The Role of Rhoa in GPR116 Mediated Alveolar Homeostasis

Home , GPR116, Surfactant protein D

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.

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.

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.

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

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

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

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-

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.

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

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 tension in the alveolar walls, surfactant provides some rigidity to the alveolar walls such that there is less risk of an airway collapse upon exhalation. Insufficient surfactant levels are typically observed in premature infants, where symptoms of neonatal respiratory distress syndrome (RDS) are present, as well as a higher risk for acute lung injury (ALI) and acute RDS (ARDS) throughout the rest of life50. Low levels or loss of surfactant is easily treated by a surfactant replacement therapy. While surfactant is crucial for proper lung function and protection against lung collapse, excess surfactant in the airways can be just as harmful as its loss.

Excess accumulation of surfactant in the airway can lead to symptoms of pulmonary alveolar proteinosis (PAP). PAP is a broad syndrome primarily caused by a lack of mature granulocyte/macrophage-colony stimulating factor (GM-CSF) receptor on the AM. Loss of this receptor prevents the ability of AM to process and clear surfactant that has been absorbed from the alveolus. Ultimately these AM become engorged with phospholipids and transition into foam cells, losing AM function and resulting in an accumulation of surfactant in the alveolus29. Although AM defects are the primary cause of PAP, this syndrome has many factors that could lead to the defining characteristic of surfactant overload in the airways. While it is the AMs job to absorb and degrade surfactant, AMs work in conjunction with ATII cells in this role. Surfactant maintenance and alveolar homeostasis is a major role for ATII cells and specifically GPR116 plays an important role in this context4.

Currently, the major therapy for PAP, and related surfactant overload diseases, is a bronchioalveolar lavage (BAL). This procedure involves sedating the patient, followed by flushing their lungs multiple times with a lavage fluid designed to extract excess surfactant and material from the airways. It is an invasive procedure that requires the patient to be put on a ventilator throughout the process. The bronchioalveolar lavage fluid (BALF) extracted from a patient may then be analyzed to determine levels of various phospholipids. This technique is also used in our lab to identify surfactant overload and potential for therapeutic rescue of wildtype phenotypes in diseased mice. Our lab has previously shown that in GPR116 whole body and ATII cell specific, conditional lung knockout mice, saturated phosphatidylcholine (satPC) levels were elevated as compared to control mice, indicating the importance of GPR116 in surfactant regulation5. These experiments were conducted with multiple knockout models, with knockout specific to lung GPR116 for our conditional models. Similar results of elevated satPC levels were observed in mice with a conditionally knocked out Gα q/11 in the lung6. These results suggest the association between GPR116 and Gα q/11 in lung epithelial tissue.

Chapter II

RhoA’s role in GPR116 activation

GPR116

GPR116 is an orphan aGPCR identified as the key regulatory receptor in surfactant homeostasis5. GPR116 is expressed in multiple cell types throughout the body, including heart endothelium, lung epithelium, and has been implicated in certain forms of breast cancer24. Although prior research has found GPR116 to play minor roles in these other cell types, GPR116 is most highly expressed in lung epithelial tissue, specifically ATII cells, where it regulates the surfactant levels of the alveoli. Though the mechanism is unclear, following GPR116 activation, surfactant secretion is suppressed, while uptake of extracellular surfactant is upregulated6. Despite being an orphan receptor, due to the nature of the aGPCR tethered agonist model, our lab has been able to identify and generate peptide from GPR116’s Stachel sequence. These synthetic peptides are sufficient to activate GPR116, providing us simple tools for studying receptor mechanics both in vitro and ex vivo.

Previous work in our lab using these peptides has identified G q/11 as the primary G- protein effector molecule activated following GPR116 stimulation5. Once activated, GTP- bound G q/11 activates phospholipase C (PLC) which cleaves phosphatidylinositol 4,5- bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglyceride (DAG). Following cleavage of PIP2, IP3 migrates to the endoplasmic reticulum, activating intracellular Ca2+ channels (figure 1). Taking advantage of this G q/11 pathway, we can track GPR116 activity by measuring intracellular Ca2+ levels. These calcium assays have been utilized to identify optimal peptides for GPR116 activation as well as to determine potential mechanistic intermediates. GAP16, a 16 amino acid stretch of the Stachel sequence, was the primary

GPR116 activating peptide used in previous studies, however further Ca2+ assays have

identified the most potent GPR116 response when stimulated with only a 10 amino acid

peptide, GAP106.

RhoA GTPases

Although we can activate the receptor and have identified its early effector

molecules, the mechanism of GPR116 following G q/11 activation up until the observed

Figure 1. Schematic of GPR116 mechanism. GPR116 activation demonstrated through GAP16, a GPCR activating peptide (GAP) derived from the Stachel sequence of the C-terminal fragment of the N-terminal tail. The GAIN domain, with its cleavage site, is marked by the red star on the receptor. GPR116 activation is shown to increase intracellular Ca2+ signaling through PIP2 conversion to IP3 and DAG. The dashed arrows represent the current gap in our understanding of the GPR116 mechanism. Current research has linked GPR116 activation with an increase in cortical F-actin which is believed to play a role in the cells decrease in surfactant secretion. This link however has not been discovered and is the core of our current research. Image taken from Brown, K., Filuta, A., Ludwig, M.-G., Seuwen, K., Jaros, J., Vidal, S., … Bridges, J. P. (2017). Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling. JCI Insight, 2(11), 1–19. https://doi.org/10.1172/jci.insight.93700

actin rearrangement remains unclear (figure 1). However, another group had previously implicated RhoA and Rac1 as necessary for GPR116 function in breast cancer metastasis and cell motility24. In this model, RhoA and Rac1 were identified as key effector molecules for GPR116 mediated lamellipodia and stress fiber formation23. RhoA and Rac1 belong to the Rho family of GTPases, an effector family essential for cytoskeletal dynamics. As this group identified in their research, Rac1 is required for the formation of lamellipodia and membrane dynamics, while RhoA regulates stress fiber formation24. This research, paired with our initial observations of cortical F-actin formation following GPR116 activation, suggests RhoA as a possible intermediate in the pathway downstream of GPR1166. To test this hypothesis, we primarily utilized two mutant forms of RhoA: RhoA T19N and RhoA

V14. RhoA T19N is a dominant negative mutant of RhoA where the inactive, GDP-bound confirmation of RhoA is stabilized. This stabilization prevents RhoA’s activation via a GEF.

Conversely, RhoA V14 is a constitutively active mutant of RhoA. This mutant has a glycine substituted with a valine near the proteins active site. This valine acts to shield the triphosphate group of an active RhoA protein from the solvent, locking the protein in an active state.

Results

Activation of RhoA downstream of GPR116 signaling

To interrogate RhoA’s involvement in GPR116 signaling, we utilized an SRE luciferase reporter assay. This assay involves transfection of a reporter plasmid, a luciferase gene regulated by a RhoA dependent serum response element (SRE), along with a receptor or other condition to be tested. In this system, the luciferase will only be active if the upstream regulator of the SRE is present. By lysing cells transfected with our various

conditions and this reporter, we can measure the luciferase levels produced from them, and discern RhoA’s role in these systems. Although the SRE used in these experiments is not specific for RhoA, it is one of few activators for the SRE and is effective in interpreting

RhoA expression based on luciferase activity.

Using both the Muscarinic 1 receptor, (M1) a common G q/11-coupled GPCR, and a constitutively active G q/11 Q209L mutant as our positive controls, we observed that our model system is functioning properly in transfected HEKT cells (figure 2 and data not shown). Although M1 receptor following carbachol stimulation does not produce the expected results in the presence of RhoA T19N, that is a marked decrease in luciferase expression, previous studies have implicated the M1 receptor to signal through other G- protein effector molecules and thus may be bypassing the need for RhoA in activation of the SRE. Moreover, our Q209L control increased luciferase activity as expected. RhoA

T19N is a dominant negative mutant of RhoA and was used throughout our experiments as both a test group and a negative control. In cells expressing GPR116, however, we observe a marked increase in luciferase expression following activation with Gap10 that is significantly decreased in the presence of RhoA T19N (figure 2). This increase in luciferase was not present when GPR116 was stimulated with a scrambled peptide. Despite GPR116 having some basal activity based on the current aGPCR signaling model, it is interesting that we did not observe an increase in luciferase expression in non-stimulated GPR116 expressing cells (figure 2).

Knockdown of RhoA in transfected cells

Having confirmed RhoA plays a role downstream of GPR116 in transfected cells, we ordered multiple shRNAs designed to knockdown endogenous RhoA and cloned them into

PLKO expression vectors containing a GFP reporter gene. A non-shRNA PLKO vector was used as a negative control. These plasmids were then used to transfect HEKT cells as well as later to generate stable RhoA knockdown cell lines. RNA was isolated 1, 2, and 3 days following RhoA shRNA transfection. cDNA was generated from the RNA and used for qPCR to detect mRNA levels of RhoA compared to a baseline of b-actin. While some of the shRNA transfected cells displayed RhoA knockdown on day 1, harvest at 3 days post transfection resulted in significant reduction of RhoA mRNA in all experimental groups (figure 3).

Figure 2. SRE Luciferase model used to determine dependence of GPR116 on RhoA. HEKT cells transfected with SRE Luciferase reporter alone or in combination with either G q Q209L (a constitutively active G q) or GPR116. Gap10 peptide was used to activate GPR116, using a scrambled peptide of Gap10 as a negative control. Dominant negative RhoA T19N transfected in to disrupt the RhoA activity of the cells. Significant loss of SRE Luciferase activity in Gap10 stimulated RhoA deficient cells containing GPR116 (n=3 wells per group, n=3 experiments). Data expressed as mean ±SD, *P<0.001 (1-way ANOVA)

After confirming by mRNA expression analysis that these shRNAs can knockdown

RhoA expression following transfection, we tested how these affected GPR116 activity in our SRE luciferase assay. ShRNA 199 and 616 were chosen along with a 2-day transfection period for these experiments based on mRNA data (figure 3). As expected, shRNA 199 knockdown of RhoA lead to a significant decrease in luciferase expression stimulating either Q209L or GPR116 Gap10 cells (figure 4a). Baseline luciferase expression with only

SRELuc transfected was decreased with shRNA 199 (figure 4a). Knockdown of RhoA expression in transfected HEKT cells also confirmed via Western blot analysis. Although not quantified, near complete loss of ShRNA 616 is observed along with potential decrease

RhoA ShRNA qPCR

2.0

* ***

1.5

l a

v 1.0

Q R *** * ** ** *** *** 0.5 * ** ***

0.0

V 9 0 2 6 O V 9 0 2 6 O V 9 0 2 6 O E 9 0 8 1 K E 9 0 8 1 K E 9 0 8 1 K 1 3 3 6 L 1 3 3 6 L 1 3 3 6 L P P P

Day 1 Day 2 Day 3

Figure 3. qPCR Time course for RhoA mRNA expression in ShRNA. mRNA levels of RhoA measured in RhoA ShRNA transfected HEKT cells. RNA isolated from cells at 1, 2, or 3 days. cDNA libraries were created from RNA isolation run in qPCR. RhoA mRNA levels decreased in all samples by three days following transfection. RhoA levels as low as 50%, 74%, 57%, and 36% of non- transfected were recorded for 199, 300, 382, and 616 ShRNA respectively (n=3 wells per group, n=1 experiment). Data expressed as mean ± SD *P<0.05 for EV day 1, **P<0.01 for EV day 2, ***P<0.05 for EV day 3 (1-way ANOVA)

in ShRNA 199 (figure 4b). Constitutively active RhoA V14 was used as a positive control for

RhoA overexpression.

Design and confirmation of stable RhoA deficient cell lines

While the transfection data described above provides a strong indication for RhoA’s

involvement in GPR116 signaling, we wanted to generate stably transfected RhoA deficient

cells to confirm our transfection data and utilize for further characterizing RhoAs potential

involvement in actin dynamics. To generate these cell lines, we used a lentiviral infection

model. Lentivirus was created for each individual shRNA in their PLKO expression vectors

as described above and used to infect low passage HEKT cells. Following multiple passes in

order to increase cell number, cell lines were sorted for GFP positive cells via fluorescence

activated cell sorting (FACS). Following sorting, each cell line remained between 60% and

A B. .

RhoA NT 199 616 V14 36 28 Wt RhoA 17

Figure 4. Loss of RhoA expression in ShRNA transfected HEKT cells via SRE Luciferase assay. A. SRE Luciferase assay to confirm knockdown of RhoA in ShRNA transfected HEKT cells with 116 following Gap stimulation. 616 and T19N not data not shown. (n=3 wells per group, n=2 experiment). B. Western blot for RhoA expression comparing basal RhoA expression with 199 and 616 ShRNA transfected HEKT cell RhoA expression. PKH3 RhoA V14 as positive control for RhoA overexpression. 30 second exposure time on film. Data expressed as mean ±SD *P<0.001 **P<0.05 (1-way ANOVA)

90% GFP positive for up to 4 weeks, or 8 passages (figure 5). Stocks were frozen down following sorting for future experiments.

Rescue of SftpcCreER Adgrf5f/f phenotype with active RhoA

Constitutively active RhoA V14 has been previously used as a positive control for

ShRNA knockdown of RhoA. Alternatively, we designed a RhoA V14 AAV to rescue

GPR116-/- phenotypes, namely increased surfactant levels in the distal lung. First, the RhoA

V14 plasmid was cloned into an AAV vector and tested for functionality in our SRE luciferase assay and by Western blot. With our Casi RhoA V14 vector transfected into

HEKT cells along with the SRELuc reporter, we observed an increase in luciferase expression that is significantly decreased in the presence of RhoA T19N (figure 6a). These results nearly mirrored that of the positive control constitutively active Gαq Q209L. These

Figure 5. FACs data for HEKT cells following lentiviral infection with RhoA ShRNAs. HEKT cells infected with RhoA ShRNA lentivirus containing GFP reporter. Cells were sorted for GFP prior to experimentation. Cell counts for GFP expression one week following cell sorting with gates for viability and size along with GFP expression. Data remained consistent up to 4 weeks and eight passages.

results were further confirmed, that Casi RhoA V14 AAV vector when transfected into cells produced high levels of RhoA protein detectable on Western blot (figure 6b). With these data we moved forward with the creation and testing of our AAV in SftpcCreER Adgrf5f/f mice.

RhoA V14 AAV was tested using a conditional knockout mouse model, SftpcCreER Adgrf5f/f mice. These mice contain a conditional GPR116 knockout. Sftpc is an ATII cell specific marker used to localize the activation of CreER. Once mice are treated with tamoxifen, a drug used to activate the synthesis of CreER, active CreER cleaves the loxP sites which are flanking Adgrf5, commonly referred to as GPR116. This conditional knockout model provides us with high levels of control in when and where these mice will exhibit GPR116 deletion and subsequent null phenotypes, namely surfactant accumulation. Following a 2-week treatment with tamoxifen

Wt RhoA

chow, mice are harvested for BALF. Lavage fluid is then analyzed for saturated phosphatidylcholine (satPC) and total phosphate levels. Following a 2-week tamoxifen treatment, control mice exhibit elevated levels of both satPC and total phosphate as compared to healthy wt mice.

To determine the role of active RhoA downstream of GPR116, we infected SftpcCreER

Adgrf5f/f mice with our RhoA V14 AAV. Virus was administered through intranasal infection and allowed one week prior to tamoxifen induced Adgrf5 knockout. Mice were then treated with tamoxifen chow for two weeks prior to harvest for BALF as described above. This conditional model and infection timeline were key to determine proper AAV infection. If attempted in a diseased mouse, where surfactant has already accumulated in the lower airways, intranasal infection would be inconsistent as virus would have difficulty infecting the proper cell type. An mcherry AAV made with the same AAV vector was used as a negative control. Both satPC and total phosphate levels of the RhoA V14 infected mice were decreased compared to mCherry infected control mice (figure 7a and b). Although a small sample size is reported, data indicate significant rescue of SftpcCreER Adgrf5f/f phenotype.

Discussion

While GPR116 has been shown to signal through G q/11, little was known about the mechanism in ATII cells. Previous studies had identified an increase in intracellular Ca2+ levels, as well as an increase in cortical F-actin, but nothing connecting these events. This left a wide gap in our understanding of how GPR116 functions in ATII to suppress secretion of surfactant into the airways, as well as increase the reabsorption of excess surfactant from the airway. Although not confirmed in the lung, RhoA and other Rho family GTPases

had been previously implicated as intermediates for GPR116 signaling in breast cancer cells. Paired with our observation of an increase in F-actin following GPR116 activation,

RhoA, commonly known to activate stress fibers and increase cytoskeletal activity, was a prime target for our mechanism.

First, a cell-based assay to determine RhoA expression and association with GPR116 had to be developed. The SRE luciferase system worked well as our model due to the SRE element being specific for RhoA, along with the ease of luciferase detection as our readout.

The system worked as expected and provided a proper baseline for RhoA dependent expression of luciferase. Although one of our positive controls didn’t respond as expected, namely the lack of a decrease in carbachol stimulated cells containing both M1 receptor and RhoA T19N, this wasn’t too concerning as the M1 receptor is known to signal through pathways other than G q/11, and this may allow for it to overcome the RhoA deficiency

(figure 2). Ultimately, we were able to confirm the dependence of RhoA in Gap10 activation of GPR116 through the decrease of luciferase expression in the presence of RhoA

A B. .

Figure 7. Phospholipid data following RhoA V14 AAV rescue of SftpcCreER Adgrf5f/f mice surfactant overload. A. SatPC levels in bronchioalveolar lavage fluid from 16-week-old SftpcCreER Adgrf5f/f mice. B. Total phosphate levels also measured from the bronchioalveolar lavage fluid. SftpcCreER Adgrf5f/f mice were infected with RhoA V14 AAV on day 0. On day 7, mice were transferred to a tamoxifen diet to induce deletion of Adgrf5. Mice were sacrificed for lavage on day 21, followed by lipid analysis of the bronchioalveolar lavage fluid (n=4 mice per group, 1 AAV-RhoA died prior to harvest, n=1 experiment). Data expressed as mean *P<0.05 (1-way ANOVA)

T19N (figure 2). While uncertain how far downstream of GPR116 activation and if this dependence on RhoA is present in vivo in the ATII cells, this data provides an initial understanding that RhoA has some role in GPR116 and that we can test this association.

Moving forward with our model for the RhoA in GPR116 activation, we wanted to analyze the effects of loss of RhoA in vitro. To knock down RhoA in vitro, we ordered multiple RhoA ShRNAs and cloned them into a PLKO expression vector containing a GFP reporter gene. These ShRNA plasmids were then used to either transiently transfect HEKT cells for experiments or to create a lentivirus which we used to stably infect HEKT cells.

While the stable cell lines are more valuable for long term experiments and potential in vitro recovery assays, many of our initial experiments to characterize our ShRNA constructs and RhoA’s role in the GPR116 mechanism. To confirm proper knock down of

RhoA in cells transfected with these RhoA ShRNAs, we first analyzed mRNA levels through qPCR. By 3 days post infection, all four RhoA ShRNAs reached at least 30% and as much as

70% knockdown of RhoA (figure 3). Both 199 and 616 ShRNAs had knockdown of greater than 50% RhoA after only two days post-infection and were chosen for further transient experiments.

When further characterizing the effects of these RhoA ShRNAs in transient experiments, some inconsistencies did arise which we cannot fully explain at this time. SRE luciferase experiments were run again, this time attempting to use our ShRNA constructs to mimic the RhoA T19N phenotype of RhoA knockdown. Although we did observe a significant decrease in luciferase cells transfected with both 199 and 616 ShRNAs, results depended on normalization of the data and for that reason we omitted the 616 ShRNA data from our results (figure 4a). Our 199 ShRNA produced much cleaner and more

representative data of the system and, while not entirely supported by our western data, these blots were difficult to identify RhoA in non-transfected cells and therefore difficult to compare between groups (figure 4b). Even blots where we omitted an active RhoA positive control to provide a cleaner picture, our data were unclear. Although all these data taken together may not paint the clearest picture of RhoA knockdown through transient transfection of our RhoA ShRNAs, these experiments provide a solid base for future stable cell line experiments to further characterize this system in vitro.

Along with determining the effects of RhoA knockdown in vitro, to properly characterize RhoA’s role in GPR116 activation in ATII cells, we wanted to determine if

RhoA expression could recover GPR116-/- phenotypes. In order to rescue these null phenotypes, we repackaged a constitutively active RhoA V14 mutant into an AAV vector in order to create an AAV for active RhoA. Once this construct was created, we tested the functionality of our RhoA V14 mutant through our SRE luciferase assay and overall RhoA protein production levels. Luciferase activity in our mutant was as expected, observing a large amount of luciferase activity with our RhoA V14 that is decreased in the presence of

RhoA T19N (figure 6a). Western blot data confirms RhoA protein levels remain consistent with those of the parent vector containing our RhoA V14 mutant (figure 6b.) Since all our initial tests came back positive for a functional active RhoA V14 mutant, we began testing our AAV for GPR116-/- rescue.

We first tested our RhoA V14 AAV in whole body GPR116-/- mice with little success.

Due to the nature of this phenotype, the lungs of these mice are overloaded with surfactant and thus our intra-tracheal infection model was unlikely to work properly from the start.

We were doubtful these mice would provide us any useable data and thus turned our focus

on a conditional GPR116-/- mouse model SftpcCreER Adgrf5f/f. These mice have an ATII specific GPR116 deletion when induced with tamoxifen chow, however prior to induction of deletion, their airways resemble wt mice and are freely infected via intra-tracheal infection. 7 days post-infection, mice were given tamoxifen chow to induce deletion of

GPR116. Following 14 days on this diet, mice were sacrificed and lavaged to test their

BALF for satPC and phosphate levels. Both satPC and phosphate levels had significantly decreased in our RhoA V14 mutant AAV group as compared to a mCherry AAV control group (figure 7). Although these data are only represented by a single experiment with a small n, the model for GPR116 rescue with active RhoA AAV is promising.

These data suggest RhoA as a key player in the mechanism downstream of GPR116 in regulating surfactant levels in the distal lung. Not only does this provide a clearer understanding of our working model of GPR116 activation but provides us with a target for potential therapies. Based on our findings that mice infected with active RhoA AAV had a significant decrease in surfactant accumulation as compared to control infected mice, active RhoA is an interesting intermediate that we would like to further interrogate.

Activation of RhoA in ATII cells may provide significant therapeutic benefits in PAP and other surfactant accumulation disease patients.

Materials and Methods

Cell Culture

Transformed human embryonic 293 (HEKT) cells were grown and maintained in

Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 0.5%

Penicillin/streptomycin, and 0.5% glutamine. HEKT cells were grown at 37°C in a humidified incubator with 95% air and 5% CO2. 10cm, 6 well, and 12 well plates were used

for various experiments and were all coated with Poly-D Lysine for up to 30 minutes prior to cell plating.

Cell Transfection

HEKT cells were plated on their respective dish size on day 0, followed by transfection using Lipofectamine 2000 transfection reagent (Thermo Fisher) on day 1.

DNA amounts transfected into cells varied between experiments and will be detailed in further sections. Lipofectamine 2000 was used at 1.2µl per well for 12 well plates and 6µl per well for 6 well plates. Transfection cocktails were mixed in 100µl of Optimem media for 30 minutes prior to transfection.

SRE Luciferase Assay

HEKT cells were plated at 1x105 cells per well in a 12 well PDL coated plates using

Lipofectamine 2000. Cells were transfected with the SRE LUC reporter DNA, along with either a negative control RhoA T19N, a positive control Q209L or test GPR116 FLV5 DNA.

All constructs were transfected at 250ng of DNA each up to a total of 750ng DNA per well.

For groups under 750ng of DNA, empty vector was added to keep amount of DNA transfected consistent. Further experimental groups included 250ng of our RhoA ShRNA

199 DNA along with either the Q209L or GPR116 FLV5. Prior to the addition of RhoA

ShRNA 199 as a group, cells were lysed using passive lysis buffer for 15 minutes on a shaker 24 hours post-transfection. These lysates were then plated at 20µl in triplicate on a black 96 well plate. The plate was then loaded into a plate reader, where100µl of luciferin was injected into the wells and luciferase expression was recorded. In experiments containing the RhoA ShRNA 199, cells were lysed at 48 hours post-transfection based on optimal knockdown of RhoA observed in qPCR studies. The remaining procedure was kept

the same. Luciferase expression data was normalized to baseline activity in HEKT cells transfected with SRE Luc reporter only. qPCR

HEKT cells were plated at 4x105 cells per well in 6 well PDL coated plates and transfected with 3µg of ShRNA construct and 1µg of empty vector in 6µl of lipofectamine.

RNA was isolated from transfected cells at 1, 2, and 3 days post-transfection using RLT+ buffer (Qiagen) to determine knockdown of RhoA over time. cDNA generated and precipitated from RNA using iScript kit (Bio-Rad). RhoA and b-Actin taqman probes ordered from Thermo Fisher.

Western Blot

HEKT cells were plated 4x105 cells per well in 6 well PDL coated plates and transfected with 4µg of RhoA ShRNA or RhoA Wt and V14 constructs. RhoA ShRNA transfected cells lysed directly in Laemelli buffer 2 days post-transfection. Cells transfected with Wt RhoA or active RhoA V14 were lysed directly in Laemelli buffer 1-day post- transfection. Lysates were sonicated for 1 minute prior to running on a gel. Precast 10-

20% Tris-Glycine gels were run for all experiments at 120V for around 90 minutes

(Thermo Fisher). Gels were transferred semi-dry to 0.2µm nitrocellulose membranes for 1 hour at 18V. Block transferred membranes in 5% milk in TBS-T for 1 hour, before incubating with primary antibody 1:1,000 in 5% milk in TBS-T overnight. RhoA (67B9)

Rabbit mAB #2117 primary antibody (Cell Signaling). Secondary antibody (goat α rabbit

HRP secondary) incubated at 1:20,000 for 1 hour in TBS-T. Blots were then washed in an

HRP chemiluminescent substrate and exposed on film for various time points. Exposure times varied between 30 seconds and 5 minutes.

Lentivirus Creation and Infection

HEKT cells were plated at 1.5x106 cells in a 10cm dish to generate lentivirus. Cells were transfected with lentiviral packaging vector along with a viral vector containing our

RhoA ShRNA with a GFP reporter. Media containing RhoA ShRNA lentivirus was then collected 3 days post-transfection. RhoA ShRNA lentivirus was then used to infect HEKT cells to create stably transfected RhoA deficient cell lines. For this, HEKT cells were plated at 1x105 per well in a 12 well plate on day 0. On day 1, media was replaced and 250µl of lentivirus was added. Media was replaced on day 3, and on day 4 the cells were passed into a 60mm dish. Infected cells that were leftover from the passage were used to screen for

GFP via FACS. On day 7, cells were passed once more into a T75 flask, where they would be cultured for experiments. If GFP expression was below 90%, cells were sorted prior to this passage, via FACS to enrich for GFP positive cells. Following passage into a T75, cells were frozen to create stocks for future use.

Adeno-Associated Virus

Genomic DNA for pCasi RhoA V14 was generated using RNA isolated from transfected cells and purified using Invitrogen PureLink Viral RNA/DNA Mini kit. As gDNA was prepared, HEKT cells were plated at .5x106 cells per dish onto 40 150mm dishes with

20ml DMEM on day 1. On day 4, cells were transfected with a DNA mixture containing

6.8µg 6.2ff packaging plasmid, 5.2µg pCasi RhoA V14 vector plasmid, and 60µl PEI MAX,

40K in 1ml of basal DMEM per dish. This mixture was vortex well to mix and incubated at room temp for 30 minutes prior to transfection. 6 hours following transfection, media was replaced with fresh DMEM. On day 8, cells were harvested via scraping and pipetting to

remove adhered cells. Cells and media were collected into 50ml conicals and frozen in a dry ice/ethanol bath before storage in -80° until purification.

Prior to AAV purification, pretreat two Amicon Ultra-4 (100,000 NMWL) centrifugal devices with 5ml HBSS (with Ca2+, Mg2+)/5% Tween 20 for 24 hours at room temperature.

Using a peristaltic pump, run the following solutions through the pump at 1-ml/minute to prime a heparin column: 20ml 1M NaOH, 100ml sterile dH2O, and 50 ml basal DMEM. After priming, remove the stopper portion of the heparin column, and run the following solutions: 50ml DMEM, 50ml HBSS without Ca2+ Mg2+, our AAV containing supernatant,

50ml HBSS without Ca2+ Mg2+, 25ml 0.5% Sarkosyl in HBSS without Ca2+ Mg2+, 50ml HBSS without Ca2+ Mg2+, 50ml HBSS with Ca2+ Mg2+, and 50ml 200mM NaCl/HBSS with Ca2+ Mg2+.

Following these washes, elute the column with 25ml of 400mM NaCl/HBSS with Ca2+ Mg2+, collecting the elution into a 50ml conical. This purified AAV was then concentrated using the Amicon tube prepared earlier. AAV was loaded into this tube and spun at 900g for 3 minutes, adding more virus until the entire 25ml is concentrated. AAV was dislodged from the Amicon concentration tube with 250µl of HBSS without Ca2+ Mg2+ and vigorous pipetting. Aliquots were stored at -80°C.

SftpcCreER Adgrf5f/f were given RhoA V14 AAV in 2 doses, totaling 2x1012 viral units in 40µl via an intranasal infection. Control mice were given a mCherry AAV under the same conditions. 7 days post-infection, infected mice were transitioned onto a tamoxifen chow diet to induce GPR116 deletion. After 2 weeks on tamoxifen diet, mice were sacrificed and their BALF was harvested to measure SatPC and total phosphate. Lipids were extracted from the lavage by the Bligh and Dyer method4. SatPC was isolated using the osmium tetroxide-based method of Mason et al27.

Chapter III

Future Directions

Characterization of RhoA stably transfected cell lines

To best understand our RhoA knockdown model for GPR116 activation in vitro, we developed stable cell lines containing ShRNAs for RhoA. Repeating some of the experiments discussed previously with these cells should provide a clear picture of RhoA in relation to GPR116. Although these cell lines have been sorted and purified for GFP, we still have not confirmed RhoA knockdown. Using the same procedure described for our transient model, we will measure mRNA of RhoA in our stable cell lines to determine RhoA knockdown efficiency. We can then further characterize these cell lines through Ca2+ assays, SRE Luciferase assays, and western blots for RhoA protein production. These RhoA

ShRNA stably transfected cell lines should provide us with a strong tool to determine

RhoA’s role in GPR116 activation, as well as potentially identify future targets that may be acting in this mechanism.

Constitutive active RhoA AAV

While repeating many of our experiments shown here will be necessary, one that shows the most promise is our constitutive RhoA V14 mutant AAV rescue of conditional

GPR116-/- mice phenotypes. This AAV is very promising in its ability to rescue satPC and phosphate levels and may be able to uncover more about through further study. In future studies, infected animals may also have tissue harvested and processed for imaging or ex vivo cell-based assays. Production of a 3xHA tagged version of this RhoA V14 AAV has already begun, and with this we can stain fixed tissue collected from infected animals to track localization of our AAV and compare this with various markers for ATII cells and

GPR116 intermediates. Lastly with this AAV, by harvesting and sorting out a pure epithelial cell population in both infected and control mice, we could run RNAseq to

identify genes that may have been rescued by our RhoA V14 AAV. This data could provide new targets for study in the pathway of GPR116 and a better understanding of the mechanism.

Bioluminescence Resonance Energy Transfer (BRET) as a model system for actin dynamics

One aspect of the current GPR116 mechanism that we are excited in pursuing is the observed, yet uncharacterized cortical F-actin stabilization following GPR116 stimulation.

This cortical actin rearrangement is something we believe may be crucial for GPR116’s role in suppressing the secretion of surfactant from ATIIs. Although easily detected through staining and fixed cell assays, a live-cell quantitative assay for F-actin formation has not been attainable. Because of this, we turned to a BRET based model to solve this issue. By modifying an existing BRET model designed for GPCR activation and tracking G-protein effectors, we were able to design our own system specific for tracking and quantifying the formation of F-actin in a live-cell plate-based readout for GFP and luciferase (figure 8a-b).

This plate-based system is key for this system as it provides a high throughput model for tracking this mechanism. In our BRET model, we tagged a renilla luciferase (rLuc) gene to

G-actin and a renilla GFP (rGFP) gene to G-actin individually. Following activation of a

GPCR that induces F-actin formation (we believe GPR116 does this), G-actin monomers will come together to form F-actin, bringing rLuc and rGFP together and close enough to promote BRET (figure 8c-d). By exciting luciferase only, if BRET doesn’t occur, we will only readout a luciferase expression. However, if this F-actin formation occurs and rLuc comes together with rGFP, BRET will occur, transferring energy to and exciting the rGFP molecule.

This results in us reading out both luciferase and GFP from our system. These data are

quantifiable representations of F-actin formation and can provide strong evidence for the importance of F-actin in our mechanism of GPR116.

A B. .

C. D .

Figure 8. Model for BRET assays. A. Sample Gαq linked GPCR. Gαq protein is tagged with a renilla luciferase (rLuc) while the Gβ and Gγ subunits are tagged with renilla GFP (rGFP) and linked to the membrane. B. Following activation of the GPCR, rLuc and rGFP disassociate, and BRET is lost. C. Model GPCR for GPR116 along with G-actin tagged with both rLuc and rGFP individually. D. Following GPR116 activation, G-actin will come together, forming F-actin and BRET between rLuc and rGFP.

References

1. Ariestanti, D. M., Ando, H., Hirose, S., & Nakamura, N. (2015). Targeted disruption of Ig-Hepta/Gpr116 causes emphysema-like symptoms that are associated with alveolar macrophage activation. Journal of Biological Chemistry, 290(17), 11032– 11040. https://doi.org/10.1074/jbc.M115.648311

2. Baak JP, Mutter GL, Robboy S, Van Diest PJ, Uyterlinde AM, Ørbo A, Palazzo J, Fianne B, Løvslett K, Burger C, Voorhorst F, V. R. (2005). In endometrial hyperplasias, the molecular-genetics and morphometry-based EIN classification more accurately predicts cancer-progression than the WHO94. Cancer, 103(11), 2304–2312. https://doi.org/10.1038/mp.2011.182.doi

3. Badr, C. E., Hewett, J. W., Breakefield, X. O., & Tannous, B. A. (2007). A highly sensitive assay for monitoring the secretory pathway and ER stress. PLoS ONE, 2(6). https://doi.org/10.1371/journal.pone.0000571

4. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911–917.

5. Bridges, J. P., Ludwig, M. G., Mueller, M., Kinzel, B., Sato, A., Xu, Y., … Ikegami, M. (2013). Orphan G protein-coupled receptor GPR116 regulates pulmonary surfactant pool size. American Journal of Respiratory Cell and Molecular Biology, 49(3), 348– 457. https://doi.org/10.1165/rcmb.2012-0439OC

6. Brown, K., Filuta, A., Ludwig, M.-G., Seuwen, K., Jaros, J., Vidal, S., … Bridges, J. P. (2017). Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling. JCI Insight, 2(11), 1–19. https://doi.org/10.1172/jci.insight.93700

7. Casado-Medrano, V., Barrio-Real, L., Wang, A., Cooke, M., Lopez-Haber, C., & Kazanietz, M. G. (2019). Distinctive requirement of PKCε in the control of Rho GTPases in epithelial and mesenchymally transformed lung cancer cells. Oncogene, 8–10. https://doi.org/10.1038/s41388-019-0796-4

8. Conkright, J. J., Bridges, J. P., Na, C. L., Voorhout, W. F., Trapnell, B., Glasser, S. W., & Weaver, T. E. (2001). Secretion of Surfactant Protein C, an Integral Membrane Protein, Requires the N-terminal Propeptide. Journal of Biological Chemistry, 276(18), 14658–14664. https://doi.org/10.1074/jbc.M011770200

9. Cui, C., Wang, X., Shang, X., Li, L., Ma, Y., Zhao, G., … Wang, H. (2019). lncRNA 430945 promotes the proliferation and migration of vascular smooth muscle cells via the

ROR2/RhoA signaling pathway in atherosclerosis. Molecular Medicine Reports, 1–10. https://doi.org/10.3892/mmr.2019.10137

10. Dai, H., Zhang, S., Du, X., Zhang, W., Jing, R., Wang, X., & Pan, L. (2019). RhoA inhibitor suppresses the production of microvesicles and rescues high ventilation induced lung injury. International Immunopharmacology, Vol. 72, pp. 74–81. https://doi.org/10.1016/j.intimp.2019.03.059

11. Demberg, L. M., Winkler, J., Wilde, C., Simon, K. U., Schön, J., Rothemund, S., … Liebscher, I. (2017). Activation of adhesion G protein-coupled receptors: Agonist specificity of Stachel sequence-derived peptides. Journal of Biological Chemistry, 292(11), 4383–4394. https://doi.org/10.1074/jbc.M116.763656

12. Francis, T. C., Gaynor, A., Chandra, R., Fox, M. E., & Lobo, M. K. (2019). The Selective RhoA Inhibitor Rhosin Promotes Stress Resiliency Through Enhancing D1-Medium Spiny Neuron Plasticity and Reducing Hyperexcitability. Biological Psychiatry, (20), 1–10. https://doi.org/10.1016/j.biopsych.2019.02.007

13. Fukata, M., Nakagawa, M., & Kaibuchi, K. (2003). Roles of Rho-family GTPases in cell polarisation and directional migration. Current Opinion in Cell Biology, 15(5), 590– 597. https://doi.org/10.1016/S0955-0674(03)00097-8

14. Fukuzawa, T., Ishida, J., Kato, A., Ichinose, T., Ariestanti, D. M., Takahashi, T., … Hirose, S. (2013). Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D. PLoS ONE, 8(7). https://doi.org/10.1371/journal.pone.0069451

15. Goupil, E., Fillion, D., Clément, S., Luo, X., Devost, D., Sleno, R., … Hébert, T. E. (2015). Angiotensin II type I and prostaglandin F2α receptors cooperatively modulate signaling in vascular smooth muscle cells. Journal of Biological Chemistry, 290(5), 3137–3148. https://doi.org/10.1074/jbc.M114.631119

16. Hall, A. (1998). Rho GTPases and the Actin Cytoskeleton10.1126/science.279.5350.509. Science, 279(5350), 509–514. Retrieved from http://www.sciencemag.org/cgi/content/abstract/279/5350/509

17. Harty, B. L., Krishnan, A., Sanchez, N. E., Schiöth, H. B., & Monk, K. R. (2015). Defining the gene repertoire and spatiotemporal expression profiles of adhesion G protein- coupled receptors in zebrafish. BMC Genomics, 16(1). https://doi.org/10.1186/s12864-015-1296-8

18. Jansing, N. L., McClendon, J., Henson, P. M., Tuder, R. M., Hyde, D. M., & Zemans, R. L. (2017). Unbiased quantitation of alveolar type II to alveolar type i cell

transdifferentiation during repair after lung injury in mice. American Journal of Respiratory Cell and Molecular Biology, 57(5), 519–526. https://doi.org/10.1165/rcmb.2017-0037MA

19. Juettner, V. V., Kruse, K., Dan, A., Vu, V. H., Khan, Y., Le, J., … Malik, A. B. (2019). VE- PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase- independent mechanism. The Journal of Cell Biology, 218(5), 1725–1742. https://doi.org/10.1083/jcb.201807210

20. Krishnan A., Nijmeijer S., de Graaf C., Schiöth H.B. (2016) Classification, Nomenclature, and Structural Aspects of Adhesion GPCRs. In: Langenhan T., Schöneberg T. (eds) Adhesion G Protein-coupled Receptors. Handbook of Experimental Pharmacology, vol 234. Springer, Cham

21. Kubo, F., Ariestanti, D. M., Oki, S., Fukuzawa, T., Demizu, R., Sato, T., … Nakamura, N. (2019). Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells. Respiratory Research, 20(1), 1–21. https://doi.org/10.1186/s12931-019-0973-6

22. La, X., Zhang, L., Yang, Y., Li, H., Song, G., & Li, Z. (2019). Tumor-secreted GRP78 facilitates the migration of macrophages into tumors by promoting cytoskeleton remodeling. Cellular Signalling, 60, 1–16. https://doi.org/10.1016/j.cellsig.2019.04.004

23. Li, L., Li, Y., Fan, Z., Wang, X., Li, Z., Wen, J., … Guo, J. (2019). Ascorbic Acid Facilitates Neural Regeneration After Sciatic Nerve Crush Injury. Frontiers in Cellular Neuroscience, 13(March), 1–17. https://doi.org/10.3389/fncel.2019.00108

24. Liu, M., Xiao, J., Li, Z., Wang, P., Wang, X., Qu, G., … Shi, T. (2013). GPR116, an Adhesion G-Protein-Coupled Receptor, Promotes Breast Cancer Metastasis via the G q-p63RhoGEF-Rho GTPase Pathway. Cancer Research, 73(20), 6206–6218. https://doi.org/10.1158/0008-5472.can-13-1049

25. Liu, Z., & Tjian, R. (2018). Visualizing transcription factor dynamics in living cells. Journal of Cell Biology, 217(4), 1181–1191. https://doi.org/10.1083/jcb.201710038

26. Lu, S., Liu, S., Wietelmann, A., Kojonazarov, B., Atzberger, A., Tang, C., … Offermanns, S. (2017). Developmental vascular remodeling defects and postnatal kidney failure in mice lacking Gpr116 (Adgrf5) and Eltd1 (Adgrl4). PLoS ONE, 12(8), 1–23. https://doi.org/10.1371/journal.pone.0183166

27. Mason RJ, Nellenbogen J, Clements JA. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J Lipid Res.

28. Mason, R. J., Lewis, M. C., Edeen, K. E., McCormick-Shannon, K., Nielsen, L. D., & Shannon, J. M. (2015). Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro. American Journal of Physiology-Lung Cellular and Molecular Physiology, 282(2), L249–L258. https://doi.org/10.1152/ajplung.00027.2001

29. McCarthy, C., Lee, E., Bridges, J. P., Sallese, A., Suzuki, T., Woods, J. C., … Trapnell, B. C. (2018). Statin as a novel pharmacotherapy of pulmonary alveolar proteinosis. Nature Communications. https://doi.org/10.1038/s41467-018-05491-z

30. Mohammadi‐Yeganeh, S., Hosseini, V., & Paryan, M. (2019). Wnt pathway targeting reduces triple‐negative breast cancer aggressiveness through miRNA regulation in vitro and in vivo. Journal of Cellular Physiology, (December 2018), jcp.28465. https://doi.org/10.1002/jcp.28465

31. Namkung, Y., Le Gouill, C., Lukashova, V., Kobayashi, H., Hogue, M., Khoury, E., … Laporte, S. A. (2016). Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nature Communications, 7, 12178. https://doi.org/10.1038/ncomms12178

32. Namkung, Y., LeGouill, C., Kumar, S., Cao, Y., Teixeira, L. B., Lukasheva, V., … Laporte, S. A. (2018). Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors. Science Signaling, 11(559), eaat1631. https://doi.org/10.1126/scisignal.aat1631

33. Namkung, Y., Radresa, O., Armando, S., Devost, D., Beautrait, A., Le Gouill, C., & Laporte, S. A. (2016). Quantifying biased signaling in GPCRs using BRET-based biosensors. Methods, 92, 5–10. https://doi.org/10.1016/j.ymeth.2015.04.010

34. Nestler EJ, Duman RS. Heterotrimeric G Proteins. In: Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999. Available from: https://www.ncbi.nlm.nih.gov/books/NBK28116/

35. Niaudet, C., Hofmann, J. J., Mäe, M. A., Jung, B., Gaengel, K., Vanlandewijck, M., … Betsholtz, C. (2015). Gpr116 receptor regulates distinctive functions in pneumocytes and vascular endothelium. PLoS ONE, 10(9), 1–25. https://doi.org/10.1371/journal.pone.0137949

36. Nie, T., Hui, X., Gao, X., Li, K., Lin, W., Xiang, X., … Wu, D. (2012). Adipose tissue deletion of Gpr116 impairs insulin sensitivity through modulation of adipose function. FEBS Letters, 586(20), 3618–3625. https://doi.org/10.1016/j.febslet.2012.08.006

37. Peng, G. E., Wilson, S. R., & Weiner, O. D. (2011). A pharmacological cocktail for arresting actin dynamics in living cells. Molecular Biology of the Cell, 22(21), 3986– 3994. https://doi.org/10.1091/mbc.e11-04-0379

38. Ren, T., Zheng, B., Huang, Y., Wang, S., Bao, X., Liu, K., & Guo, W. (2019). Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways. Cell Death and Disease, 10(4), 1–14. https://doi.org/10.1038/s41419-019-1497-1

39. Rindler, T. N., Stockman, C. A., Filuta, A. L., Brown, K. M., Snowball, J. M., Zhou, W., … Whitsett, J. A. (2017). Alveolar injury and regeneration following deletion of ABCA3. JCI Insight, 2(24). https://doi.org/10.1172/jci.insight.97381

40. Rutering, J., Ilmer, M., Recio, A., Coleman, M., Vykoukal, J., Alt, E., & Orleans, N. (2016). Characterization and Mechanism of (4S) Limonene. 5(6), 1–8. https://doi.org/10.4172/2157-7633.1000305.Improved

41. Saykali, B., Mathiah, N., Nahaboo, W., Racu, M.-L., Hammou, L., Defrance, M., & Migeotte, I. (2019). Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo. ELife, 8, 1–27. https://doi.org/10.7554/elife.42434

42. Schneberger, D., DeVasure, J. M., Kirychuk, S. A., & Wyatt, T. A. (2018). Organic barn dust inhibits surfactant protein D production through protein kinase-c alpha dependent increase of GPR116. PLoS ONE, 13(12), 1–14. https://doi.org/10.1371/journal.pone.0208597

43. Schoneberg, T.; Schulz, A.; Biebermann, H.; Hermsdorf, T.; Rompler, H.; Sangkuhl, K. Mutant G-protein-coupled receptors as a cause of human diseases. Pharmacol. Ther. 2004, 104 (3), 173−206

44. Schrage, R., Schmitz, A. L., Gaffal, E., Annala, S., Kehraus, S., Wenzel, D., … Kostenis, E. (2015). The experimental power of FR900359 to study Gq-regulated biological processes. Nature Communications, 6(May), 1–7. https://doi.org/10.1038/ncomms10156

45. Schrage, R., Schmitz, A. L., Gaffal, E., Annala, S., Kehraus, S., Wenzel, D., … Kostenis, E. (2015). The experimental power of FR900359 to study Gq-regulated biological processes. Nature Communications, 6(May), 1–7. https://doi.org/10.1038/ncomms10156

46. Tang, X. L., Wang, Y., Li, D. L., Luo, J., & Liu, M. Y. (2012). Orphan G protein-coupled receptors (GPCRs): Biological functions and potential drug targets. Acta Pharmacologica Sinica, 33(3), 363–371. https://doi.org/10.1038/aps.2011.210

47. Wang, W., Qiao, Y., & Li, Z. (2018). New Insights into Modes of GPCR Activation. Trends in Pharmacological Sciences, 39(4), 367–386. https://doi.org/10.1016/j.tips.2018.01.001

48. Yang, C., Ren, J., Li, B., Jin, C., Ma, C., Cheng, C., … Shi, X. (2019). Identification of gene biomarkers in patients with postmenopausal osteoporosis. Molecular Medicine Reports, 19(2), 1065–1073. https://doi.org/10.3892/mmr.2018.9752

49. Yang, L., Lin, X.-L., Liang, W., Fu, S.-W., Lin, W.-F., Tian, X.-Q., … Ge, Z.-Z. (2017). High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma. Oncotarget, 8(29), 47943–47956. https://doi.org/10.18632/oncotarget.18203

50. Yang MY, Hilton MB, Seaman S, Haines DC, Nagashima K, Burks CM, Tessarollo L, Ivanova PT, Brown HA, Umstead TM, et al. Essential regulation of lung surfactant homeostasis by the orphan G protein-coupled receptor GPR116. Cell Rep. 2013;3:1457–1464. doi: 10.1016/j.celrep.2013.04.019.