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The Role of Rhoa in GPR116 Mediated Alveolar Homeostasis

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The Role of Rhoa in GPR116 Mediated Alveolar Homeostasis The role of RhoA in GPR116 mediated alveolar homeostasis A thesis submitted to the Division of Graduate Studies and Research of the University of Cincinnati In partial fulfillment of the requirements for the degree of Master of Science (M.S.) In the Department of Molecular Genetics, Biochemistry, and Microbiology of the College of Medicine 2019 John J. Lawder B.S., Xavier University 2017 Committee Chair: Jim Bridges, Ph.D. i Abstract GPR116 is an orphan adhesion GPCR (aGPCR) expressed primarily in alveolar type II (ATII) lung epithelial cells. Although the mechanism in unknown, GPR116 has been identified to regulate surfactant secretion and reabsorption through a Gαq effector protein. Being an orphan receptor, no known ligand has been identified for GPR116. However, a peptide coded from the Stachel sequence contained in the GAIN domain, specific to aGPCRs, can be used to activate the full-length receptor. Previous studies have linked GPR116 activation with an increase in Rho family GTPase activity and ultimately an increase in cytoskeletal structures in various cell types. While not well characterized, a similar increase in cortical F-actin has been observed following GPR116 activation in ATII cells as well. We hypothesize that the suppression of surfactant secretion mediated by GPR116 is a direct result of an increase in RhoA signaling leading to cortical F-actin stabilization. ii iii Acknowledgements I would like to thank Jim Bridges for all the encouragement and help throughout my work. Despite many failed experiments and months of troubleshooting, he was always there to help in any way needed. I’d also like to thank Kari Brown and Alyssa Filuta for all their help in carrying out these experiments. Lastly, I’d like to thank my committee, Bill Miller and Rhett Kovall, for their help in preparing me for graduation and presenting my research. iv Table of Contents Abstract Acknowledgements Table of Contents List of Figures and Tables List of Abbreviations and Symbols Chapter I. Introduction G Protein Coupled Receptors Heterotrimeric G Proteins Alveolar Environment Pulmonary Surfactant Chapter II. Introduction GPR116 (Gq) Rho GTPases Results Discussion Materials and Methods Chapter III. Future Directions References v Figures and Tables Figure 1. Schematic of GPR116 mechanism Figure 2. SRE Luciferase model used to determine dependence of GPR116 on RhoA Figure 3. qPCR time course for RhoA mRNA expression in ShRNA Figure 4. Loss of RhoA expression in ShRNA transfected HEKT cells via SRE Luciferase assay Figure 5. FACs data for HEKT cells following lentiviral infection with RhoA ShRNAs Figure 6. Functionality of RhoA V14 construct in a pCasi AAV vector Figure 7. Phospholipid data following RhoA V14 AAV rescue of SftpcCreER Adgrf5f/f mice surfactant overload Figure 8. Model for BRET assays vi List of Abbreviations GPCR: G-Protein coupled receptor PAP: Pulmonary alveolar proteinosis aGPCR: Adhesion G-Protein coupled GM-CSF: Granulocyte/macrophage- receptor colony stimulating factor GAIN domain: GPCR autoproteolysis BAL: Bronchioalveolar lavage inducing domain BALF: Bronchioalveolar lavage fluid GEF: Guanine exchange factor SatPC: saturated phosphatidylcholine GAP: GPR116 activating peptide CTF: C-terminal fragment ATI: Alveolar type 1 cell PLC: Phospholipase C ATII: Alveolar type 2 cell PIP2: Phophatidylinositol 4,5- AM: Alveolar macrophage bisphosphate SFTPA: Surfactant protein A IP3: Inositol triphosphate SFTPB: Surfactant protein B DAG: Diacylglyceride SFTPC: Surfactant protein C SRE: Serum response element SFTPD: Surfactant protein D M1: Muscarinic 1 receptor RDS: Respiratory distress syndrome FACS: Fluorescence activated cell sorting ALI: Acute lung injury rLuc: Renilla Luciferase ARDS: Acute RDS rGFP: Renilla GFP vii Chapter I. Introduction 1 G Protein Coupled Receptors Composed of five major families, G-Protein Coupled Receptors (GPCRs) contain the largest number of human transmembrane receptors. Although split across these 5 families, Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin families, what categorizes them all as GPCRs is their highly conserved seven transmembrane regions connected by extracellular and intracellular loops46. GPCRs function by relaying an agonist activated signal into the cell, where that signal may be propagated and enhanced through G protein effector molecules. GPCRs are used across the entire human body for a wide variety of cellular signaling. Implicated in numerous diseases, including several developmental disorders as well as some cancers, GPCRs are the target for more than 40% of FDA approved drug therapies43. While ligands have been identified for many receptors, more than 15% of GPCRs have no known ligand and are classified as orphan receptors44. Despite being an orphan receptor, GPCRs such as GPR116 and other adhesion GPCRs (aGPCR) have a unique activation domain where following a self-cleavage in the extracellular region of the receptor an activation sequence is exposed and can freely bind the receptor. This domain gives us an advantage in identifying the roles and mechanisms of aGPCRs. The second largest family of GPCRs, the aGPCR family, contains 33 receptors spread across nine subfamilies. In addition to the standard seven transmembrane regions contained in all GPCRs, the aGPCRs contain a shared GPCR Autoproteolysis Inducing (GAIN) domain with extended N-terminal tails18. These GAIN domains play a key role in receptor signaling via a tethered agonist model. In this model, the GAIN domain, contained on the N- terminal tail, is autoproteolytically cleaved inside the cell as the receptor is trafficked to the membrane resulting in a N-terminal Fragment (NTF) that non-covalently attached to the C- 2 terminal fragment (CTF) receptor at the cleavage site. Upon ligand stimulation, or yet to be determined receptor activation models, the NTF is removed, exposing the extracellular portion of the CTF receptor, known as the Stachel sequence. This Stachel sequence is now free and acts as a tethered agonist for the receptor11. This agonistic Stachel sequence has been used as a template for researchers to synthesize peptide mimics, providing a synthetic activation mode for aGPCRs. Heterotrimeric G Proteins All GPCRs signal through a heterotrimeric G protein complex composed of a Gα, Gβ, and Gγ subunits. These complexes act to amplify the initial signal of the receptor through activating further downstream effectors such as protein kinases, transcription factors and/or ion channels. Following receptor stimulation, this G protein complex acts as a guanine exchange factor (GEF) to exchange the GDP bound to the Gα subunit for a GTP. As this exchange occurs, the Gα dissociates from the Gβ/γ, and signals downstream based on the type of Gα protein it is. Although we will focus on the Gα signaling, the Gβ/γ subunits also have downstream effectors following release from the heterotrimeric complex34. Over thirty-five Gα proteins have been identified; the major types being Gα q/11, Gα i, Gα s, Gα t, Gα 12/1334. While each GPCR is typically associated with one specific type of Gα protein, they may exhibit promiscuity with respect to their Gα effector proteins. This is often in cases where the same GPCR is expressed in different tissues, requiring different mechanisms. Our lab has previously identified and synthesized multiple GPR116 activating peptides (GAP) modeled from the Stachel sequence. Using these peptides, we have found that GPR116 signals through the Gα q/11 pathway6. We continue to use this peptide-based activation approach to examine mechanisms of GPR116 signaling. 3 Alveolar Environment In order to best understand the role of GPR116 in the human body, the environment in which it resides must be well understood. Although expressed in many tissues, GPR116 is found primarily in lung epithelial cells, specifically alveolar type II cells (ATII). ATII cells, along with alveolar type I cells (ATI) and alveolar macrophages (AM), compose the major cell types of the alveolus. In the alveoli, ATI cells provide the structure for the alveolar walls, as well as facilitate gas exchange of CO2 and oxygen between the alveoli and the blood. The AM main function is to keep the alveolus clear of foreign pathogens that may have escaped earlier detection and elimination. However, AM have another important role in aiding the clearance and catabolism of surfactant in the distal lung. Along with AM, data shows that ATII cells contribute to roughly 50% of surfactant clearance and catabolism. ATII cells synthesize surfactant proteins, packaging them along with high amounts of phospholipids, and some neutral lipids, into vesicles called lamellar bodies6. These lamellar bodies are then secreted into the alveoli where surfactant can function to protect the airways. Along with secretion of surfactant, ATII are responsible for surfactant reabsorption. ATII cells can reabsorb surfactant into endosomes, where it is either targeted for degradation, or recycled back into lamellar bodies and secreted once again6. Pulmonary Surfactant Pulmonary surfactant is an important lipid-protein matrix that is synthesized and released from ATII cells into the alveoli5. Produced in lamellar bodies inside the ATII cells, surfactant is composed of 80% phospholipids along with neutral lipids and lipid-associated 4 proteins surfactant proteins A, B, C, and D (SFTPA, SFTPB, SFTPC, and SFTPD). When secreted into the alveolus, surfactant adheres to alveolar wall, decreasing the surface tension of the alveolus. Along with decreased surface
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