pre-CaaX adjacent to the prenylated-cys in the CaaX motif in Gγ provided the highest PM-affinity for Gβγ. Gγ9 which lacks such hydrophobic character shows the lowest PM-affinity, signified by its ability to elicit a faster Gβγ translocation rate. This fast and reversible Gβγ9 distribution between the PM and IMs was exploited to develop a universal assay for real-time detection of GPCR-G protein activation, both qualitatively, quantitatively, and in subcellular regions of living cells. Since GPCR-G protein signaling is universally conserved and Gβγ signaling pathways are major drug targets, mechanisms we identified and described here are likely to have a wide influence in studying and controlling cellular signaling. Also, our newly engineered light activatable chimeric receptors which can signal like chemically sensitive GPCRs with better signaling efficacy than currently available ones will allow to facilitate in vivo optogenetics to study and control numerous GPCR-controlled processes including immune system function, pain, addiction, and many other physiological and behavioral responses with much higher spatial and temporal control. vi Acknowledgements I would like to express my highest gratitude to my advisor, Dr. Ajith Karunarathne, for his support, guidance, and encouragement during the past four years of research. I would like to extend my deepest gratefulness to my committee members Dr. Donald R. Ronning, Dr. Dragan Isailovic, and Dr. Song-Tao Liu. Also, I want to thank Dr. John L. Payton and Dr. Deborah N. Chadee for their contribution and assistance in computational modeling and Western blotting, respectively, in my projects. I would like to thank all my lab members for all their support, advice, words of encouragement, and for all the insightful scientific discussions throughout the past few years. I want to thank Elise Harmon for proof reading the abstract of the thesis for me. Lastly, I would like to thank the Department of Chemistry and Biochemistry, the University of Toledo for providing me the opportunity to pursue my studies. vii Table of Contents Abstract ......................................................................................................................... iii Acknowledgements .......................................................................................................vii Table of Contents ........................................................................................................ viii List of Tables ...............................................................................................................
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