The Pennsylvania State University
The Graduate School
ANALYSIS OF NATURALLY OCCURRING GENETIC VARIANTS IN THE
SPHINGOSINE–1–PHOSPHATE RECEPTOR FAMILY
A Dissertation in
Biochemistry and Molecular Biology
by
Jacob T. Hornick
2019 Jacob T. Hornick
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
May 2019
ii The dissertation of Jacob Hornick was reviewed and approved* by the following:
James Broach Professor and Chair Department of Biochemistry and Molecular Biology Director Penn State Institute for Personalized Medicine Dissertation Advisor Chair of Committee
Ralph L. Keil Chair, Biomedical Sciences Graduate Program Associate Professor of Biochemistry and Molecular Biology
Ira Ropson Assistant Dean for Medical Student Research Associate Professor of Biochemistry and Molecular Biology
Richard Mailman Professor and Distinguished Senior Scholar of Pharmacology and Neurology
Thomas Spratt Director Biochemistry and Molecular Biology Graduate Program Professor Biochemistry and Molecular Biology
Charles Lang Associate Dean for Graduate Studies Distinguished Professor Department of Cellular and Molecular Physiology Distinguished Professor Department of Surgery
*Signatures are on file in the Graduate School
iii
ABSTRACT
The field of personalized medicine has significantly expanded with the advent of gene sequencing and the decreasing cost and increasing availability of whole genome sequencing data. This dissertation focused on a family of sphingosine-1-phosphate receptors, members of the G protein-1-coupled receptor (GPCR) superfamily. GPCRs, have been of interest because they are the largest superfamily in the human genome, occur across species, and play a diverse role in signaling and biological function. The sphingosine–1–phosphate receptor (S1PR) family has five members that are broadly expressed throughout human tissues and mediate signaling critical to vascular maturation, hair cell formation and repair, T and natural killer – cell trafficking and function. My goal was to examine how naturally occurring genetic variants affected
S1PR function and signaling capabilities.
For the analysis, I have developed a yeast model system adapting the yeast,
Saccharomyces cerevisiae, pheromone signaling pathway to permit a simple and elegant system to perform single receptor analysis. For example, the S1PR1 variant
R13G leads to an increase in potency (EC50) of S1P when compared to the wild type receptor. In S1PR2, the R60Q variant, decreases the potency of S1P compared to the wild type receptor. Most interestingly, the S1PR5 variant L318Q had little effect on S1P signaling mediated by Gαi2–chimeric G protein, yet abolished signaling via Gα12- chimeras.
To begin to translate these findings, human cells either transiently or stably transduced with S1PR5 and the L318Q variant were studied. Compared to the wild type
S1PR5, the L318Q variant caused decreased f-actin accumulation and less stress fiber and filopodia formation when treated with S1P. This region in S1PR5 was predicted to
iv be post–translationally modified with the addition of a palmitoyl group at two cysteine residues at positions 322 and 323. Acyl–biotin exchange experiments confirmed the
S1PR5 L318Q variant, unlike the wild type receptor, is unable to be palmitoylated. To test if the cysteine residues were essential for the Gα12 interaction, the C322A and
C323A double mutant was created. This receptor replicated the unusual G protein coupling pattern observed in L318Q receptor.
I also performed experiments here where the variant receptors were examined for any dysfunction when treated with current and experimental drugs for relapsing remitting multiple sclerosis (RRMS). I discovered minimal effects caused by the variants compared to the signaling via activation of S1P, however I do observe significant differences between the drugs when comparing potency and efficacy. A major finding from those studies is that the experimental drug RPC1074 displays higher efficacy and potency of S1PR1 signaling when compared to the current standard treatment of fingolimod. Examination of the other drugs ponesimod, siponimod, and MT1303p showed similarities in signaling effects when compared to fingolimod.
One important finding of this work was discovered when I examined the allelic distribution of the S1PR L318Q polymorphism and found it to be highly enriched in South
Asian populations compared to the average population frequency. I also observed an enrichment of another S1PR variant, S1PR1 A11D, in people of African descent. Since
S1P metabolism and signaling has been found to be involved in both resistances to and survival from malarial infection, I hypothesize that the L318Q variant by affecting the expression of S1P lyase, an important mediator of S1P metabolism, may affect the response to malarial infections. These ideas are worthy of future experimentation.
v TABLE OF CONTENTS
LIST OF FIGURES ...... viii
LIST OF TABLES ...... x
ABBREVIATIONS ...... xi
ACKNOWLEDGEMENTS ...... xii
Chapter 1 Introduction ...... 1
Personalized Medicine in the Age of Genomic Sequencing ...... 1
Personalized vs Precision Medicine ...... 1
Pharmacogenomics and Personalized Medicine ...... 5
GPCR Variants and Disease ...... 7
G Protein-Coupled Receptor Signaling ...... 8
Intracelluar G Protein Signaling ...... 11
Yeast Pheromone Pathway and Applications for Personalized Medicine…...13
Post-Translational Modifications of G Protein-Coupled Receptors ...... 16
Glycosylation ...... 17
Phosphorylation...... 18
Ubiquitination...... 19
Palmitoylation...... 19
Sphingosine-1-Phosphate Signaling System...... 21
S1PR1...... 22
Targeting S1PR1 in Relapsing Remitting Multiple Sclerosis...... 24
S1PR2...... 24
S1PR3...... 25
S1PR5...... 26
vi
Summary...... 28
Chapter 2 Natural Human Genetic Variants Affect Efficacy and Coupling of Sphingosine-1-Phosphate Receptors ...... 29
Preface...... 29
Introduction...... 29
Results ...... 33
Some Naturally Occurring Variants of S1P Receptors Exhibit Altered Ligand Response ...... 33
S1PR5 Variant L318Q Fails to Activate Gα12/13...... 36
S1PR5 L318Q Normally Signals through Gαi...... 39
The L318Q Variant Eliminates Receptor Palmitoylation...... 40
S1PR5 L318Q Variant Does Not Exhibit Defective Localization...... 41
Elimination of Receptor Palmitoylation Sites Alters Gα Coupling...... 42
S1PR5-L318Q and S1PR1-A11D are Under Positive Selection in Certain Populations...... 45
S1PR5-L318Q Fails to Induce Expression of SGPL1...... 45
Discussion...... 48
Chapter 3 Yeast as Platform for Multiple Sclerosis Drug Analysis and S1P Receptor Antagonists...... 51
Preface...... 51
Introduction...... 51
Results...... 54
Minimal Differences of Activity in S1P Variants Treated with Fingolimod...... 54
Ponesimod Exhibits Altered Signaling in S1PR5 L318Q Variant...... 56
Siponimod Exhibits Selectivity for S1PR1...... 58
MT1303p Exhibits S1P-Like Signaling Activity...... 59
vii
Selectivity and Activity of RPC 1074 Shows Promise for RRMS Treatment..61
Discussion...... 63
Chapter 4 Conclusions and Future Directions ...... 68
Appendix A Yeast as a Platform For Drug Discovery ...... 76
Preface...... 76
Introduction...... 76
Results...... 78
Spectrum2000 High Throughput Screening...... 78
Molecular Modeling to Aid in Compound Selection...... 80
Candidate Drugs Show S1PR5 Selectivity...... 81
Discussion...... 82
Appendix B Methods and Materials ...... 84
References...... 91
viii LIST OF FIGURES
Figure 1-1. From a diseased population to personalized medicine ...... 2
Figure 1-2. Schematic of the general pheromone response pathway of Saccharomyces cerevisiae ...... 15
Figure 2-1. Saccharomyces cerevisiae system to analyze signaling dysfunction of variants in the S1P receptor family ...... 34
Figure 2-2. Loss of Gα12 coupling is observed in S1PR5 L318Q variant utilizing HIS3 induction system ...... 36
Figure 2-3. S1PR5 WT and L318Q transiently transfected HEK293T cells show significant differences in f-actin accumulation after 1 hour of S1P treatment ...... 38
Figure 2-4. S1PR5 L318Q variant exhibits minimal F-actin accumulation after treatment with S1P ...... 39
Figure 2-5. Gαi signaling via Akt phosphorylation of S1PR5 is observed in the L318Q variant ...... 40
Figure 2-6. Post translational modification of S1PR5 L318Q is altered compared to S1PR5 WT ...... 42
Figure 2-7. S1PR5 L318Q localization is not altered...... 43
Figure 2-8. Cysteine residues 322 and 323 are evolutionarily preserved and mutation to alanine replicates the S1PR5 L318Q coupling phenotype ...... 44
Figure 2-9. S1PR5 L318Q fails to induce SGPL1 expression compared to S1PR5 WT ...... 46
Figure 3-1. S1PR1 and S1PR5 Receptor specificity during Fingolimod treatment ... .56
Figure 3-2. Ponesimod exhibits lower efficacy in signaling through S1PR5 L318Q compared to WT ...... 57
Figure 3-3. Siponimod exhibits S1PR1 selectivity ...... 59
Figure 3-4. MT1303 has minimal to no receptor activity ...... 60
Figure 3-5 MT1303p has higher efficacy in S1PR5 WT and L318Q compared to S1PR1 ...... 61
Figure 3-6 RPC1074 has high efficacy in S1PR1 and S1PR5 and exhibits binding with S1PR2 ...... 62
ix Figure A-1. Molecular docking aids in confirmation of potential S1PR5 inhibitors .... .80
Figure A-2. Select S1PR5 inhibiting molecules exhibit S1PR5-selectivity ...... 82
x LIST OF TABLES
Table 2-1. Published and experimental EC50 and Emax values for S1P receptors and variants ...... 35
Table 2-2. S1PR1 A11D and S1PR5 L318Q variants exhibit population specific enrichment of causative minor alleles ...... 47
Table 3-1. EC50 and Emax normalized to S1P for S1P receptors and variants of RRMS drugs ...... 55
Table A-1. Screening of Spectrum2000 molecule set yields potential S1PR5 specific antagonist compounds ...... 79
Table B-1. Genotype of strains used for yeast coupling and luciferase assays...... 84
xi ABBREVIATIONS
ApoM apolipoprotein M BBB blood brain barrier cAMP cyclic-adenosine monophosphate CDK cyclin dependent kinase CML chronic myelogenous leukemia ECL extracellular loop ExAC Exome Aggregation Consortium GBM glioblastoma GDP guanosine diphosphate GnomAD Genome Aggregation Database GPCR G protein-coupled receptor GTP guanosine triphospate GWAS genome wide association study Gα G protein alpha Gβγ G protein beta gamma HAM hydroxylamine HDL high density lipoprotein ICL intracellular loop IFNγ interferon gamma KSR kinase suppressory of Ras LGL large granular lymphocyte MAF minor allele frequency MAP mitogen-activated protein mTOR mammalian target of rapamycin NDI nephrogenic diabetes insipidus NEM N-Ethylmaleimide NK natural killer nM nanomolar NMR nuclear magnetic resonance PKC protein kinase C PKU phenylketonuria PP1/2A protein phosphatase 1/2A PTM post-translational modification relapsing remitting multiple RRMS sclerosis S1P sphingosine-1-phosphate SHH sonic hedgehog SNP single nucleotide polymorphism T2D Type II diabetes TM transmembrane domain
xii uM micromolar ACKNOWLEDGEMENTS
First and foremost, I would like to thank my family for support through the time
I’ve spent in Hershey. My mom and dad who instilled in me at a young age a love of science and nature, which has been paramount for my path in life and though may not always understand the science, but always understood what I was and am capable of and knew how to help me visualize and achieve the goals I set for myself. My brother and sister who are so talented and driven have been such inspirations to me without them even knowing. My extended family, who I can always count on to have a cold beverage waiting for me in the garage and a warm embrace when I was lost. To Emily and Nancy, for being the best of friends when I needed them. And to my partner Kelsey, for being my rock and ultimate supporter, without her I would certainly have failed to finish this work. Without the support of my family and loved ones I’m convinced I would have ended this endeavor and regretted it many years down the road.
To Jim, thank you for the opportunity to work with you and for all of the years of support and the understanding only a truly great mentor could give to a student who may not have always known what he wanted to do in life. To my lab mates, past and present,
I thank you so much for the input and being a shoulder and an ear when I needed it most. From the midafternoon Troegs meetings with Mark to the cave discussions with
Vasudha, Kim, Mariano, and Joel, I thank you for always being a strong support system and making the many years I’ve been in school seem so short. To Kenny, Jess, and
Scott, the lab managers through the years, thank you for the help when I needed it.
A special thank you is due to the other mentors in my life, Robbie Iuliucci and
Ralph Keil. Robbie, you helped me to find my passion and Ralph, you helped me to
xiii realize it was ok to strive to do what I love as a profession. Without either of you, I genuinely don’t know what I would be doing with my life.
To the administrative staff of the Biochemistry Department and the BMS, thank you for helping me navigate the years of changes and requirements I needed to get to the end. Finally, thank you to the Penn State Hershey Department of Biochemistry and
Molecular Biology for the opportunity to study and become a problem solver and scientist. Thanks to the NIH for funding of my research.
Chapter 1
Introduction
Personalized Medicine in the Age of Genomic Sequencing
Personalized medicine in the modern age has been driven by the ever lowering cost of DNA sequencing and the understanding of how small genetic variation between individuals can have extensive repercussions when it comes to treatment options.
Although some may use personalized and precision medicine interchangeably, others feel there are some differences in the origins. Separating personalized from precision medicine has become even harder as the lines between individual treatments and treatments based upon population studies evolve and elucidate more important information every day. The work presented in this dissertation offers small but impactful findings that contribute to personalized medicine. To begin understanding why these studies are essential we first must understand the history and evolution of personalized medicine through modern day.
Personalized vs precision medicine
Personalized medicine has somewhat evolved from the concept of precision medicine but amplified by the specificity of the genetics and/or environment of the patient. In precision medicine, the ultimate goal is identification and treatment avoiding misdiagnosis wherein the patient is treated correctly at the initial diagnosis leading to an improved outcome (Ashley, 2016). Currently the definition of personalized medicine centers on the application of therapeutic approaches for an individual, rather than a population or group of individuals (specifically a subset of people with the same
2 disease/disorder), based on that person’s genetic composition (Ashley, 2016). In Figure
1-1 is a generic flow chart diagramming the movement from a diseased population to a targeted treatment based on the physiology of the cells affecting the disorder and finally how a persons’ genetics can affect the treatment outcome. As long as humans have been treating medical disorders, we have sought to differentiate diseases to improve treatments. One of the early examples of personalized medicine is the use of arsenic to treat leukemia, specifically chronic myelogenous leukemia (CML) in the early 1900’s
(Antman, 2001). Because this treatment worked for a few cases it was adopted as a universal remedy for those patients exhibiting leukemic characteristics, however this treatment was only effective for ~ 20% of patients. Interestingly this use of arsenic based drugs for leukemia treatment has continued and is a standard treatment for patients with acute promyelocytic leukemia (Lo-Coco et al., 2013). The discovery of the many types of leukemia lead to the treatment modifications through the years.
Techniques for diagnosis are ever improving and contributing to better patient diagnosis.
Figure 1-1. From a diseased population to personalized medicine. In personalized medicine the goal is to move from a diseased population (A) to disease subtypes (B) through diagnostic analysis to finally creating specified treatments (C) for patients based on the patients’ having or not having specific genetic composition.
3 Many diagnostics used today in current medical analysis are geared toward this outcome, to correctly identify the patients’ ailment for expedient and effective treatment.
Use of metabolomics, proteomics, and highly sensitive imaging equipment has been pivotal to the improvements to precision medicine seen in the recent decades (Li et al.,
2017a). Analysis using mass spectrometry, nuclear magnetic resonance (NMR) imaging, and simple protein analysis via electrophoresis allows for metabolomics to aid in the identification of different diseases such as phenylketonuria (PKU), diabetes, and cancer (Nagana Gowda et al., 2008). Sadly, due to the inescapable difficulty of diagnosis, there is still much work to be done in honing precision medicine to correctly identify and treat the disease from the first onset of symptoms.
Metabolomics allows for the analysis of the over 100,000 components that make up the human metabolome for a physiological snapshot of the human body (Wishart et al., 2007, 2018). In addition to these classical metabolic analysis, oncologists can use cell-surface markers to determine type and treatment susceptibility for complex cancers.
Large granular lymphocyte (LGL) leukemia, which is intimately tied to sphingosine metabolism, is divided into T and natural kill (NK) cell types along with acute and chronic subtypes beyond the cell type (Lamy et al., 2017). In general, LGL leukemia presents with characteristics of enlarged lymphocytes with splenomegaly, neutropenia, and sometimes lymphopenia. However, classification into T or NK requires flow cytometry analysis to distinguish between cells (Sokol and Loughran, 2006) . Unfortunately, the acute subtype of either T or NK – LGL leukemia are treatment resistant, and no viable treatment is currently available (Nazarullah et al., 2016). Work presented in Appendix A aims to find a potential treatment aimed at acute NK cell LGL leukemia. The addition of genomic sequencing has also boosted the quality of precision medicine patients
4 currently experience and the addition of this relatively new technology is where precision medicine transitions to personalized medicine.
Medical professionals specializing in multiple sclerosis (MS) have high incidence of misdiagnosis, upwards of 25%, and sometimes aren’t able to correctly identify the patient as suffering from MS for longer than five years after their initial referral (Hansen and Okuda, 2018). These misdiagnoses are physically and psychologically harmful to patients as they worry and stress over what is the source of their illness, and the fact they cannot seem to get healthier, regardless of patients’ effort to obtain treatment.
Although there are numerous treatments for MS, if doctors are not correctly identifying the cause of the patients’ disorder, then those available treatments are moot. These types of multi-symptom diseases require more sophisticated analysis to delineate and determine the exact ailment for treatment in the clinic.
Driving the use of genomics in personalized medicine has been the ability to identify naturally occurring genetic variation between humans from inherited to de novo variants within families. Initial discoveries of these single nucleotide polymorphisms
(SNPs) were done largely through the implementation of genome wide association studies (GWAS) to identify regions in the genome responsible for a specific phenotype.
Currently there have been over 3600 GWASs completed looking at various disease states in an effort to identify a genetic fingerprint for further investigation into the origin of the disease of interest (NHGRI-EBI GWAS Catalog). From these studies over 70,000
SNPs have been compiled with implications in multitudes of phenotypes. These initial studies have created a platform that we have built on here to investigate how common variants in a biologically important G protein coupled receptor (GPCR) family may or may not affect biological function. However, GWAS certainly have limitations. The analysis, as it’s names indicates, identifies genomic regions associated with the specific
5 trait in question. The research presented in this dissertation seeks to achieve the basis for a pipeline of reverse genetic studies wherein I assess common variants of GPCRs for altered biological functions and determining the mechanism of dysfunction. By doing this, I certainly found natural variants having no or minimal functional relevance, however, as will be discussed, I have also identified naturally occurring variants causing drastic alterations to the receptor function and may in fact contribute to a positive selection for some ethnic backgrounds. Investigating of the pharmacological consequences of receptor variants is also critical to personalized medicine and has created the field of pharmacogenomics.
Pharmacogenomics and Personalized Medicine
With the access, ease of implementation, and decreasing cost of genetic sequencing and high-throughput analysis, the ability to analyze the interactions of pharmacological agents and the target protein variants led to the creation of a new scientific field, pharmacogenomics. Creating a comprehensive assessment of how a pharmacological agent interacts with its target, and possible variant/mutant targets, is critical to the high specificity required of personalized medicine. The combination of pharmacology and genomics to create pharmacogenomics has added an extra layer to personalized medicine. It is a critically important facet of personalized medicine because of the information gained on how to treat disease and how a patient’s genetic variation in the drug target may affect potential treatment options. The research presented in this dissertation is driven by the principles of pharmacogenomics.
One method to assess the biological consequences of GPCR variation is the use of in silico modeling of variants based on the domain and sequence location within the receptor. As will be described later, all GPCRs share similar functional domains with specific and general functions based on the amino acid sequence at those regions. Kim
6 et al performed an analysis of 367 GPCRs from the five GPCR families for protein domains (N-terminus, TM1-7, ICLs, ECLs, C-terminal helix, and C-terminal tail), the total number of variants, and the number of receptors with variants in the specific domain.
Variation in all GPCRs analyzed fell in the N-terminus, C-terminal tail, and transmembrane domains (Kim et al., 2018). However, the regions where few non- synonymous variations were observed were the extra (ECLs) and intracellular loops
(ICLs). This is likely due to the importance of those regions to receptor-ligand and receptor-G protein interactions, respectively. Interestingly, the C-terminal helix, Helix 8, exhibits lower natural variation than any other helix in the average GPCR, possibly indicating an evolutionarily conserved functional importance. Those results provide a map to guide investigations into which regions of the protein induce the functional effects of non-synonymous natural variants.
Considering over one third of small molecule compounds targets GPCRs for treatments of disease, the importance of monitoring how genetic differences alter the effects of prescribed drugs on those receptors is critical to successfully treating patients
(Santos et al., 2017). Certain variants can contribute to economic and societal burden on patients having drug targeted receptors with altered drug function. An individual can have around 68 missense variants within the coding region of a third of all drug targeted
GPCRs (Hauser et al., 2018). These numbers have provided a significant impetuous for other researchers to thoroughly examine the effects of variants on GPCR biology. It was also determined that de novo variants in drug targeted GPCRs occur in one out of every
300 births, meaning every year there are 13,000 new variants in targeted GPCRs appearing in the United States alone, considering just under 4 million babies born in
2016 (CDC.gov, 2018; Hauser et al., 2018). This would equate to de novo missense variants occurring at a rate of 0.00003 per drug targeted GPCR per person,
7 approximately 2.3 times higher than would be seen for an average protein coding region with a rate of 0.000013 missense variants per gene per person (Turner et al., 2017).
Although this is a minor increase, the rate of de novo missense mutations occurring is higher than would be expected and indicates GPCRs are sites of high genetic diversity.
Analyzing the current and future variants for functional consequences is of high importance and it must be approached in a highly collaborative manner.
GPCR Variants and Disease
A question many people studying personalized medicine must address is how one achieves the end goal of helping the patient. Disease prediction and treatment can be viewed in a shared lens because potential treatment information can be gleaned from disease prediction. My work is aimed to bridge this divide utilizing a multi-model- organism system to create a network of information to identify how variants affect GPCR biology and ultimately treatments targeting receptors. This endeavor is based on years of pharmacogenomics and personalized medicine studies of which provide a great amount of information for analysis and interpretation of data for the work done in this dissertation. A recent study performed a meta-analysis looking at the different families of receptors, the numbers of variants and how many lead to functional effects. To date, only 218 variants in 41 GPCRs have been studied for the effect on receptor biology from out of the 1440 total non-synonymous variants from 269 receptors (Kim et al., 2018).
That means only 15% of all currently known non-synonymous variants have been studied for deleterious effects and there is a significant need to examine the remaining
85%.
Many common diseases, like heart disease, are caused or influenced by a multitude of factors leading to many different patient presentations and outcomes. Other disorders can be related to a signaling pathway where the disease appearance is
8 similar, but the genetic cause could be due to a failure of function in a set of proteins, such as retinitis pigmentosa and xeroderma pigmentosum, where the rhodopsin signaling, a well-studied GPCR pathway, or DNA repair pathway, respectively, are altered resulting in the disease state. However other disorders are monogenic including cystic fibrosis. One such monogenic disease caused by a GPCR variant is nephrogenic diabetes insipidus (NDI) caused by single amino acid changes in the vasopressin V2 receptor H80R, S167K, and C319Y each of which result in impaired receptor localization and ligand binding and cause excessive urination, thirst, and potential for dehydration
(Wüller et al., 2004). Although these variants are located in different transmembrane
(TM) regions of the receptor, TM2, TM3, and TM7, respectively, each confer dysfunction and lead to NDI. Instances like these are why research like the work presented here is important to gain a global understanding of the potential defects natural occurring variants could be contributing to human disease states. GPCRs, as will be detailed, are a very large super-family of proteins and are ubiquitously expressed throughout human cells. GPCR signaling is also critical to yeast biology. This fact has been utilized for the creation of a yeast-human but before I can describe the yeast pheromone response pathway, understanding the components of GPCR signaling cascade is needed.
G Protein-Coupled Receptors
GPCRs constitute one of the largest super-families of proteins in the human body. The receptors were originally described as a transmembrane protein comprised of an extracellular N-terminus with seven transmembrane (7TM) helices ending with an intracellular C-terminus usually containing an eighth alpha helix. The N-terminus is critical for many GPCR functions including interacting with ligands and other receptors/proteins as well as being a site of post-translational modifications for proper receptor function in the extra-cellular space. The C-terminus of many GPCRs contains
9 another alpha helix, commonly referred to as helix-8. For some receptors this extra helix is critical for regulating cellular localization, a site of post-translational modification, and modulating interactions with another known intracellular signaling protein, β-arrestins.
Originally GPCRs were classified into seven different families, but are now reclassified into five major groups of receptors (Fredriksson et al., 2003). An acronym based on Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin families the classification was termed GRAFS. Rhodopsin-like receptors make up almost 80% of all
GPRCs with around 670 members, and are the most well studied family (Gloriam et al.,
2007). The family is called rhodopsin-like because the structures resemble that of rhodopsin, the first structurally determined GPCR (Ovchinnikov, 1987). Rhodopsin-like family members most commonly have short N-termini although with some exceptions
(Lagerström and Schiöth, 2008). Structurally the S1P receptors are unique because the
N-terminus contains an alpha helix forming a cap over the central pocket of the receptor, and it is believed to play a role in how S1P is bound to the receptor (Hanson et al.,
2012). Olfactory receptors make up a large portion of the rhodopsin-like receptor family with ~ 400 of the almost 700 rhodopsin-like members (Gloriam et al., 2007). Olfactory receptors are an interesting subgroup of receptors because of the wide range of molecules the receptors are known to be activated. A single receptor can bind multiple odorants, and single odorants can bind multiple receptors creating a highly complex and plastic system (Malnic et al., 1999).
Secretin, Adhesion, Glutamate, and the Frizzled/Taste2 receptor families make up the remaining 20% of all GPCRs expressed in the human genome. The Secretin family is comprised of 15 receptors with ligands comprised of peptides and proteins, and specifically hormones such as calcitonin, growth-hormone, and parathyroid hormone
(Harmar, 2001). The Adhesion family of receptors comes contains the second most
10 receptors with 33 in the group (Foord et al., 2005). As the family name suggests, these receptors are important regulators of cellular interactions. A hallmark of this family is the many glycosylation patterns and residue diversity of N-termini both of which are important for the receptor family functions in the extra-cellular space (Baud et al., 1995;
Bjarnadóttir et al., 2004). The metabotropic glutamate family, comprised of 22 receptors, is targeted by ligands of amino acids, small organic molecules, ions, and carbohydrates.
These receptors are known to bind the ligands via the N-terminus and not within an inner-protein binding pocket, like rhodopsin type receptors. The glutamate family of receptors are essential to various functions including neurotransmission, learning and pain sensation (Ohashi et al., 2002). Finally the Frizzled/Taste2 family technically has
11 frizzled/smoothened and 25 Taste2 receptors in the combined class of GPCRs
(Lagerström and Schiöth, 2008). Frizzled and smoothened receptors were first identified in Drosophila melanogaster (fruit fly) (Vinson et al., 1989). This discovery showed the receptor family was an essential component of cellular and embryonic development via the Wnt signaling pathway for Frizzled and the sonic hedgehog (SHH) pathway for
Smoothened (Bhanot et al., 1996; Murone et al., 1999). Taste2 receptors, as indicated by the name, are involved in the sensation of taste and originally of bitterness, an essential evolutionary trait for aversion to poisonous plants in early hominids, potentially helping to explain the significant variation within receptor sequences of members in the family (Lagerström and Schiöth, 2008).
Understanding how each receptor functions and interacts with the respective ligands is essential to developing and/or identifying compounds with modifying or antagonistic activity for targeting receptors for treatment of potential diseases. Even though the sequence and biological functions have been known for many decades with the first identification of receptors in the early 1900’s and the first radio-ligand
11 experiments performed in the 1960’s it is only in the last 2 decades GPCR structure resolution has been possible (Hill, 2006) . The GPCRs are amphipathic transmembrane proteins, making it difficult to make the preparations required for structural studies by x- ray crystallography and NMR. The first GPCR structure from the rhodopsin family was determined only in 2000 (Okada et al., 2000). Recent advances in both solubilization and imaging of receptors has allowed for the determination of 42 individual GPCR structures, most of which are from the Rhodopsin – like family (Gacasan et al., 2017).
Obviously, this means there are hundreds of others yet to be resolved, however, due to the receptors previously described amphipathic nature, obtaining structures of all receptors will be a difficult task.
Intracellular G Protein Signaling
Even though there are hundreds of GPCRs, adding to the complexity of signaling are the intracellular signaling G-proteins. The main intracellular signaling complex is the heterotrimeric G protein. Comprised of an α, β, and γ subunit, these complexes interact with the intracellular loops and C-terminus of the GPCR. The specificity of the GPCR-G protein interaction is determined by the subtype of the Gα subunit. The four families of
G proteins are the Gαs, an activator of cAMP production, Gαi/o, leading to inhibition of cAMP production and neuronal specific signaling, Gαq/11, known to initiate activation of phospholipase C and regulate calcium homeostasis, and the Gα12/13 G proteins, primary regulators of cytoskeletal rearrangement and sodium/hydrogen exchange.
With four groups and 18 genes coding for G proteins, the GPCR-G protein system is one of the most complex signaling systems in the body. Contributing to this is the fact GPCRs can also exhibit promiscuity in G protein interactions. By binding to multiple Gα family subtypes this makes the signaling context extremely important to maintaining proper pathway activation inside specific cell types. Promiscuity within the
12 system mostly correlates with a single family of G proteins. Typically, if a GPCR is known to couple to the Gα12/13 family of G proteins, there is high probability the receptor exhibits promiscuous G protein signaling (Choi, 2018). This enrichment for promiscuity by Gα12/13 coupling GPCRs is most likely due to the ubiquitous expression of the Gα12/13 family of G proteins. The family has been found to interact and influence a wide range of downstream signaling cascades and biological pathways. Many are related to cytoskeletal rearrangement and cell motility such as cell adhesion, proliferation, and stress fiber formation via activation of the GTPase RhoA. The Gα12/13 family has been a critical protein family to this research as it is a central focus in the S1PR5 variant studies in Chapter 2.
Gα12/13 was originally discovered in mice and there is 67% shared homology between 12 and 13 with less than 50% homology to the other G proteins (Strathmann and Simon, 1991). Gα12/13 is pertussis and cholera toxin insensitive, while Gαi is pertussis toxin sensitive and Gαs is cholera toxin sensitive. The sensitivities of Gαi and
Gαs has allowed for analysis of downstream signals that are specific to the Gα12/13 cascade. In addition to having a relatively broad biological action, Gα12/13 over- expression contributes to uncontrolled cell growth and is overexpressed in some breast, oral, esophageal, and colon cancers. However, compared to other G proteins, there are very few Gα12/13 inactivating mutations in cancers (O’Hayre et al., 2013).
Another important class of intracellular proteins, related to GPCR function, are arrestins. Comprised of four subtypes, arrestins are critical for preventing GDP-bound G protein rebinding to activated GPCRs. Arrestins bind to the phosphorylated C-terminal tail of the activated GPCR therefore preventing further propagation of GPCR signal
(Kang et al., 2015). In addition to preventing further propagation signal, arrestins mediate internalization and GPCR sorting from the endosomes (Moore et al., 2007).
13 Whether the GPCR is recycled back to the plasma membrane for further signaling or sent for degradation depends on the specific GPCR-arrestin interaction (Oakley et al.,
2000; Zhang et al., 1999).
Along with mediating cellular localization of GPCRs post-activation, arrestins, specifically β-arrestin 1 (βarr1) and β-arrestin 2 (βarr2), modulate intracellular signaling cascades. βarr, along with G proteins, mediate ERK signaling pathways, although in a mechanistically different manner (Smith and Rajagopal, 2016). Also, the activation of p38 MAPK is mediated by βarr, specifically βarr2 (Sun et al., 2002). The signaling of
βarr can be preferentially activated by certain ligands, leading to βarr-biased signaling.
The drug carvedilol, a β2AR inverse agonist, mediates activation of ERK, but in a Gαs independent, βarr2 dependent manner (Wisler et al., 2007). This dissertation does not address the potential implications of βarr function, but it is an important component of
GPCR biology.
Yeast Pheromone Pathway and Applications for Personalized
Medicine
The common baker’s yeast, Saccharomyces cerevisiae, has been a model organism for the study of eukaryotic cell and molecular biology. Yeast have a well annotated genome as it was the first eukaryotic cell to have the entire genome sequenced, and have high genetic and proteomic overlap to human cells. Yeast express just over 6000 genes, of which 20% are orthologous to disease causing genes in humans. This high similarity combined with yeast’s ease of growth and manipulation for biological assays make it ideal for high-throughput assay drug development.
Recently the Boeke lab has succeeded in creating artificial yeast chromosomes where genes are rearranged and fused with the goal of creating an entire functioning synthetic genome (Mitchell et al., 2017; Wu et al., 2017). Another lab has succeeded in creating a
14 yeast strain with a single chromosome that behaves and grows in a very similar manner to a wild type laboratory strain (Shao et al., 2018). These advances are pushing the boundaries of what questions can be asked and how the basic building blocks of life are organized to create a single cell organism and give insight into more complex biology like that of humans. Before these perfectly annotated and genetically manicured yeast are available to be used for translational medicine, I have to utilize the yeast in its natural state.
The yeast pheromone response is a GPCR activated MAP kinase cascade that results in halting of the mitotic processes as cells prepare for fusion and mating. Yeast can access two sexual states, a and alpha (α), when in the haploid state. Cells that are a release an a – factor and α release α - factor. These mating factors bind two GPCRs
Ste2 (α) and Ste3 (a) activating the downstream cascade via the βγ subunit of the heterotrimeric complex (Figure 1-2). After binding the specific mating factor, the
Ste4/Ste18 complex (Gβγ complex orthologue) is released from Gpa1 (Gαi orthologue)
G protein alpha. This induces the activation of the MAP kinase cascade by Ste20 via activation by Cdc42. Following Cdc42 activation there is downstream phosphorylation and activation of the MAPKKK, Ste11, which is bound to the essential scaffolding protein, Ste5. This scaffolding protein is required for pheromone response induction and contributes to the specificity of the pathway signaling. There is subsequent activation of
Far1, a cyclin dependent kinase (CDK) inhibitor, leading to the halt of mitotic processes and induction of cytoplasmic rearrangement leading to shmooing, where the yeast physically stretches toward regions of higher concentration of mating factor. Yeast will
15
Figure 1-2. Schematic of the general pheromone response pathway of Saccharomyces cerevisiae. Binding of mating factor, a or α, to the Ste2 (α) or Ste3 (a) GPCR causes release of the Ste4/Ste18, yeast βγ complex, and activation of a MAPK cascade inducing cell cycle arrest and morphological changes wherein the yeast begins to stretch the cell membrane toward the gradient of mating factor and activates transcription of genes involved in the mating of yeast. continuously monitor the gradient of mating factor, relocating mating factor receptors to the region in the membrane where the factor concentration is highest. This ensures the yeast cell direction is always towards the other cell in the vicinity.
I harnessed this tightly regulated signaling pathway for my own uses and created yeast strains unable to shmoo and still able to progress through the cell cycle and divide normally and harness a luciferase construct driven by the pheromone response element
16 (PRE) pFUS1. Those changes are described in more detail in Chapter 2. Other researchers have accomplished this previously, however the use of a plasmid-borne luciferase expression is unique to my system. Yeast are an ideal candidate organism for the study of these variant effects because they are known to have only two GPCRs pathways. In addition to the Ste2/Ste3 mating factor sensing receptors, Gpr1, a glucose sensing GPCR, is vital to survival for detecting of one of the main food sources for yeast
(Liu et al., 2016). Because of the lack of diverse GPCRs in yeast there have been studies harnessing the pathway to study the functional domains of GPCRs such as probing for allosteric modulating sites, domain conservation, and novel signaling modulators. Studies like these have been done investigating the adenosine, somatostatin, muscarinic acetylcholine, neurotensin, and olfactory receptors to name a few (Liu et al., 2016). The use of a yeast based reporter system is not necessarily novel, but our application as a screen for discovering dysfunction and utilizing known receptor functions for probing mechanisms associated with altered signaling due to naturally occurring variants, many of which have no known biological perturbation or disease is quite novel, and offers a new avenue for what these types of yeast systems of capable to analyze.
Post-Translational Modifications of G Protein-Coupled Receptors
A major component of protein biology, post-translational modifications (PTMs) can affect a multitude of protein functions, both inter- and intra-protein interactions, activation status, localization, and whether or not a protein is degraded or recycled. A major post-translational modification, palmitoylation, is a major factor of S1PR5 biological function.
17 Glycosylation
Glycosylation is most commonly associated with proteins with activities and location in the extra-cellular matrix and varying regions of the cellular membrane.
Glycosylation is the covalent addition of single, or chains of, sugars to certain residues through, most commonly, N or O-linked modification. The amino acid residue asparagine is required for the N-linked glycosylation of proteins, while O-linked glycosylation occurs through the hydroxyl groups of serine, threonine, tyrosine, and the rarer hydroxylysine and hydroxyproline. Glycosylation is an important post-translational modification for a few key functions, especially for Adhesion-type GPCRs.
Down regulation of the β1 adrenergic receptor (β1AR), critical for cardiac function, has been shown to be mediated by N-terminal cleavage (Hakalahti et al.,
2010). This cleavage is regulated by O-linked glycosylation of the N-terminus (Goth et al., 2017). The β2AR exhibits activity in a homodimer form, which is regulated through N
– glycosylation of asparagine residues at positions 6 and 15 of the N-terminus (Li et al.,
2017b). As previously stated GPCRs can exhibit G protein coupling promiscuity in the downstream signaling pathways they activate. The N-linked glycosylation of the extracellular loop 2 (ECL2) of the protease-activate receptor 1 (PAR1) mediates the selective coupling to Gαq and Gα12/13 family of G proteins. Glycosylation induces preferential signaling through Gα12/13 compared to unglycosylated PAR1, which prefers signaling through Gαq (Soto et al., 2015). The S1P receptors have not been investigated for functional effects of glycosylation, but a study from 2003 shows S1PR1, and likely
S1PR3, require N-linked glycosylation of the N-terminus for proper cellular localization to the cell membrane (Kohno and Igarashi, 2003) .
18 Phosphorylation
Phosphorylation of proteins is a major post-translational modification that alters the activation status of the target protein. Many pathways leading to transcriptional activation or repression are driven by the phosphorylation status of a protein. Even within the residues of a protein, phosphorylation of certain residues induce activation while others result in inhibition. Within the super family of GPCRs, phosphorylation is the first step to receptor desensitization and internalization. Serine and threonine residues on the C-terminal tail are phosphorylated by G protein receptor kinases (GRKs) and allow for binding of another secondary messenger, β-arrestin. The binding of β- arrestin prevents the activated GPCR from binding heterotrimeric G protein complexes and further propagating the signaling cascade. In addition to the desensitization, β- arrestin binding allows for the β-arrestin bound GPCR to be internalized through β- arrestin interaction with clathrin internalization machinery. Besides β-arrestin binding,
GPCR phosphorylation can also mediate other receptor activities.
In S1PR1, the phosphorylation of serine 351 of the C-terminal tail is required for receptor internalization, and without this particular phosphorylation, polarization of the
TH17 population of T cells in the nervous system can lead to enhancement of autoimmune neuroinflammation (Garris et al., 2013). Protein-protein interactions can also be affected by the phosphorylation status of the receptor. Phosphorylation of serine
339 allows the C-X-C chemokine receptor type 4 (CXCR4) receptor, a chemokine receptor important for immune cell function, to form a heterodimer with T cell antigen receptor (TCR) allowing for production of cytokines (Dinkel et al., 2018). So not only does phosphorylation of CXCR4 allow for the TCR protein – protein interaction to form, it also is critical to a signaling pathway essential to T cell function.
19 Ubiquitination
The covalent addition of ubiquitin to proteins is important for protein degradation, protein-protein interactions, trafficking, and stability among other functions. The ubiquitination of a protein occurs at exposed lysine residues where ubiquitin is covalently attached via a thioester bond. With a single ubiquitin added, the post-translational modification can alter localization, protein-protein interactions, and/or activation status.
The linking of other ubiquitin molecules to the initial site most commonly results in targeted degradation of the polyubiquitinated protein by the proteasome. The mechanism for the downregulation of S1PR1 by the drug fingolimod is mediated by enhance polyubiquitination and increased degradation when compared to S1P binding alone (Oo et al., 2007).
Palmitoylation
Palmitoylation is a PTM important to multiple aspects of GCPR signaling. The free thiol group of cysteine residues are targeted by acyl-palmitoyl transferase enzymes via a poorly defined recognition motifs comprised mostly of hydrophobic and basic amino acid residues. Like the other PTMs, palmitoylation of a protein can alter the ability of the target to interact with other proteins. However, palmitoylation is unique in the fact that it can alter the interaction of the protein with the membrane. Because GPCRs are transmembrane proteins, any PTM affecting this interaction takes on significant importance. In addition, GPCR internal signaling effectors are also proteins that can be modified by lipidation.
All major G protein alpha effector family members are palmitoylated on the N- terminal domain (Escribá et al., 2007). This modification is reversible, and important for the function of the Gα subunits. GDP-bound Gα subunits are tightly associated with the membrane and thus in the vicinity of the GPCRs. Upon GPCR-ligand binding and GDP-
20 GTP exchange, there is high turnover of the palmitate, presumably allowing for movement from being membrane-associated to cytoplasmic and able to interact with other signaling molecules or translocate. However, this correlation has not been found to have a direct or required mechanism associated with palmitate turnover on activated
Gα’s (Escribá et al., 2007).
The first receptor found to be modified in this manner was a rhodopsin family
GPCR (O’Brien and Zatz, 1984). Since then, many GPCRs have been discovered as targets of palmitoylation including V2 vasopressin, μ – opioid, endothelin, and S1P receptors (Charest and Bouvier, 2003; Horstmeyer et al., 1996; Ohno et al., 2009; Zheng et al., 2012). These all are critical to the cellular functions of the receptors. Considering the context of the work presented here, the palmitoylation of the S1P receptors is of significant importance. The work by Ohno, et. al. shows all members of the S1P receptor family are palmitoylated. Specifically S1PR1 palmitoylation is critical for signal transduction but not necessarily the localization of the receptor (Ohno et al., 2009). The
S1PR1 palmitoylation then mediates the interaction with the intracellular G protein. This is in contrast to the melanocortin-4 receptor, a GPCR important for feeding and satiation signaling, where palmitoylation of the C-terminus is critical for both cellular localization and function (Moore and Mirshahi, 2018). The effect of the GPCR endothelin receptor,
ETA, palmitoylation is unique and quite interesting. ETA was found to be palmitoylated on the C-terminus. However, the loss of the palmitoylation site resulted in the loss of phospholipase C stimulation while the production of cAMP was not impeded meaning the palmitoylation is essential to activating Gαq signaling but not Gαs activation
(Horstmeyer et al., 1996). As important as palmitoylation is to GPCR biology, the mechanism by which the modification affects different GPCRs in different ways is yet to be fully understood.
21 Sphingosine-1-Phosphate Signaling System
Sphingosine-1-phosphate is an amphipathic bioactive signaling lipid. Synthesis and degradation of S1P are tightly regulated and vital processes to cellular functions.
The lipid is the ligand for the S1P family of G protein receptors with five members:
S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. Each of these receptors regulates a wide variety of biological processes and the family is ubiquitously expressed throughout all cell types of the human body. Synthesis of S1P begins with ceramide cleavage through
N-deacylation via the enzyme ceramidase to a single fatty acid chain sphingosine.
Sphingosine kinase then phosphorylates sphingosine at the serine head group hydroxyl group creating S1P. S1P is carried via high density lipoproteins (HDLs) through the blood plasma for biological function with erythrocytes acting as the main site of synthesis and reservoirs for S1P (Bode et al., 2010; Hänel et al., 2007). Within HDLs, apolipoprotein M (ApoM) is believed to be the main binding partner of S1P
(Christoffersen et al., 2011).
The ratio of S1P to ceramide regulates two opposing activities. Colloquially called the ceramide-S1P rheostat, shifting the balance of this “ceramide – sphingosine rheostat” towards ceramide activates pro-apoptotic signaling pathways, while higher levels of S1P is considered pro-mitogenic (Baran et al., 2007; Van Brocklyn and
Williams, 2012). S1P activates the mammalian target of rapamycin (mTOR) pathway and induces protein levels of the anti-apoptotic Bcl2, leading to the pro-growth effects of
S1P signaling (Justin R. Sysol et al., 2015; Sauer et al., 2005). Ceramide activates protein phosphatase 1(PP1) and 2A (PP2A), protein kinase C (PKC), kinase suppressor of Ras (KSR), and cathepsin D, all of which contribute to the pro-apoptotic effect of high levels of ceramide in cells (Hannun and Obeid, 2008).
22 While the levels of S1P can be a determinant of cell fate, it also has many other effects via the five receptors of the S1P family of receptors. The concentration of S1P signaling is location dependent with varying levels across the body ranging from low nanomolar concentrations in the lymphatic system and marrow, where many immune cell types develop, to hundreds of nanomolar to micromolar in concentration in blood
(Hla et al., 2008). This concentration gradient is essential to the function of the biological molecule and if altered can alter immune cell trafficking (Rosen et al., 2013).
Recently, Guo et al established that S1P exerts significant effects on cell proliferation of pancreatic cancer cells in the 100 nM to 1 uM range, a range consistent with that used in this work and with overall S1P signaling (Guo et al., 2015). The S1P pathway is of significant interest to cancer biologists.
Altering the S1P gradients has been the target for many treatments for cancer.
As previously stated, when the ceramide-S1P rheostat tips to higher S1P levels, there is a pro-mitogenic response. Turning the rheostat to be more ceramide rich and pro- apoptotic is being achieved by inhibition of Spk1 and Spk2, the two main kinases responsible for phosphorylation of sphingosine for treatments of certain cancers (Lamy et al., 2017). S1P overall is a critical signaling molecule for the proper function of human cells and tissues. Although S1P can exert effects through other pathways, the main pathway is through the five receptors that make up the S1P receptor family.
S1PR1
S1PR1 is a developmentally essential gene in mammals that was initially discovered as an essential endothelial cell differentiation gene (EDG) which also include another set of lipid binding GPCRs, the lysophosphatidic acid receptors (LPA).
Knockout of S1PR1 in mice results in embryonic lethality from severe hemorrhaging due to the lack of vascular maturation and differentiation (Im et al., 2001; Liu et al., 2000).
23 The importance of S1PR1 to embryonic vascular maturation has been well established, however no other S1P receptor has been found to be essential for embryo viability.
While S1PR1, S1PR2, and S1PR3 exhibit near ubiquitous expression in human tissues,
S1PR4 and S1PR5 are more selectively expressed. The essential nature of S1PR1 may a consequence of being the only receptor in the S1P receptor family that transduces signals via a single G protein, Gαi. Although initially discovered as a gene essential for vascular differentiation, S1PR1 is a target of pharmacological research for disease treatment because of the importance of the receptor to the migration of T cells from the lymphatic system into circulation. This cell mobility is driven by a gradient of S1P between the circulatory and lymphatic system. As previously stated, S1P concentrations are at the low nanomolar level in the lymph while reaching hundreds of micromolar in the blood (Hla et al., 2008) . Drugs targeting this signaling response have been of high priority for manipulation of the human immune system.
In addition to the vascular development, S1PR1, along with S1PR5, is critical for brain development and blood brain barrier maintenance (Di Pardo et al., 2018; Yanagida et al., 2017). S1PR1 signaling through Gαi activates both lowering cAMP levels and cell migration pathways via the Rac1, Src, and MAP kinases cascades (Huang et al., 2011).
This signaling is critical to the lymphocytic migration of T cells for which S1PR1 has been targeted for treatment of multiple sclerosis (Fujino et al., 2003). Because S1PR1 is important to migration, it’s involvement with cancer has been studied as well., and
S1PR1 promotes invasion in Wilms tumor, fibrosarcoma and glioblastoma cell lines while promoting tumor vascularization and inflammation in mouse models of cancer when signaling is altered (Patmanathan et al., 2017).
24 Targeting S1PR1 in Relapsing Remitting Multiple Sclerosis
The modification of S1PR1 signaling in relapsing remitting multiple sclerosis is highly investigated because of the significant impact of S1PR1 on T-cell function.
Fingolimod was the first oral drug treatment approved for RRMS (Fujino et al., 2003).
The drug acts as a mimetic of S1P, being phosphorylated by the same kinases and binding in the orthosteric binding pocket of S1P receptors (Brinkmann et al., 2010).
Since the discovery of fingolimod, researchers are developing more selective drugs for the treatment of RRMS. This is because, although fingolimod is an effective attenuator of T-cell migration, there are off-target and side effects because fingolimod non- specifically binds to other S1P receptors and S1PR1 is ubiquitously expressed. These effects include bradycardia and hypertension/pulmonary fibrosis propagated via S1PR1 and S1PR3 signaling, respectively (Fryer et al., 2012).
To combat these effects, finding alternatives with greater receptor specificity for treating RRMS is of high importance. Many compounds, both pro-drug and direct- acting, have been tested since the approval of fingolimod. In Chapter 3, I evaluate some compounds for efficacy and potency across the family of S1P receptors and analyze whether selected naturally occurring variants could potentially alter the ability of the selected drugs to treat RRMS. These drugs are discussed in greater depth in Chapter 3.
S1PR2
S1PR2, like S1PR1, is widely expressed in human tissues. S1PR2 couples to
Gαi/o, Gαq, and the Gα12/13 families of G proteins making it, and S1PR3, the most promiscuously coupled receptors of the family. Because of this promiscuity, S1PR2 can initiate a wide array of signaling pathways. Although the receptor is not required for growth or embryonic viability, it plays significant roles in certain biological processes. In zebrafish the receptor was originally discovered and named Miles-apart because of the
25 phenotype when morpholino knockdown of S1PR2 resulted in loss of myocardial migration to the midline of developing embryos resulting in cardia bifida which was linked to a loss of Gα13 signaling (Ye and Lin, 2013).
In mice the receptor is an essential gene for hair cell development and maintenance (Herr et al., 2007). Further, a spontaneous mutation observed in mice,
T289R, resulted in profound deafness and the finding that genomic markers for deafness in humans were in fact located around the S1PR2 loci (Ingham et al., 2016). That same year a highly consanguineous Pakistani family was found to possess two novel inactivating variants of S1PR2 causing hereditary deafness and some lower limb deformation (Santos-Cortez et al., 2016). Both the R108P and Y140C variants from the
Santos-Cortez paper are analyzed in work presented in Chapter 2.
A naturally occurring variant of S1PR2 is implicated in the incidences and age of onset of type II diabetes. The receptor was linked to insulin signaling modulation, making S1PR2 a potential candidate as a target for diabetes treatment (Tu et al., 2009).
Expanding on the Tu et al, another study analyzed two variants, N10K and V286A, and found the V286A variant to be carried by nearly 50% of the experimental cohort (Kozian et al., 2010). The V286A mutant confers a supposed signaling defect via disruption of the conserved NPXXY motif at the C-terminal end of TM-7. The NPXXY motif directs structural remodeling of the C-terminal 8th helix conferring an active-state conformation in the C-terminus (Fritze et al., 2003). This specific variant was also examined during my research with contrasting results from the Fritze et al. publication concerning the
V286A variant.
S1PR3
S1PR3, the other S1P receptor exhibiting high promiscuity, coupling to three of the four family of intracellular G protein effectors is important for functions in the lung,
26 cardiovascular system, kidney, intestines, spleen, and cartilage and muscle. There are few known effects from knocking out S1PR3, but the primary effect is a worsening of sepsis outcome. S1PR3 is up-regulated in macrophages during bacterial infections and knockout of the receptor leads to a decrease in bacterial clearance and an increase in mortality due to sepsis (Hou et al., 2017).
S1PR3 has also been linked to proper lung function. Sun et al (2012) found that elevated plasma levels of S1PR3 were a reliable biomarker for not only sepsis but also acute lung injury. S1PR3 contributes to lung-dysfunction when patients are undergoing treatment with S1P receptor agonists, such as fingolimod. As previously mentioned it was discovered S1PR3 is responsible for some off-target effects of fingolimod treatment for RRMS, specifically fibrosis of the lung. This pro-fibrotic signaling in the lung is linked to Smad dependent S1PR3 signaling (Keller et al., 2007; Sobel et al., 2013a).
S1PR5
An important component of my work focuses on the effect a naturally occurring variant has on the biological response of S1PR5. S1PR5 couples to the Gαi/o and Gα12/13 family of G protein effectors and is important to myriad biological processes, within oligodendrocyte progenitor cells, peripheral blood mononuclear cells including T and NK cells, and the endothelial cells of the blood brain barrier (Doorn et al., 2012; Jaillard et al., 2005; Kothapalli et al., 2002; Walzer et al., 2007). The receptor was identified, via similarity, to the rat Edg8 gene, now known as S1PR5 (Im et al., 2001). S1PR5 is a protein significantly increased in expression in large granular lymphocytic (LGL) leukemia (Kothapalli et al., 2002).
The receptor is essential to the proliferation of the leukemic cells and was identified as a potential target for drug discovery, specifically for LGL leukemia (Shah et al., 2008). LGL leukemia is characterized by enriched lymphocyte populations,
27 splenomegaly, anemia, and rheumatoid arthritis. There are two subtypes based on the amplified cell type stratified into either CD3+ T-cell or CD3- NK-cell LGL leukemia
(Zhang and Loughran, 2012). Fingolimod acts as a potent inducer of apoptosis in LGL cells in culture (Shah et al., 2008). Even though fingolimod is an effective promoter of apoptosis in LGL cell cultures, it has not been approved for use as a LGL leukemia treatment. It is currently unknown the reason this drug has not been tested for treatment for LGL leukemia.
S1PR5 is currently increasing in interest to sphingosine-signaling researchers.
The expression of S1PR5 was identified as the only sphingosine-signaling pathway component, included sphingosine kinases, transporters, phosphatases, lyases, and
S1PR1-4, to be negatively correlated with the outcome of patients with glioblastoma
(GBM) (Quint et al., 2014) . Patients with low expression levels of S1PR5 tended to live10-20% longer with GBM. Although there still is no significant treatment for GBM, any potential treatments for remission or life extension are of significant interest. The most prevalent biological function of S1PR5 is regulating the amount of circulating NK cells in the body (Walzer et al., 2007). NK cells are critical to a fully functional immune system. They are responsible for viral clearing and maintenance and surveillance of immunologically stressed cells. NK cells complete these functions without needing cell surface marker initiation like other immune cells. The knockout of S1PR5 in mice inhibits bone marrow egress of NK cells into circulation a critical component of the receptor to NK cell function (Mayol et al., 2011). In addition to initiating egress from the bone marrow, NK homing is mediated by S1PR5 as well (Drouillard et al., 2017). The biology of S1PR5-dependent NK homing is still not entirely understood. The results presented in this dissertation will contribute to understanding the function of S1PR5 on
NK cell biology.
28 Summary
The work in my dissertation will contribute information to the fields of personalized medicine, pharmacogenomics, and S1P receptor biology. By utilizing both human cell and yeast systems I have successfully analyzed the function of single GPCR populations and the interactions with ligands, approved and novel drugs, and intracellular G protein effectors. Additionally, the use of these systems for the analysis of
GPCR variants was informative. With importance to human cell biology and diseases including RRMS, diabetes, deafness, sepsis outcome, LGL leukemia, and potentially malaria, the S1PR family is becoming of more interest to scientists.
Chapter 2
Natural Human Genetic Variants Affect Efficacy and Coupling of Sphingosine-1-Phosphate Receptors
Preface
This work began as a result of the antagonist studies in Appendix A leading to investigating how naturally occurring variants in the S1PR family could alter receptor biology. When attempting to create yeast carrying variants of the S1PRs I found a variant of S1PR5 result in a unique phenotype, a loss of coupling to one of two cognate G proteins. To investigate that phenotype, I applied a modified yeast pheromone signaling pathway and transduced human cells to uncover how signaling pathways are altered between the WT and variant receptors. The impact of the variant on cellular function.
And the molecular mechanism responsible for the functional differences.
Introduction
The connection between single nucleotide polymorphisms and human phenotypes has been extensively explored through genome wide association studies, in which genotypes of groups of individuals possessing a particular trait are compared to those of individuals not exhibiting that trait. To date, more than 3600 such GWAS have identified more than 70,000 human SNPs, each of which is associated with one or more of several hundred different traits. I have taken a different approach to assessing the relationship between SNPs and phenotype. Specifically, I have explored the physiologic consequences of relatively common SNPs in the coding regions of a subset of genes, namely those encoding G-protein coupled receptors, to gain insights into the possible phenotypic consequences of the variant.
30 GPCRs comprise a class of approximately 850 seven transmembrane proteins in humans that generally reside on the plasma membrane and that mediate cellular signaling of a significant fraction of hormones, neuropeptides, chemokines, odorants, tastants and other modulators of cellular physiology. Given the size of this family, their ubiquitous expression, and the diversity of their agonists, it is not surprising that GPCRs participate in most physiological and pathophysiological processes and are a major target for drug therapy (Oprea et al., 2018). Whole genome and exome sequencing has identified SNPs in many of the genes encoding these GPCRs that alter the protein’s amino acid sequence and that occur in greater than 1% of the general population. I have examined the pharmacological properties of several variant GPCR proteins that respond to sphingosine-1-phosphate.
The sphingosine-1-phoshpate receptor family is a ubiquitously expressed, developmentally critical family of receptors responsible for angiogenesis, vascular maturation, cell mobility, and tissue barrier integrity (Drouillard et al., 2017; Liu et al.,
2000; Mendelson et al., 2013; Rosen et al., 2009). A variety of drugs targeting these receptors are currently in use or clinical trials for a number of neurodegenerative diseases including multiple sclerosis, amyotrophic lateral sclerosis, acute stroke and schizophrenia as well as a variety of proliferative diseases (Choi and Chun, 2013; Liu et al., 2012; Nagahashi et al., 2018; O’Sullivan and Dev; Takabe et al., 2008).
The five S1PRs are members of the endothelial differentiation gene (EDG) receptor family, which also includes lysophosphatidic acid (LPA) receptors (Massé et al.,
2010; Sanchez and Hla, 2004). These receptors are distinguished structurally by the presence of an N-terminal cap formed by the first alpha helix in all members of the EDG family that obstructs cytoplasmic access to the ligand binding pocket and likely restricts
31 ligand entry to between TM domains 1 and 7 (Hanson et al., 2012). The cap is conserved in both LPA and S1P receptor subtypes.
S1PR1 is an essential, ubiquitously expressed gene. S1PR1-/- mice die within 14 days of conception due to severe defects in vasculature and in neuronal development, including failure of neural tube closure (Liu et al., 2000; Mizugishi et al., 2005; Rivera and
Chun, 2008). Similarly, morpholino treatment targeting the S1PR1 homolog in zebrafish results in substantial vascular defects (Mendelson et al., 2013). S1PR1 is the dominant receptor on leukocytes and regulates their egress from lymphatic organs. In addition, the primary pharmacologic treatment for RRMS targets S1PR1 to restrict T-cell-mediated myelin sheath degradation by preventing T-cell egress from the thymus (Brinkmann et al.,
2010; Huwiler and Zangemeister-Wittke, 2018; Matloubian et al., 2004). S1PR1 couples to a single G protein effector family, G alpha i/o (Gαi/o) (Rivera and Chun, 2008).
S1PR2 and S1PR3 exhibit similar cellular location to each other, namely the brain, heart, spleen, liver, lung, thymus and kidney, and are both involved in protection of cardiomyocytes to ischemia reperfusion. Both exhibit promiscuous signaling and coupling (Gαi/o, Gαq, Gα12/13). However, S1PR2 alone is essential to hair cell formation in the ear (Ingham et al., 2016; Rosen et al., 2009). Two variants have been identified,
R108P and Y140C, each of which co-segregates in consanguineous families with profound deafness and severe lower limb defects when homozygous (Santos-Cortez et al., 2016). In addition, a relatively common variant of S1PR2, V286A, is linked to incidence and early onset of type II diabetes (Kozian et al., 2010).
The role of S1PR4 in physiological processes is poorly understood. Expression is restricted to lymphocytes and tissues of the immune system and S1PR4 null mice or zebrafish have reduced levels of neutrophils (Allende et al., 2011; Gräler et al., 1998).
Consistent with those results, the Cohorts for Heart and Aging Research in Genomic
32 Epidemiology (CHARGE) consortium recently reported that individuals who were carriers of a rare variant in S1PR4, R365L, had reduced levels of circulating neutrophils
(CHARGE Consortium Hematology Working Group, 2016).
Expression of S1PR5 is highly restricted to the brain, blood and spleen. The receptor is critical for blood brain barrier function and oligodendrocyte progenitor cell survival (Di Pardo et al., 2018). Mice lacking S1PR5 have significantly reduced levels of circulating natural killer (NK) cells, demonstrating a critical role of the receptor in innate immunity (Drouillard et al., 2017; Walzer et al., 2007). Moreover, S1P5R is elevated in
NK cells in LGL leukemia and reduction in receptor levels or treatment with the functional antagonist, fingolimod, results in a significant increase in apoptosis of the leukemic NK cells (Shah et al., 2008).
I investigated a series of relatively common naturally occurring variants as well as several suspected pathogenic variants within this family to examine pharmacologic consequences of the variants. This study provided a biochemical rationale for several of the pathogenic variants, although I failed to identify a functional deficit in others previously associated with disease states. This study also revealed a novel functional defect of a variant affecting the C-terminal tail of S1PR5 that results in significant loss of receptor signaling activity. Finally, several of these variants are present at significantly high levels in certain subpopulations, suggesting that they are under positive selective pressure, potentially providing some protection from lethal diseases in those populations.
33 Results
Some naturally occurring variants of S1P receptors exhibit altered ligand
response
I used a yeast-based assay system to evaluate the pharmacology of naturally occurring variants of members of the S1PR family (Broach and Thorner, 1996; Dowell and Brown, 2009). In these strains, the yeast pheromone responsive GPCR is replaced by one of the selected S1P receptors (Figure 2-1 A). A collection of strains, with chimeric
Gα containing a yeast derived portion to interact functionally with the yeast Gα subunits and a human derived portion to interact with the human GPCR. Different strains carry different versions of these chimeric Gα’s, corresponding to different subclasses of human
Gα’s, allowing us to match each receptor with its appropriate G protein. Ligand binding induces the liberation of Gα, which initiates a MAP kinase cascade resulting in activation of the Ste12 transcription factor and transcription emanating from the pheromone responsive FUS1 promoter. The FUS1 promoter drives expression of either HIS3, stimulating growth in the his3 background of the strain, or luciferase, allowing a quantitative measure of the level of receptor activation. Prior studies with other receptors demonstrated that pharmacologic responses to various agonists and antagonists of a human receptor expressed in yeast mirrored that of the receptor in its normal context
(Campbell et al., 1999).
34
A
B S 1 P R 1
1 0
W T n
o 8 A 1 1 D
i
t a
l R 13G
u 6
m
i
t S
4
d
l o
F 2
0 -6 -4 -2 0 2 [S 1 P ] (lo g u M )
S 1 P R 2
5 W T
N 1 0 K n
o 4
i R 60Q
t a l R 1 0 8 P u 3
m Y 1 4 0 C
i t
S V 2 8 6 A
2
d
l o
F 1
0 -6 -4 -2 0 2 [S 1 P ] (lo g u M )
S 1 P R 3
5
W T n
o 4
i R 15 3H
t a l T 1 67M
u 3
m R 2 4 3Q
i
t S
2
d
l o
F 1
0 -6 -4 -2 0 2 [S 1 P ] (lo g u M )
S 1 P R 5
5
W T n
o 4 i
t V 2 8 4 I a l L 3 1 8 Q
u 3
m
i
t S
2
d
l o
F 1
0 -6 -4 -2 0 2 [S 1 P ] (lo g u M ) Figure 2-1. Saccharomyces cerevisiae system to analyze signaling dysfunction of variants in the S1P receptor family. A) Harnessing the yeast pheromone response. HIS3 or luciferase for monitoring dose dependent S1PR-G protein interactions activation to select ligands or, respectively. System previously described in Klein et al. (B) Dose response curves measuring luciferase fold stimulation by S1P of S1PR1, S1PR2, S1PR3 and S1PR5 (sequences to the right). All curves are normalized to no drug controls for each receptor and variant. Each point is n = 9 and error bars are standard deviation. Fold change was performed using Microsoft Excel and graphing and sigmoidal curve analysis performed using GraphPad Prism 6.
35
Published Experimental Emax (fold Receptor Variant Gα EC50 (nM) EC50 (nM) stimulation) S1PR1 WT GPA1 4.3-5.6 4.6 5.3 A11D GPA1 3.9 5.8 R13G GPA1 40 6.8 S1PR2 WT GPA1 1.3-2.9 1.3 2.8 N10K GPA1 1.5 2.8 R60Q GPA1 >1000 >3 R108P GPA1 N/A 1 Y140C GPA1 N/A 1.3 V286A GPA1 2.4 3.2 S1PR3 WT GPA1 3.9-79 15.2 3.3 R153H GPA1 21.2 2.7 T167M GPA1 118.4 4 R243Q GPA1 20 2.6 S1PR5 WT i2 1.8-120 6 3.4 V284I i2 6 2 L318Q i2 ~10 >2.6
Table 2-1. Published and experimental EC50 and Emax values for S1PRs and variants. Receptor wild type EC50 from published studies along with experimentally calculated EC50 and Emax for both WT and variants. Gα column indicates which Gα is present in strains. N/A: experimental EC50 not calculable
Dose response curves of four S1PRs, their relatively common variants as well as certain select rare variants are shown in Figure 2-1 B as fold activation over the unstimulated strain. The results are summarized in Table 2-1 (Bolli et al., 2010;
Clemens et al., 2005; Deng et al., 2013; Evindar et al., 2013; Horan et al., 2016). For all four receptors carrying the major alleles, which for convenience we refer to as wild type, the EC50s I determined were quite comparable to those reported for receptors present in human cells (Table 2-1). The A11D variant of S1PR1 and the N10K and
V286A variants of S1PR2 exhibited pharmacologic properties essentially equivalent to those of the wild type receptors, while the R153H and R243Q variants of S1PR3 exhibited similar EC50s but somewhat reduced Emax. In contrast, the N60Q variant of
S1PR2 had reduced efficacy with an EC50 value at least 50 fold greater than that of the
36
Figure 2-2. Loss of Gα12 coupling is observed in S1PR5 L318Q variant utilizing HIS3 induction system. Plate spotting showing L318Q coupling to CY12946 and CY17751 (Gαi2) and but not CY12949 and CY13395 (Gα12) strains. Summarized table showing coupling results of 18 different strains of chimeric G protein alphas in the Gαi/o and Gα12/13 families. S1P was spotted at 4, 40, and 400 uM (top center, bottom left, bottom right, respectively). wild type. Thus, the N60Q variant is unlikely to elicit a response at physiological levels of S1P.
S1PR5 Variant L318Q Fails to Activate Gα12/13
My initial qualitative growth screen for functional coupling of the L318Q variant of
S1PR5 demonstrated that, while it coupled efficiently in strains carrying Gαi2, it failed to couple through Gα12 and poorly through Gα13 (Figure 2-2). The loss of coupling is hallmarked by no halo of growth around the spotting of S1P which can be seen in all WT coupling plates and the two Gαi2 strains under the L318Q variant in Figure 2-2. I confirmed that initial observation with a number of different Gαi, Gα12 and Gα13 strains.
This analysis also demonstrated that wild type S1PR5 couples essentially exclusively
37 through Gαi subtype Gαi2, with relatively little efficacy through subtypes Gαi1, i3 or o, in addition to the Gα12/13 coupling.
To assess whether the failure of the L318Q variant to couple through Gα12/13 was unique to my yeast system or was a fundamental property of the variant, I evaluated the ability of the variant to elicit pathway specific responses in cultured human cells. Since
S1P signaling through Gα12/13 activates the rho pathway and subsequent cytoskeletal remodeling. I assessed the actin organization by confocal imaging of f-actin strained
HEK293T cells transiently transfected with either S1PR5 wild type or S1PR5 L318Q followed by treatment with 100 nM S1P. S1P treatment of S1PR5 WT transfected cells resulted in increased formation of filopodia and intracellular actin filaments as well as deposition of f-actin at cell-cell boundaries (Figure 2-3 A). In contrast, S1P treatment of cells transfected with the S1PR5 L318Q construct elicited little change in filopodia, actin filaments or actin deposition. I measured the level of F-actin by quantitative microscopy.
There is a significant increase in f-actin upon S1P treatment in the S1PR5 WT transfected cells (p < 0.0001, n = 79 cells) but significantly less in S1PR5 L318Q transfected cells (Figure 2-3 B). These results are consistent with a reduced capacity of the variant receptor to signal through Gα12/13 in yeast.
To confirm the diminished f-actin response upon signaling by S1PR5 L318Q, I examined f-actin accumulation over time following treatment with 100 nM S1P of stably transduced cells expressing either WT or L318Q variant of S1PR5. At the indicated times, cells were lysed by homogenization and f-actin separated from G-actin by
38
Figure 2-3. S1PR5 WT and L318Q transiently transfected HEK293T cells show significant differences in f-actin accumulation after 1 hour of S1P treatment. A) Rhodamine-phalloidin staining and representative images of serum-free, no S1P and 100 nM S1P treatment for 1 hour of S1PR5 WT and S1PR5 L318Q. B) Quantitation of f-actin volume in 25% of cell image stacks to enrich for coverslip contact. Images were taken with a Leica SP8 scanning confocal microscope with a white light laser. Actin volume measured using Imaris Imaging Software and quantitated with GraphPad Prism (Untreated EV (n=67), S1PR5 WT (n=108), S1PR5 L318Q (n=85), Treated EV (n=88), S1PR5 WT (n=79), S1PR5 L318Q (n=112)). Statistical analysis 2-way ANOVA with multiple comparisons to Untreated EV cells with Bonferroni correction in GraphPad Prism 6 (* p = 0.0027, ** p < 0.0001) sedimentation following centrifugation of cell extracts at 100K x g. S1P treatment of stably transduced cells harboring the wild type receptor resulted in increased f-actin accumulation while the same treatment of cells expressing the variant receptor led to no
39
Figure 2-4. S1PR5 L318Q variant exhibits minimal F-actin accumulation after treatment with S1P. Centrifugal separation of F and G-actin fractions of stably transduced HEK293T cells with either S1PR5 WT or L318Q variant. A) Representative western blot probing for F and G-actin fractions of 0, 30, and 60-minute time treatments with S1P. B) Quantitation of F/G actin ratio normalized to the 0 minute time point for both WT and L318Q. Each dot is an experiment. Students t-test performed by GraphPad Prism (* - p = 0.002, n=4). increase in f-actin (Figures 2-4A and B). These results support my conclusion that the variant has diminished capacity to signal through Gα12/13.
S1PR5 L318Q Normally Signals Through Gαi
To determine whether the failure of S1PR5 L318Q to elicit Gα12/13 mediated processes in human cells was due to a complete inactivation of the receptor or whether it was due to a specific defect of the variant signaling through Gα12/13, I tested the variant receptor’s ability to elicit Gαi mediated responses. Andrieu et al (2017) previously demonstrated that S1P-induced phosphorylation of Akt was mediated by Gαi signaling downstream of S1PR5. Accordingly, I treated cells stably expressing either S1PR5 WT or the L318Q variant with S1P and harvested them at 0, 20 and 40 min post-treatment.
40
Figure 2-5. Gαi signaling via Akt phosphorylation of S1PR5 is observed in the L318Q variant. A) A representative western blot showing pAkt S473 and total Akt in stably transduced HEK293T cells of S1PR5 WT and L318Q variant over a time course of 0, 20, and 40 minutes of S1P treatment. B) Quantification of total Akt normalized pAkt induction compared to the 0-minute time point. 2-way ANOVA with multiple comparisons performed in GraphPad Prism (** - p < 0.05, n=4).
Treatment with S1P elicits an increased level of Akt phosphorylation reaching a statistically significant level of stimulation by 40 min (Figure 2-5A and B). These results demonstrate the S1PR5 L318Q variant compares closely to WT with respect to Gαi signaling, thereby establishing the variant is specifically defective in coupling to Gα12/13.
The L318Q Variant Eliminates Receptor Palmitoylation
Previous studies have demonstrated all the S1P receptors are, in fact, palmitoylated (Ohno et al., 2009). To test whether the variant alters receptor palmitoylation, I performed the assay diagrammed in Figure 2-6 A. Protecting the free cysteine residues is required for the proper biotin-BMCC labeling. I observed a similar
41 signal when probing for Myc-tagged S1PR5 in both WT and L318Q cell lines (Figure 2-6
B). Of note is that there is significant loss of myc signal after hydroxylamine (HAM) treatment of the protein bound beads (Figure 2-6 B). The loss of myc signal is significant, because the null-HAM treated samples contained only 10% of the total bead volume.
Presumably this was caused by the drastic pH change resulting from the HAM treatment.
The decrease reinforces the results of the lower blot as there is robust streptavidin-HRP signal only in the WT HAM treated lane.
Quantitation of the anti-myc probing indicates a similar expression of Myc-tagged
S1PR5 WT and L318Q within all experiments (Figure 2-6 C). However, the streptavidin-
HRP probing shows significantly less biotin signal in HAM treated S1PR5-L318Q (Figure
2-6 C). This indicates a near elimination of S1PR5-L318Q to be post-translationally modified by palmitoylation.
S1PR5 L318Q Variant Does Not Exhibit Defective Localization
Since receptor localization may be affected by the palmitoylation status of the target GPCR, using fluorescence microscopy, I investigated if localization of the S1PR5
L318Q variant is altered compared to WT S1PR5. Targeting the N-terminal Myc-tag of both S1PR5 constructs shows no substantial difference in cellular localization between the WT or the L318Q variant of S1PR5 (Figure 2-7.) Although there are no cell- membrane specific markers, I observe broad cellular localization with some concentration which appears to be near the nucleus, most likely the endoplasmic reticulum, the primary site of translation for membrane-bound proteins.
42
Figure 2-6. Post translational modification of S1PR5 L318Q is altered compared to S1PR5 WT. A) Acyl-biotin exchange diagram. S1PR5 WT and L318Q are pulled down via a Myc-tag attached to the N-terminus of the receptor. After isolation and protection of free cysteines by N- ethylmaleimide, extracts are treated with hydroxylamine to cleave the thioester bond of the palmitoylation. Free cysteines are then exposed to biotin-BMCC where covalent linkages occur biotinylating S1PR5 where palmitoylation was previously. B) Representative western blot of the acyl- biotin exchange, the top blot is probed with anti-Myc-mouse and the bottom is the same blot stripped and probed with streptavidin-HRP. C) Quantification of the cell line specific relative density of the total biotin signal (** - p < 0.05, n=4). D) Quantification of the cell line specific Myc-tag signal density (** - p < 0.05, n=4). One-way ANOVA performed using GraphPad Prism.
Elimination of Receptor Palmitoylation Sites Alters Gα Coupling
Failure of the variant receptor to couple to Gα12/13 may result from the absence of the C-terminal palmitoylation. Palmitoylation prediction and comparison of zebrafish and
43
S1PR5 WT S1PR5 L318Q
Figure 2-7. S1PR5 L318Q localization is not altered. Immunofluorescence microscopy of Myc – tagged receptors. Cells were grown to 70% confluence and fixed to coverslips before immunofluorescently tagged with Anti-myc GFP tagged antibody. Images taken using DeltaVision Elite high resolution microscope at 63x magnification. Nuclei are counter stained using DAPI. mouse S1PR5 orthologues reveals the two cysteine residues in human S1PR5 at positions 322 and 323 are conserved in mouse and the S1PR5a zebrafish orthologues, while the S1PR5b zebrafish orthologue is missing cysteine 323 (Figure 2-8 A). In addition, the mouse shares 100% identity from the histidine residue at position 315 to the second cysteine at position 323. Since the palmitoylation is linked to the conserved C- terminal cysteine residues, I constructed a mutant form of the receptor in which those two cysteine residues were converted to alanine residues (Figure 2-8 A). I introduced the mutant receptor, called S1PR5-C2A, into my reporter yeast strains carrying either Gα12 or
Gαi2 and tested the resultant transformants for their ability to grow on histidine limiting medium after stimulation by S1P. The S1PR5-C2A mutant strain mediated S1P stimulated signaling via Gαi2 but not through Gα12 (Figure 2-8 B). The S1PR5-C2A mutant duplicates the S1PR5 L318Q strain phenotype. Thus I conclude that
44
Figure 2-8. Cysteine residues 322 and 323 are evolutionarily preserved and mutation to alanine replicates the S1PR5 L318Q coupling phenotype. A) CSSPalm predicts cysteine residues 322 and 323 are palmitoylated in S1PR5. In addition, the prediction software shows conservation of predicted sites downstream of the conserved leucine residue. B) Alanine mutants of cysteine 322 and 323 in S1PR5 replicate loss of Gα12 and maintenance of Gαi2 coupling associated with the S1PR5 L318Q variant. Yeast pheromone coupling assay selecting for HIS3 production at increasing concentrations of S1P (4, 40, and 400 uM S1P).
palmitoylation of the receptor is required for coupling to Gα12 but not to Gαi. In addition, the defective signaling observed with the L318Q is most likely due to the lack of C- terminal palmitoylation.
45 S1PR5-L318Q and S1PR1-A11D are under positive selection in certain
populations.
The allele frequency of S1PR5-L318Q in the overall population is 3%, as reported by the Genome Aggregation Database (GnomAD) (Table 2-2), with values ranging from
1% to 3% in European, African, East Asian and Latino populations. However, the minor allele frequency (MAF) value is 10% in the South Asian population, a frequency confirmed in the ongoing Genome Asia 100K study (Table 2-2). This suggests that the heterozygous genotype is under positive selection in this population. Moreover, and notwithstanding the failure of the receptor to couple to Gα12/13, the expected frequency of individuals homozygous for the variant allele conforms to Hardy-Weinberg expectations.
Thus, the absence of S1PR5 signaling through Gα12/13 does not appear to have a serious effect on viability. Rather, heterozygosity may confer a selective advantage in the South
Asian environment.
Similarly, the allele frequency of S1PR1-A11D in the overall population examined to date is 0.47%, as reported by GnomAD (Table 2-2), with values ranging from 0 to 0.2% in European, South Asian, East Asian and Latino populations. However, the MAF is
4.8% in the African population, twenty times greater than that of the other populations.
S1PR5 L318Q Variant Alters Sphingosine-1-Phosphate Lyase Transcription
Studies investigating the links between sphingolipid metabolism and signaling in malarial resistance have shown promise for manipulation of the pathway for treatments
(Finney et al., 2011; Punsawad and Viriyavejakul, 2017). Because of the loss of Gα12/13 signaling in the S1PR5 L318Q variant and the additional data supporting enrichment of the variant in South Asian populations I examined if the variant had any potential effect on S1P metabolism or signaling. Previously, it was discovered maintaining high serum
46 S1P concentrations through genetic knockdown of sphingosine-1-phosphate lyase 1
(SGPL1), an enzyme responsible for the irreversible breakdown of S1P, allowed for 95% survival of hSGPL1 -/- mice with experimental cerebral malaria. SGPL1 is highly expressed in the brain and bone marrow, areas where sphingolipid metabolism and signaling regulation are critically important (Human Protein Atlas). This expression also coincides with areas where S1PR5 signaling is critically important.
I used stably transduced HEK293T cell lines expressing either WT or L318Q
S1PR5 and found SGPL1 expression differs significantly when comparing S1PR5 WT to
S1PR5 L318Q cell lines after treatment with S1P (Figure 2-9). S1PR5 WT induced an average of 2.5-fold increase in SGPL1 expression after 2 hours of treatment with 1.6 μM
S1P compared to a 50% decrease in expression in the S1PR5 L318Q cells (Figure 2-9
A
B
Figure 2-9. S1PR5 L318Q fails to induce SGPL1 expression compared to S1PR5 WT. A) Representative western blot showing SGPL1 and histone H3 (HH3), as loading control, comparing S1PR5 WT and L318Q at 0, 1, and 2 hours of treatment with 1.6 uM S1P. B) Quantification of SGPL1 signal normalized to HH3 signal, GraphPad Prism 6, multiple t-tests, n = 4,* - p = 0.03.
47
A11D Allele Total Allele Allele Observed Expected Population Count Number Freq. Homozygotes Homozygotes
Total (GnomAD) 1310 281976 0.0046 22 3
South Asian 3 30534 0.0001 0 0
African 1198 24914 0.048 22 29
European (Non-Finnish) 9 128686 0.0001 0 0
East Asian 0 19932 0 0 0 Latino 85 35368 0.0024 0 0 Finnish 0 25030 0 0 0
Ashkenazi Jewish 0 10312 0 0 0
Other 15 7200 0.0001 1 0
L318Q Allele Total Allele Allele Observed Expected Population Count Number Freq. Homozygotes Homozygotes
Total (GnomAD) 8290 274786 0.03 214 125
South Asian 3065 30472 0.101 158 154
African 761 24164 0.031 6 12
European (Non-Finnish) 2468 123582 0.02 21 25
East Asian 328 19478 0.017 4 3 Latino 388 35184 0.011 4 2 Finnish 762 24468 0.031 11 12
Ashkenazi Jewish 291 10194 0.029 3 4
Other 227 7064 0.032 7 4
South Asian (Genome Asia) 142 1382 0.103 5 7
Table 2-2. S1PR1 A11D and S1PR5 L318Q variants exhibit population specific enrichment of causative minor alleles. Data compiled from GnomAD for S1PR1 A11D and S1PR5 L318Q show enrichment in African and South Asian populations, respectively, significantly higher than the average minor allele frequency. Genomic data from the Genome Asia cohort for S1PR5 L318Q confirms GnomAD minor allele frequencies and enrichment in the South Asian population.
48 B). Interestingly, I observe an overall higher initial level of SGPL1 in the S1PR5 L318Q cells (Figure 2.9 A). However, over the time course there is a decrease of SGPL1 expression. Although the data is preliminary, it appears the S1P metabolic pathways are perturbed.
Discussion
My analysis revealed an unusual signaling defect in the S1PR5 L318Q variant. In particular, whereas the common S1PR5 variant couples to both Gαi2 and Gα12/13, this variant showed normal signaling through Gαi2 but no detectable signaling through Gα12 or
13 in either yeast or human cells. This coupling defect is due to a loss of C-terminal palmitoylation of the L318Q variant receptor on at least one of two cysteine residues lying in the C-terminus. Moreover, a mutant receptor lacking both of these cysteine residues also fails to signal through Gα12 or 13 although couples normally to Gαi2. Previously Ohno et al (2009) showed that all S1P receptors are palmitoylated and the palmitoylation of the
S1PR1 C-terminal tail is essential to signaling function and internalization. Also Moore and Mirshahi (2018) recently showed the melanocortin receptor 4 is palmitoylated on one of the two adjacent cysteine residues located in the C-terminus of the protein and the absence of the PTM substantially reduces cell surface localization and eliminates receptor signaling. The defect I observe with the L318Q variant is subtler, in that the variant receptor lacking palmitoylation retains substantial cell surface localization and activity for signaling through one G-protein but completely fails to signal though the second G-protein. Accordingly, I surmise that Gα12/13 but not Gαi coupling requires interaction either directly with the palmitoyl group or with the palmitoylation-driven anchoring of the receptor C-terminus to the plasma membrane.
The L318Q variant is significantly enriched in the in South Asian population relative to its occurrence in other subpopulations. Similarly, the S1PR1 A11D variant is
49 enriched twenty fold in African populations. These findings suggest that each of these variants may be under positive selection in the two different populations, providing a selective advantage under conditions unique to that population. In other cases of positive selection for a gene variant that otherwise confers a defect in the functioning of the gene, the variant imparts resistance to an infectious disease. In the case of sickle cell, the trait is enriched in the sub-Saharan African population, where it confers resistance to malaria.
I hypothesize the variants may also confer resistance to malaria in the two different populations. The fact the two variants are enriched in two different populations is consistent with the fact the major malaria causing agent is different in the two regions –
Plasmodium vivax in South Asian and Plasmodium falciparum in sub-Saharan Africa.
This hypothesis is fortified by previous data from several studies that established a connection between S1P and malaria. In particular, in mouse models, the levels of S1P modify the ability of the malaria parasite to propagate with lower levels of circulating S1P associated with more severe malaria (Finney et al., 2011). In children with cerebral malaria, severity of the disease correlated with decreasing levels of plasma S1P
(Punsawad and Viriyavejakul, 2017). Treatment of mice with fingolimod, an orally available functional antagonist of S1PR1 and S1PR5 used for treatment of relapsing or
RRMS (Chun and Hartung, 2010), reduces the severity of experimental cerebral malaria in mice (Finney et al., 2011). Accordingly, the altered signaling activity of S1PR5 L318Q may mimic the effect of fingolimod treatment. However, I do not observe a reduced activity of the S1PR1 A11D, so I cannot conclude there is a protective phenotype to this variant on the basis of my observed pharmacologic properties. Nonetheless, these population enrichments in conjunction with the effects of fingolimod and the correlation between S1P and malarial severity call for further analysis of the role these receptors and their variants contribute to malaria pathogenicity.
50 In addition, I examined the pharmacologic effects of a select number of genetic variants in the S1P receptor family. Most of these variant receptors showed little deviation in their response characteristics from those of the common form of the receptor.
Consistent with that observation, individuals homozygous for the variant allele are generally present in the population at the frequency expected from the overall allele frequency in the population. One variant I examined that exhibited a significant defect in receptor function, S1PR2 R60Q, is a relatively rare variant in the population most likely due to the deleterious nature of the variant.
My assay system allowed for the examination of the pharmacologic properties of receptor variants that had previously been associated with various disease phenotypes.
Both S1PR2 R108P and S1PR2 Y140C have been linked to rare cases of severe deafness and limb defects in homozygous individuals (Santos-Cortez et al., 2016).
Consistent with that association, I found that these variants have significantly reduced
S1PR2 function. On the other hand, Kozian et al (2010) previously reported results from a small study indicating an increased incidence of early onset type II diabetes in individuals heterozygous for S1PR2 V286A. My analysis showed a minimal decrease in
EC50 for the variant, which would be unlikely to significantly affect the S1PR2 signaling capacity in a heterozygous individual. The variant S1PR1 R13G, reported to be enriched in patients prone to cardiovascular risk, but seemingly offers carriers protection from coronary artery disease, showed a decrease in S1P potency, shifting the EC50 right and increasing it by 10-fold. Investigating the functional significance of the S1PR1 R13G variant on a rodent model system would be of interest considering the intriguing protection and decreased potency. S1PR3 T167M induces a higher S1P efficacy achieving an Emax of 4-fold stimulation, but a decrease in potency, this variant hasn’t been implicated in a disease.
Chapter 3
Yeast as Platform for Multiple Sclerosis Drug Analysis and S1P Receptor Antagonists
Preface
This chapter can be summarized as a pharmacogenomic analysis of selected
S1PR variant and WT receptors to compare responses when treated with current or experimental therapeutics for the treatment of RRMS. Because research and discovery of novel RRMS treatments are becoming more prevalent I partnered with a company to examine A) how my yeast expression system carrying a single population of either the
WT or variant receptors could be used to analyze altered response of the variants to the selected RRMS treatment molecules and B) how the new drug they provided compared to other approved or experimental treatments for RRMS with respect to potency and efficacy of the activation of the S1P receptors.
Introduction
RRMS affects approximately 400,000 people per year in the United States
(Dilokthornsakul et al., 2016). It is an autoimmune disorder hallmarked by infiltration of the brain by activated CD8+ and CD4+ T cells and subsequent degradation of myelin sheaths (Lucchinetti et al., 2000). Many treatments have been utilized in the past including immunosuppressives, mitoxantrone, cyclophosphamide, and more recently, monoclonal antibodies (Lyamani et al., 2017). Currently, there are minimal oral treatments for RRMS. The S1PR family of GPCRs are the main protein target for treatment of RRMS (Chaudhry et al., 2017).
52 S1PR family of receptors is comprised of five receptor subtypes, S1PR1-5. The
S1PR family of receptors are ubiquitously expressed and are essential to a multitude of biological functions, including but not limited to vascular maturation, hair-cell function, blood brain barrier function, Natural Killer (NK) and T cell migration and function, and oligodendrocyte progenitor cell maturation (Di Pardo et al., 2018; Ingham et al., 2016;
Liu et al., 2000; Walzer et al., 2007; Yanagida et al., 2017). S1PR1 is the target of interest in the disease of RRMS (Matloubian et al., 2004). S1P:S1PR1 signaling drives
T-cell egress from the thymus, and by blocking this egress with the functional antagonist fingolimod, amelioration and repair of myelin breakdown can be achieved (Balatoni et al., 2007; Fujino et al., 2003).
Previously, I’ve reported alterations in S1P signaling in naturally occurring genetic variants of S1PR1, S1PR2, S1PR3, and S1PR5. Here, I examine a similar subset of variants and a group of current and experimental RRMS treatments for altered signaling effects. Fingolimod, or FTY720p (in the phosphorylated active state), is the only prescribed oral treatment for RRMS but has off-target effects resulting in unwanted side-effects for the patient (Anastasiadou and Knöll, 2016; Sobel et al., 2013).
Fingolimod was the first drug for oral application approved for RRMS (Fujino et al.,
2003). It is a pro-drug, meaning it is activated via metabolism, specifically phosphorylation by sphingosine kinase 2 (Brinkmann et al., 2002). Although S1PR1 is the pharmacological target for RRMS treatment, fingolimod also targets receptors
S1PR4, S1PR5, and to a lesser extent S1PR3 (Brinkmann et al., 2002; Mandala et al.,
2002). Because of the lack of selectivity there are off target effects associated with fingolimod treatment including first use bradycardia and lung fibrosis (Pelletier and
Hafler, 2012). RRMS is the primary disease for which fingolimod can be used for treatment, but there have been alternative uses proposed including rheumatoid arthritis,
53 large granular lymphocyte (LGL) leukemia, and malaria (LeBlanc et al., 2015;
Oggungwan et al., 2018; Yoshida et al., 2013).
Ponesimod (ACT-128800) is a S1PR1 specific functional antagonist with a similar mechanism of action as fingolimod, causing persistent internalization of S1PR1 and subsequent sequestration of T-cells to the thymus (D’Ambrosio et al., 2016).
Siponimod (BAF312) is a functional antagonist targeting S1PR1 and S1PR5, and is currently completing Phase III clinical trials for treatment of secondary progressive multiple sclerosis (Gentile et al., 2016; Kappos et al., 2018). Unlike fingolimod, both ponesimod and siponimod are direct acting drugs. They do not need to undergo metabolic pathways to achieve biological function. MT-1303 was designed to limit the cardiovascular off-target effects observed when MS patients are treated with fingolimod.
MT-1303, like fingolimod, is also a prodrug (Sugahara et al., 2017a). Siponimod has been observed to target both S1PR1 and S1PR5 and also limits the off-target activation of S1PR3 (Gergely et al., 2012). RPC1074, a direct acting drug, has potent and efficacious function via S1PR1 and S1PR5, however there is limited data surrounding this compound as it is still in development at Celgene. RPC1074 is related to another drug, ozanimod (RPC1063), which is currently in Phase III clinical trials. Ozanimod reduces brain lesions and relapses of MS, and like RPC1074, binds via S1PR1 and
S1PR5 (Scott et al., 2016). It also diminishes some of the off target effects of fingolimod treatment (Scott et al., 2016).
Here, I use the yeast expression and pheromone inducible luciferase signaling system to address whether some natural S1P receptor variants alter the receptor function when treated with approved or novel RRMS treatments. These compounds act as functional antagonists, normally silencing the cellular response and signaling of target receptors. However, because the yeast-expression system is purely a measure of the
54 activation of the pheromone response pathway the receptor-ligand interaction appears as an immediate agonist. This immediate activation is due to the pheromone response element inducible luciferase construct. This is the reason the potency is tabulated as an
EC50 value instead of IC50, the typical potency calculation for an antagonist. Even though the system does not distinguish the compounds as functional antagonists, it still allows for the determination if genetic variants detailed contribute to altered drug responses. Understanding if these variants affect drug signaling is important to recognizing whether a treatment will be effective or not and ultimately the improve the health of the patient.
Results
Minimal Differences of Activity in S1P Variants Treated with Fingolimod
To calculate Emax each receptor and variant RRMS drug response was normalized to the fold stimulation of the equivalent concentration of S1P (values from
Chapter 2). Then using a normalized 0-100% scaled S1P curve, for each compound I calculated the percent difference between S1P and the drug fold stimulation to reflect the percent difference at each equivalent concentration. For example, an Emax value of
100% for a RRMS drug means there is no difference from the Emax of S1P treatment of the receptor, or if the drug has an Emax of 70% then it exhibited 30% less fold stimulation compared to S1P. EC50 values were calculated to be the concentration of ligand inducing 50% of the maximal signal after normalization to S1P fold stimulation.
Fingolimod treatment of S1PR1 WT resulted in no change to Emax with a slight increase in EC50 from 6 nM for S1P to 8 nM for fingolimod (Figure 3-1 A, Table 3-1).
S1PR1 A11D treatment with fingolimod shows no change to EC50 compared to S1P,
55
FTY720p Ponesimod Siponimod MT1303 MT1303p RPC 1074 Receptor Variant EC50 (nM) Emax % vs S1P EC50 (nM) Emax % vs S1P EC50 (nM) Emax % vs S1P EC50 (nM) Emax % vs S1P EC50 (nM) Emax % vs S1P EC50 (nM) Emax % vs S1P WT 8 100 25 120 40 130 26 23 6 110 10 140 S1PR1 A11D 6 84 20 140 18 96 DNC 22 4 110 6 140 WT 7 24 7 25 11 27 13 31 8 25 1600 330 N10K 7 29 6 20 6 23 10 27 6 21 2900 240 S1PR2 R60Q >1000 35 >1000 32 >1000 40 >1000 44 >1000 35 1800 77 V286A 56 31 4 29 96 32 120 36 71 32 4800 210 WT 110 27 >1000 39 140 26 120 24 110 23 130 25 S1PR3 R153H 190 38 >>1000 45 340 29 >>1000 48 280 32 1000 33 R243Q 120 28 >1000 43 >>1000 32 250 26 >>1000 30 >>1000 32 WT 10 120 23 190 630 230 25 36 20 170 >>1000 340 S1PR5 L318Q 44 180 >>1000 100 90 150 >>1000 73 180 200 >>1000 150
Table 3-1. EC50 and Emax normalized to S1P for S1P receptors and variants of RRMS drugs. All EC50 values were calculated using GraphPad nonlinear regression analysis of dose response curves of each drug listed. Emax was calculated as percent of fold induction at each concentration compared to S1P for the specific receptor in question (ie S1PR1 A11D to S1PR1 A11D).
however there is a decrease in fingolimod efficacy when compared to S1P dropping to
84% (Figure 3-1A, Table 3-1).
Neither S1PR2 nor S1PR3, or the variants of either, exhibit fold stimulation from
treatment with fingolimod. Emax and EC50 values calculated are not true values, simply
an output of the sigmoidal dose response algorithm in GraphPad Prism 6.0 (Figure 3-1 B
C, Table 3-1).
S1PR5 WT treatment with fingolimod yields a small increase in efficacy with a
calculated Emax of 120% (Figure 3-1 D, Table 3-1). There is a small increase in
fingolimod EC50 compared to S1P treatment of 4 nM (Figure 3-1 D, Table 3-1). S1PR5
L318Q response to fingolimod treatment shows marked differences from S1P treatment.
I observe a greater efficacy of fingolimod fold stimulation, compared to S1P, having an
Emax of 180%, equating to a near doubling of the S1P maximal fold stimulation (Figure 3-
1 D, Table 3-1). Although Emax is increased for S1PR5 L318Q fingolimod treatment, I
56
Figure 3-1. S1PR1 and S1PR5 Receptor specificity during fingolimod treatment. Percent stimulation is relative to corresponding S1P concentration for each receptor and variant relative to 100% stimulation of the highest S1P concentration. A) S1PR1 B) S1PR2 C) S1PR3 D) S1PR5. Graphical representations are n = 9 +/- SD. Graph and EC50 calculations performed using GraphPad Prism 6.0.
observe a decrease in potency with a fingolimod EC50 of 44 nM, a near 7.5-fold increase from a S1P EC50 of 6 nM (Figure 3-1 D, Table 3-1).
Ponesimod Exhibits Altered Signaling in S1PR5 L318Q Variant
Treatment of S1PR5 WT with ponesimod shows an increase in both Emax and
EC50 when compared to S1P treatment increasing from 6 nM to 25 nM and 120%, respectively (Figure 3-2 A, Table 3-1). A similar result was observed with ponesimod treatment of S1PR5 A11D. The potency of ponesimod is lower than that of S1P with a higher EC50 of 20 nM compared to 4 nM, respectively (Figure 3-2 A, Table 3-1).
Although ponesimod exhibits lower potency compared to S1P for S1PR1 A11D, the drug showed greater fold stimulation, increasing Emax to 140% (Figure 3-2 A, Table 3-1).
57
Figure 3-2. Ponesimod exhibits lower efficacy in signaling through S1PR5 L318Q compared to WT. Percent stimulation is relative to corresponding S1P concentration for each receptor and variant relative to 100% stimulation of the highest S1P concentration. A) S1PR1 B) S1PR2 C) S1PR3 D) S1PR5. Graphical representations are n = 9 +/- SD. Graph and EC50 calculations performed using GraphPad Prism 6.0
Ponesimod, like fingolimod, is not believed to interact with S1PR2 or S1PR3, and
I observe no appreciable fold stimulation in either of the WT receptors or the select genetic variants (Figure 3-2 B, Figure 3-2 C, Table 3-1).
Ponesimod treatment of S1PR5 WT and A11D lead to differences between the receptors. S1PR5 WT exhibits a slight decrease in drug potency with an EC50 value of
23 nM compared to an EC50 of 6 nM for S1P, however this decrease in potency is countered by a 2-fold increase in efficacy (Figure 3-2 D, Table 3-1). S1PR5 WT achieves an Emax of 190% relative to S1P treatment of the same dosage, nearly doubling the maximal fold stimulation of S1P (Figure 3-2 D, Table 3-1). However, S1PR5 L318Q exhibits significantly different signaling properties when treated with ponesimod compared to the WT receptor. The dose response curve never fully reaches a sigmoidal
58 response, and I observe a significant rightward shift of the curve equating to a very large increase in EC50 to well over 1000 nM (Figure 3-2 D, Table 3-1). However, I do observe an equivalent fold stimulation level compared to the maximal stimulation of S1P (Figure
3-2 D, Table 3-1).
Siponimod Exhibits Selectivity for S1PR1
I next examined how S1PR1 WT and A11D signaling differed upon treatment with siponimod and I observed slight differences between the receptors. I see a greater
Emax for the WT when compared to S1P treatment, achieving 127% stimulation while the
A11D variant signals to nearly the same Emax as S1P 96% (Figure 3-3 A, Table 3-1).
However, both WT and A11D exhibit a decrease in potency with higher EC50 values than
S1P at 40 and 18 nM compared to 5 and 4 nM, respectively (Figure 3-3 A, Table 1).
Again, receptors S1PR2 and S1PR3 show minimal activation when treated with siponimod as would be expected (Figure 3-3 B, Figure 3-3 C, Table 3-1).
Siponimod treatment of both S1PR5 WT and L318Q result in different potency and efficacy compared to S1P. Neither of the receptor dose response curves achieves a sigmoidal response, continually increasing fold stimulation up to 3.33 uM siponimod.
S1PR5 WT has a large rightward shift in the curve leading to an increased EC50 value of
630 nM, two orders of magnitude higher than the EC50 of S1P (Figure 3-3 D, Table 3-1).
However, siponimod has a greater Emax, topping out at 230% for the highest concentration (Figure 3-3 D, Table 3-1). The S1PR5 L318Q also exhibits a rightward shift of the response curve, but not as large as WT, increasing the siponimod EC50 value
59
Figure 3-3. Siponimod exhibits S1PR1 selectivity. Percent stimulation is relative to corresponding S1P concentration for each receptor and variant relative to 100% stimulation of the highest S1P concentration. A) S1PR1 B) S1PR2 C) S1PR3 D) S1PR5. Graphical representations are n = 9 +/- SD. Graph and EC50 calculations performed using GraphPad Prism 6.0
to 90 nM (Figure 3-3 D, Table 3-1). Siponimod also achieves an Emax greater than S1P increasing to 150% (Figure 3-3 D, Table 3-1).
MT1303p Exhibits S1P-like Signaling Activity
Across the S1P family of receptors there was no to minimal response to the unphosphorylated pro-drug MT1303 (Figure 3-4). Only S1PR5 L318Q exhibited any appreciable activity in response to MT1303 treatment achieving an Emax of 73%.
Treatment of the receptors and variants with the active MT1303 phosphate
(MT1303p) shows activity in both S1PR1 and S1PR5 and the respective variants (Figure
3-5). S1PR1 treatment of MT1303p exhibits the most S1P-like activity of all RRMS drugs examined. MT1303p has an EC50 value of 6 nM, nearly identical to the 5 nM EC50 value of S1P (Figure 3-5 A, Table 3-1). The efficacy of MT1303p is 10% higher than
60
Figure 3-4. MT1303 has minimal to no receptor activity. Percent stimulation is relative to corresponding S1P concentration for each receptor and variant relative to 100% stimulation of the highest S1P concentration. A) S1PR1 B)S1PR2 C)S1PR3 D)S1PR5. Graphical representations are n = 9 +/- SD. Graph and EC50 calculations performed using GraphPad Prism 6.0
S1P for S1PR1 WT (Figure 3-5 A, Table 3-1). The variant, S1PR1 A11D, has nearly identical EC50 and Emax values as the WT for MT1303p, at 4 nM and 110%, respectively
(Figure 3-5 A, Table 3-1).
Similar to the other RRMS drugs examined here, there is minimal fold stimulation of S1PR2 and S1PR3 and the respective variants after treatment with MT1303p (Figure
3-5 B, Figure 3-5 C, Table 3-1)
After MT1303p treatment of S1PR5 WT and L318Q I observed differences in the efficacy and potency of the drug between the two receptors. S1PR5 WT has a lower
EC50 value of 20 nM compared to a value of 180 nM for the L318Q variant (Figure 3-5 D,
Table 3-1). Both receptors exhibit greater fold stimulation compared to S1P achieving
61
Figure 3-5 MT1303p has higher efficacy in S1PR5 WT and L318Q compared to S1PR1 Percent stimulation is relative to corresponding S1P concentration for each receptor and variant relative to 100% stimulation of the highest S1P concentration. A) S1PR1 B)S1PR2 C)S1PR3 D)S1PR5. Graphical representations are n = 9 +/- SD. Graph and EC50 calculations performed using GraphPad Prism 6.0
170% and 200% Emax values for S1PR5 WT and L318Q, respectively (Figure 3-5 D,
Table 3-1).
Selectivity and Activity of RPC 1074 Shows Promise for RRMS
Treatment
RPC 1074, the novel drug compound from Celgene, exhibits high potency and efficacy when S1PR1 WT and A11D are treated. After treatment with RPC1074 I see very high potency with EC50 values of 10 and 6 nM for S1RP1 WT and A11D, respectively (Figure 3-6 A, Table 3-1). When the efficacy is analyzed, I observe an increase in Emax values for both WT and A11D to 140% (Figure 3-6 A, Table 3-1).
RPC1074 is believed to be selective for S1PR1 and S1PR5, however treatment of S1PR2 WT, N10K, R60Q, and V286A show activity in those receptors (Figure 3-6 B).
62
Figure 3-6 RPC1074 has high efficacy in S1PR1 and S1PR5 and exhibits S1PR2 activity. Percent stimulation is relative to corresponding S1P concentration for each receptor and variant relative to 100% stimulation of the highest S1P concentration. A) S1PR1 B) S1PR2 C)S1PR3 D)S1PR5. Graphical representations are n = 9 +/- SD. Graph and EC50 calculations performed using GraphPad Prism 6.0
The activation of S1PR2 WT and N10K, R60Q, and V286A give Emax values of 330, 240,
76, 210%, respectively (Figure 3-6 B, Table 3-1). However, there was not a similar result for potency. RPC1074 has limited potency when the S1PR2 receptors are treated shown by significant rightward shift of the response curves, and uM level EC50 values for the WT and variant receptors (Figure 3-6 B, Table 3-1).
RPC1074 treatment of S1PR3 WT, R153H, and R243Q exhibited minimal fold stimulation and low activity (Figure 3-6 C).
Dose response analysis for S1PR5 WT and L318Q proved to be challenging.
The data does not follow an exact sigmoidal curve with a very high response for the 3.33 uM dose of RPC1074 (Figure 3-6 D). Regardless, RPC1074 exhibits high efficacy when treating both S1PR5 WT and L318Q receptors with Emax values of 340% and 150%, respectively (Figure 3-6 D, Table 3-1). However, because of the large increase in fold
63 stimulation signal between 1.11 uM and 3.33 uM of RPC1074 treatment, the data does not follow a sigmoidal curve and drives the dose response curve rightward driving the
EC50 values to greater than 1000 nM for both S1PR5 WT and L318Q (Figure 3-6 D,
Table 3-1).
Discussion
Determining potential effects of naturally occurring genetic variation on drug targets is essential to understanding not only the pharmacogenomics of the variants but also to give insight into protein structure-function and the implications of protein modifications that lead to altered protein function. Here I have performed an analysis of selected S1P receptor natural variants to assess if any receptor signaling deficiencies could affect the treatment of persons afflicted with multiple sclerosis.
Fingolimod, FTY720, is the most well-known and studied pro-drug of the group of drugs examined. When comparing the four receptors and genetic variants, I observe minimal changes in receptor signaling with respect to the S1PR1 and S1PR5 receptors and the variants examined. Expectedly, I do not observe appreciable signal efficacy in
S1PR2 or S1PR3. The lack of S1PR3 stimulation by fingolimod was curious because the receptor is known to induce side effects resulting in lung fibrosis in certain patients
(Sobel et al., 2013). This work utilized label-free impedance measurements to account for the promiscuity of GPCR–G-protein interactions and were able to detect S1PR2 activity by fingolimod in addition to S1PR3 activity by ponesimod. This contradicts the results I observe where there is not any appreciable signaling via the native yeast GPA1, a Gαi orthologue. This doesn’t preclude a scenario where fingolimod or ponesimod signals biasedly through an alternative G protein. Since S1PR2 and S1PR3 are known to promiscuously couple to Gαi/o, Gαq, and Gα12/13 G proteins it is possible this phenomenon could be occurring (Jiang et al., 2007; Rosen et al., 2009). Future studies
64 for more comprehensive analysis of potential biased signaling will need to be performed to determine whether the activation of S1PR2 and S1PR3 by fingolimod and ponesimod, respectively, is an example of biased agonism or simply the complex nature of biology.
Treatment of S1PR5 with fingolimod results in an increase of efficacy in the
S1PR5 L318Q variant achieving 180% signal stimulation compared to an equivalent dosage of S1P, however there is a notable decrease in potency with an EC50 value increased to 44 nM, 10-fold higher than for S1P. The WT receptor when treated with fingolimod, proved to have both potent and efficacious signaling compared to S1P activation. Currently, disorders where targeting S1PR5 for therapeutic purposes are not well established save T and Natural Killer cell types of LGL leukemia. However, recent investigations into the physiological activity of S1PR5 signaling have shown the receptors importance to the blood brain barrier, NK cell migration, and oligodendrocyte progenitor cell survival (Di Pardo et al., 2018; Doorn et al., 2012; Jaillard et al., 2005;
Walzer et al., 2007). In addition to the NKLGL and TLGL leukemia, S1PR5 mRNA levels were identified as negatively correlated with survival in end stage glioblastoma, meaning treatment for those patients with either an LGL disorder or glioblastoma could stand to benefit from further drug discovery of modifying compounds of S1PR5.
Comparing the activation curves of ponesimod and siponimod I found similar activation dynamics of the two compounds across all the receptors. There is minimal activation of receptors S1PR2 and S1PR3 and the associated variants, however I observe some differences with respects to S1PR1 and S1PR5. S1PR1 exhibits similar efficacy and potency when treated with ponesimod compared to S1P with both S1PR1
WT and A11D having low nanomolar EC50 values. However, analysis of S1PR5 yields an interesting result where I see dose dependent activation of S1PR5 WT to an Emax and
EC50 which are very similar when compared with S1PR1. This is interesting because it
65 has been reported ponesimod is specific for S1PR1 alone (Bolli et al., 2010). Here I clearly show a very robust and potent activation of S1PR5 WT, but also a significant loss of signal transduction in S1PR5 L318Q. Although the L318Q variant S1PR5 achieves an equal efficacy compared to S1P, I observe an over 100-fold increase in EC50 compared to S1P alone. This is the first time activation of S1PR5 by ponesimod has been observed to be of equivalent stimulation of S1PR1. Most current drugs targeting
S1PR1 for MS treatment have overlapping activation with S1PR5 most likely due to the high sequence similarity between the two receptors, with 47% identical and 64% positive residue compatibility (BLAST Alignment), so having activation of S1PR5 by a supposed
S1PR1 specific compound is surprising. Siponimod activates both S1PR1 and S1PR5, however my system shows there is less potency in S1PR5 compared to S1PR1 with increased EC50 values for both S1PR5 WT and L318Q (Pan et al., 2013). This is interesting considering siponimod is a derivative of fingolimod but does not require phosphorylation via sphingosine kinase (Pan et al., 2013).
MT1303 is a pro-drug and must be phosphorylated via sphingosine kinases to gain activity and MT1303p was identified as a highly selective and potent ligand for activation of S1RP1 and S1PR5 (Sugahara et al., 2017). Here I used the unphosphorylated MT1303 as a negative control to control for aberrant signaling and for comparison with MT1303p. There is minimal activation of all four receptors and the affiliated variants by MT1303. Comparing the S1PR1 WT and A11D variant responses to MT1303p treatment I observed minimal difference between the two receptors. Both achieve an Emax nearly identical to S1P and exhibit excellent potency with EC50 values nearly identical to S1P. However, when I compare the WT and variant receptor L318Q of the S1PR5 subtype, I see a considerable difference between the two. S1PR5 WT exhibits reasonable low nM potency, but the potency is less when compared to S1PR1.
66 Although S1PR1 shows higher potency, there is almost a doubling in S1PR5 WT efficacy compared to S1P treatment of equal dosage. Clearly MT1303p is highly efficacious in the S1PR5 as the variant L318Q exhibits a near doubling of signal output compared to S1P treatment, however I do observe a decrease in potency in the L318Q
S1PR5 variant.
A novel drug in line for medical trials has been designed by the biopharmaceutical company Celgene. The drug, identified as RPC1074, is a potent and efficacious activator of S1PR1 both the WT and the A11D compared to S1P activation.
The gains in efficacy compared to the standard of oral treatment, fingolimod are quite significant. Fingolimod achieves Emax values to similar values as S1P while RPC1074 provides significantly increased efficacy while maintaining the required low nanomolar affinity, albeit slightly higher than fingolimod, for S1PR1. Here, I report both selectivity increase and overall better properties for RPC1074 compared to industry standards.
In addition to the robust stimulation of S1PR1 by RPC1074, I also observe great increases in efficacy when S1PR5 WT is treated with the compound. The analysis of all twelve doses of RPC1074 leads to a non-sigmoidal curve and drives the curve rightward increasing the EC50 value calculations to be extremely high, greater than 1000 nM, and very high efficacy with Emax values of 340% and 150% for WT and L318Q, respectively.
Therefore, even though RPC1074 is billed as a S1PR1-specific functional antagonist, it may be prudent to investigate the druggability of S1PR5 for LGL leukemia and glioblastoma.
It was concerning to see a robust stimulation of S1PR2 and the N10K, R60Q, and V286A variants when treated with RPC1074, but with EC50 values in the uM level, there would most likely be minimal effects from treatment with low nanomolar concentrations of RPC1074 in patients. However it would be critical to monitor for side –
67 effects previously ascribed to S1PR2 during treatment with fingolimod. Overall,
RPC1074 appears to be a potentially effective treatment for the treatment of RRMS and should also be investigated for alternative treatments of other S1P receptor targeting disorders.
Chapter 4
Conclusions and Future Directions
Reflecting back to when these projects were first conceived there were two lofty goals established. One was to use the yeast-luciferase system to find novel molecules targeting the S1P receptor family for manipulation of diseases. Discovering the first
S1PR5 antagonist for the treatment of LGL leukemia was a primary goal (see Appendix
A), with the additional goal of creating a pipeline from yeast to other model organisms to investigate the cellular and organismal effects of deleterious naturally occurring genetic variants. Obviously the work presented here has but scratched the surface of those goals, but the scratches are enlightening none the less.
The primary phenomenon observed in these studies was the seemingly unique dysfunction I observed when examining which yeast strains were capable of being used for analysis of S1PR5 and the variants. The L318Q variant exhibits some unique genetic enrichment in South Asian populations. Overall the MAF is 3%, which would not be considered a particularly rare variant, but since South Asian populations have a 4-fold higher frequency it would indicate there is some interesting biology at play. When the coupling experiments were performed and I observed coupling to the Gαi2 strains, but not to the Gα12 carrying strains, I decided this would be an extremely interesting avenue to pursue. The loss of a single G protein signaling in a promiscuous GPCR was something only observed previously after mutation of intracellular regions (Horstmeyer et al., 1996). To determine the biological consequences of the S1PR5 L318Q variant I needed to address whether there were signaling pathways altered downstream of the G proteins. Transitioning to human cells and probing pathways commonly associated with
69
Gα12 signaling, I was able to show the L318Q variant had significantly diminished actin dynamics compared to the WT receptor when treated with S1P but maintained time- dependent phosphorylation of Akt. This decrease in actin signaling could be an indicator the variant would alter NK cell function. S1PR5 is essential to circulation of NK cells
(Debien et al., 2013). This supported my hypothesis that the variant was causing a defect preventing signaling through Gα12 while maintaining activation of the other cognate G protein Gαi2.
My attempts to utilize the zebrafish model system for physiological analysis of the consequences of the S1PR5 L318Q variant by creating a CRISPR knock in of the variant did not work so I was left to at least establish the mechanistic origin of the effect.
The two cysteine residues downstream of the variant position which were predicted to be palmitoylated, a post-translational modification central to general GPCR signaling biology, and I hypothesized this change from a hydrophobic leucine to a larger polar glutamine, could interfere with the palmitoyl-transferase enzymes required for the lipidation of S1PR5. After analysis using biotin-exchange I could say there was a complete loss of palmitoylation in the S1PR5 L318Q variant. Initially I believed there would be an intermediate result, where there would be a significant decrease in palmitoylation of S1PR5 L318Q, but not complete loss. Analysis using the yeast coupling assay of a double mutant S1PR5 with alanine substitution of the predicted palmitoylated cysteine residues reinforced the results of the biotinylation experiments. I observed an identical coupling pattern when compared to the S1PR5 L318Q variant strains. I also attempted to create mutants with either residue 322 or 323 substituted to alanine in place of the native cysteine. However, these mutants were not successfully transformed into the yeast system. For human cells I attempted to create mutants for analysis and reexamine the palmitoylation status of the mutants but the site directed
70 mutagenesis attempts were not successful either. That series of experiments would have identified which of the two cysteine residues are palmitoylated and whether a single palmitoylation site is sufficient for signaling via Gα12.
As was discussed in Chapter 2, there is potential for this L318Q variant to behave similar to sickle-cell trait in African populations, conferring some resistance to a parasite, possibly the less virulent strain of malaria in South Asia, Plasmodium vivax.
Research has shown a significant interaction between sphingosine metabolism, S1P serum concentration, and resistance to malaria (Finney et al., 2011; Punsawad and
Viriyavejakul, 2017). We attempted to probe how the variant could confer resistance by monitoring the induction of SGPL1 in the stably transduced cells. The inhibition of
SGPL1 was previously shown to prevent death from cerebral malaria in a mouse model
(Finney et al., 2011). One specific interaction is the preservation of the blood brain barrier (BBB) and the decrease in interferon-γ (IFNγ). When mice were treated with fingolimod, there was marked decrease in BBB permeability compared to untreated mice, and as well there was a decrease of IFNγ induction in all treated mice. The L318Q variant may actually be influencing these and inducing a fingolimod treatment like condition in carriers of the variant. This potential protection may be supported because there is data to suggest that NK cell IFNγ induction can be mediated through S1PR5 but not explicitly required for the IFNγ response (Fang et al., 2017). Examining whether this
S1PR5-IFNγ dynamic is driven by Gαi/o or Gα12 would be extremely enlightening to whether this hypothesis has any credence. If data support a Gα12 mediated IFNγ response, it would logically follow the L318Q variant would diminish that response and potentially confer resistance to P.vivax infection, helping explain the allelic enrichment in
South Asian populations.
71 The BBB integrity is critical to fending malarial infiltration of the brain. S1PR5 activation is a critical component of this integrity (Doorn et al., 2012). S1P is the endogenous activator of the S1PRs and so maintaining high serum and cellular levels of
S1P around and inside the BBB would be a critical to its integrity. The S1PR5 L318Q variant altered SGPL1 expression after treatment with S1P, where I saw a decrease in expression after treatment. In addition, Finney et al (2011) did establish that knockout of
SPGL1 confers resistance to cerebral malaria in a mouse model, presumably through maintenance of the BBB. This may also be a mechanism conferring resistance to invasive parasites, wherein the BBB cells of people with the S1PR5 L318Q variant express lower levels of the lyase and therefore have higher S1P levels allowing for a higher reservoir of S1P for autocrine activation of S1PRs on the BBB endothelial cells.
Examining the BBB of mice that are homozygous WT, heterozygous, and homozygous
L318Q for S1P levels and SGPL1 levels would be a way to determine if the S1PR5
L318Q variant would increase BBB integrity.
Another explanation is a functionally altered NK cell population due to the L318Q variant. In humanized mouse models of P. falciparum infection, it was found NK cells will preferentially interact and clear the infected red blood cells in a contact dependent manner (Chen et al., 2014). The Chen et al (2014) system would be ideal for examination of this because instead of the mouse specific P. berghei, most commonly used for mouse-malaria studies, it would be possible to examine human NK cells harboring the L318Q variant and monitor the NK cell response to both the more virulent
P. falciparum and the South Asian specific P. vivax. These studies would allow for a comprehensive analysis of immune cell populations, IFNγ response, BBB integrity, and whether the L318Q alters localization or signaling. This would be the most logical next
72 step for understanding any potential influence the S1PR5 L318Q variant would have over conferring resistance to malaria
Although analysis of the other S1PR family variants didn’t yield another biologically interesting path like S1PR5 L318Q, there was important information gathered from the studies. Some of the genetic variants examined in the S1P receptor family did have known biological consequences such as S1PR2 R108P and Y140C, both of which result in a dead receptor and lead to profound deafness and minor lower limb defects (Santos-Cortez et al., 2016). The variants in S1PR1, A11D and R13G, have higher prevalence in populations at risk for cardiovascular disorders, but that study showed no significant defect when comparing the WT S1PR1 to the R13G variant
(Obinata et al., 2014). However, using my simplified yeast system, where there is but a single human receptor expressed, I observed no discernable difference in A11D compared to WT S1PR1, but a rightward shift in the EC50 of the R13G variant. The
Obinata study used a high density lipoprotein (HDL) bound form of S1P which alters the exact concentration the cells are exposed during the experiments. In addition, they used a HUVEC line, which may express other S1P receptors which all are known to activate
Gαi like S1PR1. The apparent protective effect may be due to some other mechanism, potentially through lymphocyte-endothelial cell interaction dysfunction, as they discuss in the publication. This is very plausible, as S1PR1 function is highly important to lymphocyte function and specifically trafficking, the rationale for the targeting of S1PR1 in RRMS. Also the activation of S1PR1 is known to down regulate the β1AR in cardiomyocytes, so there may be some complex interplay with a variant where S1P exhibits similar efficacy in signaling but decreased potency (Cannavo et al., 2013).
These variants, specifically R13G, should be investigated from a systemic analysis using a rodent model system. Although there was no significant effect on signaling with
73 S1PR1 A11D, the receptor MAF is higher in African populations compared to other populations. This enrichment may be due to a fitness contribution or perhaps a genetic bottleneck. If beneficial, the variant effect may be the result of some other biological interaction than altered S1P signaling. This could not be assessed in the yeast system, and whether that is protein-protein complexes with other GPCRs or membrane associated proteins remains to be seen.
I analyzed the most genetic variants in the S1PR2 subtype. The R108P and
Y140C de novo variants found in the consanguineous Pakistani family served as ideal negative controls because of the known signaling defects from the residue substitutions.
I also analyzed some variants of S1PR2 which were previously studied as markers for early onset of Type II diabetes (T2D). The N10K and V286A variants were both analyzed as potential markers for T2D and they discovered the V286A variant was implicated in the incidences and age of onset for Type II diabetes (Kozian et al., 2010).
My results indicated there is minimal signaling defect in neither the S1PR2 N10K nor
V286A variants. The S1PR2 R60Q variant caused the greatest change in S1P activation. The variant nearly abrogates receptor function shifting the EC50 by many orders of magnitude. Eventually at the highest concentrations of S1P tested I observed a near WT-like efficacy, but this is not biologically normal and it would be expected to have some potential significant effect. There is currently no known deleterious function in individuals carrying the S1PR2 R60Q variant, however it is a very rare variant having with an average MAF of 0.023 % (GnomAD). To understand the function of S1PR2 it would be interesting to see how the R60Q variant altered biological pathways, because unlike the R108P and Y140C variants which eliminate receptor function, the R60Q maintains some functionality. The region on S1PR2 where the R60Q variant occurs is at the beginning of the first intracellular loop which interacts with Gαq in thyrotropin receptor
74 (Kleinau et al., 2010). Therefore, this effect may be partly due to ineffective transductance of the S1P binding to S1PR2 through to the intracellular G protein effector.
The variants in S1PR3 exhibited limited functional disruption, with the T167M variant exhibiting a marginal decrease in potency with respect to S1P. However, there is no indication this variant has any potential significant biological consequence.
Utilizing the yeast system for discovery of a S1PR5 specific antagonist (Appendix
A) and for probing the functional differences of variants on current and potential RRMS drugs provided a mix of results. I probed for S1PR5 antagonists as a treatment for
NKLGL leukemia however, more sensitive methods for the analysis of competitive inhibitors are required as I experienced significant signal-to-noise issues leading to the shelving of experimentation. Analyzing compounds for direct antagonistic properties requires a very stable and established system wherein the agonist can be added to achieve a certain level of activation as to not be overpowering or under stimulating.
Those experiments were performed in a naïve manner and most likely resulted in diminished results. The S1PR5 antagonist discovery studies were performed before I utilized the most effective method of S1P solubilization. Since adjusting my methods and decreasing the signal-to-noise it would be prudent to reexamine the top hits from that work. Again, I do not believe these data are worthless but are in fact worth further exploration, by potentially adjusting the yeast system to produce a more robust or alternate signal which could be more sensitive for antagonist studies. Also, as noted, the RRMS treatments serve as immediate agonists for the S1P receptors, allowing for my system to be used for a single receptor/single drug response analysis. Although there was limited differential effects of variant activation by RRMS drugs, fingolimod, ponesimod, siponimod, MT1303p, and RPC1074 when compared to S1P, I did observe
75 how those drugs selectively activated the receptors and how the efficacy and potency of the treatments compare to S1P. The most interesting aspect of the drug-variant studies are the results of RPC1074. This is a novel drug in the first stages of testing, and supposedly exhibits selectivity for S1PR1 and S1PR5, as most RRMS drugs. However,
I observed a highly reproducible high dose-activation of S1PR2, which was unexpected.
The company developing the drug will hopefully examine this result and establish whether the activation is specific to the yeast system or in fact a legitimate concern.
This effect may be a consequence of the promiscuity of S1PR2 and the activation may be a result of biased agonism. Utilizing the other G protein subtype yeast strains to analyze whether the RPC1074 activation of S1PR2 is related to the GPA1 G protein or if changing the G protein effector diminishes the activity would be of interest.
.
76 Appendix A Yeast as a Platform For Drug Discovery
Preface
The work presented in this dissertation derived from the initial project with the goal to discover specific antagonists to S1PR5 for the treatment of large granular lymphocyte (LGL) leukemia. I developed an assay using yeast carrying a single S1P receptor 1,2,3, or 5 to perform a screening of biologically relevant molecules in the
Spectrum2000 library. Although the work was never able to be used for translational studies, it began the ground work for the research presented in Chapters 2 and 3.
Introduction
Targeting S1PR5 with a receptor specific antagonist would be incredibly beneficial to individuals suffering from both T and NK cell LGL leukemia, but most specifically of the natural killer cell origin. Inhibition of S1PR5 via a receptor specific antagonist could induce leukemic LGL apoptosis and the first curative disease for the malignancy. LGL leukemia is marked by dysregulated growth of CD3+ cytotoxic T (CT) cells and CD3- natural killer (NK) cells (Kothapalli et al., 2002). Many pathways have been implicated in the progression of LGL leukemia including IL-15 signaling, JAK/STAT signaling, constitutive Ras/Raf/MEK/ERK activation, and sphingolipid metabolism and signaling dysregulation (Lamy et al., 2017).
S1PR5 expression is up-regulated in T cell LGL (TLGL) leukemia compared to the other receptor family members in both naïve and activated PBMCs and normal T cells (Kothapalli et al., 2002). In NK cells S1PR5 has been identified to be crucial to mobility and trafficking of mature cells and for certain functions (Drouillard et al., 2017).
Targeting sphingolipid signaling and metabolism has recently been shown to be a potential therapeutic option via treatment of TLGL leukemia cells with fingolimod, a
77 S1PR1,3,4, and 5 functional antagonist causing induced sensitization to Fas mediated apoptosis. NKLGL leukemia exhibited increased apoptosis dependent on Mcl-1 lysosomal degradation upon FTY720 treatment (Liao et al., 2011) . Treatment of a rat model of NKLGL leukemia with fingolimod resulted in complete remission in 5 of 16 rats treated with the remaining 11 rats experiencing improvements of decreased white blood cell count and maintained platelet cell counts for multiple weeks post treatment when compared to controls. Fingolimod, although an important immunomodulator, is not selective for S1PR5 or entirely effective for the treatment of LGL leukemia. Aggressive
NKLGL leukemia results in a limited life expectancy of 2 months and to date there is no effective treatment (Lima, 2013). Identification of a receptor specific antagonist to
S1PR5 will create a needed treatment for patients suffering from all LGL leukemia types, especially aggressive NKLGL leukemia.
Sampling a small scale Spectrum2000 (MicroSource Discovery Systems Inc) molecule collection I seek to identify S1PR5 specific antagonists for the treatment of
LGL disorders. The Spectrum collection is comprised of approved and unapproved drugs, bioactive compounds and natural products spanning a broad chemical structure panel for economic small scale high-throughput experimentation. Utilizing my established yeast-luciferase assay I identified potential candidates for LGL treatment with partial S1PR5 specificity. Unlike current RRMS treatments, this work seeks to discover direct antagonists of S1PR5. Because of this goal, I used a classical antagonist approach co-treating the cells with a dose of S1P to achieve 50% maximum signal output (EC50), described in Appendix B, and then examining whether any compounds decreased the signal compared to S1P treatment alone.
78 Results
Spectrum2000 High Throughput Screening
Screening the Spectrum2000 drug discovery library, detailed in Appendix B:
Methods and Materials, I found several compounds where I observed significant reduction in luciferase activity compared to S1P treatment (Table A-1). The values represent the fold change of luciferase signal output compared to S1P alone. Control strains (Rec Null) transformed with empty vector were used to eliminate false positives for antagonistic properties. S1PR1, S1PR2, and S1PR3 were then treated with the 42 candidate compounds to determine selectivity of the antagonists, after the initial screening against S1PR5. I observed antagonist activity for bleomycin in all receptors, likely because this compound is a cancer treatment drug that inhibits cellular growth via
DNA damage (Hecht, 2000). Methyl-parathion, an insecticide, has some antagonist activity for S1PR2 while not inhibiting activation of S1PR1 or S1PR3.
Triacetylresveratrol, a derivative of the resveratrol found in red wine, known to induce p53 activity also exhibited antagonist activity for S1PR2 but not S1PR1 or S1PR3. The remaining 39 candidate compounds did not antagonize S1P activation of the receptors.
79
Compound Rec Null S1P1 S1P2 S1P3 S1P5 4,6-DIMETHOXY-5-METHYLISOFLAVONE 1.06 1.06 1.17 1.04 0.49 DIHYDRO-OBLIQUIN 1.14 1.06 0.86 1.05 0.60 ATROPINE 1.09 1.02 1.33 0.88 0.64 BETAMETHASONE VALERATE 1.25 1.04 1.25 0.90 0.66 DIOSMETIN 0.96 1.04 1.38 0.89 0.68 Dichlorophene 1.09 1.35 1.29 0.99 0.70 ARTHONIOIC ACID 1.14 1.02 0.94 0.87 0.71 LITHOCHOLIC ACID 1.09 0.84 1.29 0.86 0.71 BLEOMYCIN (bleomycin B2 shown) 1.13 0.62 0.57 0.64 0.73 THIOPENTAL SODIUM 1.18 1.09 0.87 1.09 0.75 3,7-DIHYDROXYFLAVONE 1.04 1.08 1.00 0.88 0.75 beta-AMYRIN 1.25 1.44 1.23 1.03 0.75 MEPHENESIN 1.25 1.07 1.44 0.93 0.76 AZASERINE 1.09 1.12 1.44 1.75 0.76 SODIUM FLUOROACETATE 1.03 1.12 1.05 1.00 0.77 METHYL PARATHIONE 1.11 1.09 0.78 0.99 0.77 AUSTRICINE 1.01 1.10 1.17 0.90 0.77 LARIXINIC ACID 1.17 1.36 1.04 0.98 0.77 BENZALKONIUM CHLORIDE 1.25 1.36 1.13 1.04 0.77 GLICLAZIDE 1.05 1.11 1.16 1.02 0.78 2,6-DI-t-BUTYL-4-METHYLPHENOL 1.04 1.06 0.97 1.01 0.78 CEFUROXIME SODIUM 1.17 1.06 1.15 1.02 0.78 DISULFOTON 1.10 1.10 1.05 1.30 0.78 TROLEANDOMYCIN 1.16 1.10 1.08 1.10 0.78 1,3,5-TRIMETHOXYBENZENE 1.13 1.04 1.39 0.95 0.78 ETHYL 1-BENZYL-3-HYDROXY- 2-OXO[5H]PYRROLE-4-CARBOXYLATE 1.15 1.12 1.02 1.05 0.78 TRIACETYLRESVERATROL 1.17 1.02 0.79 1.02 0.78 PIROMIDIC ACID 1.17 1.14 1.82 1.06 0.78 PASINIAZID 1.09 0.94 0.85 1.06 0.78 APHYLLIC ACID 1.10 1.07 1.01 0.96 0.78 PINOCEMBRIN 1.05 1.07 0.94 0.97 0.79 PIRACETAM 1.13 1.05 1.36 0.95 0.79 ANGOLENSIN ® 1.13 1.20 1.00 0.94 0.79 ORTHOTHYMOTINIC ACID 1.17 1.02 1.31 0.93 0.79 CLENBUTEROL HYDROCHLORIDE 1.17 0.97 0.91 0.99 0.79 METHYLMETHANE SULFONATE 1.08 1.09 1.08 1.04 0.80 DIMETHYL 4,4-o-PHENYLENE-BIS (3-THIOPHANATE) 1.06 1.07 1.01 1.00 0.80 RANITIDINE 1.16 0.97 1.00 0.94 0.80 URSOCHOLANIC ACID 1.16 1.35 1.09 0.96 0.81 SALSOLINE 1.16 0.96 0.97 1.01 0.81 SULFAMETHOXYPYRIDAZINE 1.17 1.08 1.29 1.01 0.81 BRUCINE 1.17 1.47 1.03 0.99 0.81
Table A-1. Screening of Spectrum2000 yields potential S1PR5 specific antagonist compounds. Each compound is tested against a control strain to account for cell death and then each receptor type evaluated for a decrease in luciferase signal compared to a S1P treatment alone. Values are an average of n = 3.
80 Molecular Modeling to Aid in Compound Selection
In addition to the high throughput screen performed using the Spectrum2000 set,
Dr. Shen-Shu Sung performed a molecular docking analysis of the Spectrum2000 set to cross reference and determine whether the yeast assay results were theoretically plausible. Combining the docking score and screening data I narrowed the group to 18 compounds that were comprised of the best docking score of the 42 original candidate compounds and continued to examine those select compounds for S1PR5 antagonistic effects (Figure A-1 B). Of the 18 compounds tested I found four that showed antagonist activity for S1PR5 (Figure A-1 A). Compounds 1 (angolensin), 11 (pinocembrin), and 14
(arthonionic acid) all decreased S1P stimulation levels by 55%, 50%, and 60%, respectively (Figure A-1 A). Compound 15 (dichlorophene), showed the greatest effect decreasing the luciferase signal by nearly 80% (Figure A-1 A). However, arthonionic acid and dichlorophene both exhibited decreased cell counts post assay which contributed to the S1P treatment luciferase signal inhibition (data not shown). I
Figure A-1. Molecular docking aids in confirmation of potential S1PR5 inhibitors. A) Comparison of 10 μM drug treatment with indicated drugs on S1PR5 luciferase signaling. Values are normalized to S1P treatment alone, n = 3 +/- standard deviation. B) Compound list with tabulated docking score. The more negative the score, the better the docking prediction.
81 observed moderate antagonist activity in compounds 4 (atropine), 7 (mephenesin), 13
(rosuvostatin), 16 (3,7-dihydroxyflavone), and 18 (4,6-dimethoxy-5-methylisoflavone)
(Figure A-1 A). Compounds 3,7-dihydroxyflavone and 4,6-dimethoxy-5-methylisoflavone exhibited similar decreased cell counts contributing to the decrease in S1P signal (data not shown).
Candidate Drugs Show S1PR5 Selectivity
After the retesting of the docking score selected compounds on S1PR5, I examined selected compounds on the remaining S1P receptors. When S1PR1 and
S1PR3 were treated with angolensin I observe antagonistic activity but less so than angolensin treatment of S1PR5 (Figure A-2, Compound 1). Atropine appears to have minimal cross reactivity with the other receptors but is not an effective inhibitor of S1PR5 compared to the other compounds tested (Figure A-2, Compound 4). Mephenesin behaves similar to atropine, having marginal S1PR5 inhibition and seemingly little to no inhibition of S1P signaling in S1PR1, S1PR2, or S1PR3 (Figure A-2, Compound 7).
Pinocembrin exhibits a 50% decrease in S1PR5 signal output, consistent with both the preliminary screen and the docking screen (Figure A-2, Compound 11). In addition, pinocembrin appears to exhibit S1PR5 selectivity with minimal signal inhibition of
S1PR1, S1PR2, or S1PR3. Rosuvostatin shows limited inhibition of S1PR1, S1PR2, and S1PR3, but also exhibits limited inhibition of S1PR5 (Figure A-2, Compound 13).
82
Figure A-2. Select S1PR5 inhibiting molecules exhibit S1PR5-selectivity. Percent stimulation relative to the S1P alone treatment of Spectrum2000 candidate compounds. Compound identifiers can be found in Figure A-1 B. All data points are n = 3 plotted as the average with standard deviation using GraphPad Prism 6.0.
Discussion
This limited work was expected to produce significant translational results and the hope was to begin experiments examining the effects of the S1PR5 antagonists in
LGL leukemia cell lines and animal models. These experiments were never undertaken and therefore the potential for angolensin and pinocembrin as LGL leukemia treatments is yet to be analyzed.
Angolensin is a relatively understudied compound, an isoflavonoid formed during human gut microbiota metabolism. Angolensin binds with estrogen receptors and can help to inhibit cells over expressing estrogen receptors (Pfitscher et al., 2008; Powell et al., 2012). In addition, angolensin and isoflavones exert anti-inflammatory activity via agonist activation of PPAR α/γ (Medjakovic et al., 2010). Although there is no documented interaction between S1P receptors and angolensin, the docking score and
83 antagonist activity against S1PR5 signal transduction should prompt investigation into angolensin as a potential treatment for LGL leukemia. In addition, because angolensin is a metabolite in the human digestive tract this compound is already naturally occurring and could be manipulated via diet or microbiome alteration.
Pinocembrin is another flavonoid compound, found in extracts of honey, ginger, and other plants. Like angolensin, pinocembrin exerts anti-inflammatory, neuroprotective, and cardio-protective effects. Interestingly, pinocembrin helps with
Parkinson’s disease via decreasing reactive oxidative species and increasing Nrf2 protein levels (Jin et al., 2015). Because sphingolipid metabolism and signaling were also recently tied to the survival of dopaminergic neurons and the prevention of
Parkinson’s disease via reduction of ROS in an S1PR1 dependent manner
(Sivasubramanian et al., 2015).
Although there is no literature to suggest pinocembrin acts as an antagonist of
S1PR5 signaling or modifier of S1P metabolism or other related pathways, my research presented here clearly shows that investigation of the flavonoids angolensin and pinocembrin as inhibitors of S1PR5 could be fruitful.
Appendix B
Materials and Methods
Strains, Plasmids, and Primers
To construct yeast strains functionally expressing S1PR1, S1PR2, and S1PR3, we PCR amplified each of the coding regions of the corresponding receptors from human genomic DNA and cloned them into the yeast expression vector CP1651 as previously described (Klein et al., 1998). For S1PR5, I obtained a synthetic version of the gene with optimized yeast codons from GeneArt and cloned it into CP1651.
Plasmids were introduced by transformation into appropriate yeast strains as indicated along with plasmid B2943 (URA3 pFus1-luciferase) as needed. Variant receptor plasmids were created using QuikChange XL Site-Directed Mutagenesis Kit (Agilent).
The sequences of all the wild type and variant receptors were confirmed by Sanger sequencing. Functional expression of the constructs was confirmed by growth of the
Strain Genotype CY9623 FUS1p-HIS3 GPA1p-G@i1 can1 far1*1442 his3 leu2 lys2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY9624 FUS1p-HIS3 GPA1p-GPA1(41)-G@i1 can1 far1*1442 his3 leu2 lys2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY17751 FUS1p-HIS3 GPA1-G@i2(5) can1 dap2*6253 far1*1442 his3 leu2 lys2 sst2*2 ste13*6077 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY11701 FUS1p-HIS3 can1 cyh2 far1*1442 gpa1(41)-G@i2 his3 leu2 lys2 ste14::trp1::LYS2 ste18g6-3841 ste3*1156 stp22::TRP1 tbt1-1 trp1 ura3 CY12946 FUS1p-HIS3 GPA1G@i2(5) can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura CY4925 FUS1p-HIS3 can1 far1*1442 gpa1(41)-G@i3 his3 leu2 lys2 ste14::trp1::LYS2 ste3*1156 trp1 ura3 CY7965 FUS1p-HIS3 can1 far1*1442 gpa1(41)-G@i3 his3 leu2 lys2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY18906 FUS1p-HIS3 can1 far1*1442 gpa1(41)-G@i3 his3 leu2 lys2 mf@1*6180 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY9554 FUS1p-HIS3 can1 far1*1442 gpa1(41)-G@i2-G@oA his3 leu2 lys2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY9556 FUS1p-HIS3 can1 far1*1442 gpa1(41)-G@i2-G@oB his3 leu2 lys2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY18043 FUS1p-HIS3 GPA1G@o(5) STE18g6-3841 can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY18045 FUS1p-HIS3 GPA1G@o(5) can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY12949 FUS1p-HIS3 GPA1-G@12(5) can1 far1*1442 his3leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 CY13395 FUS1p-HIS3 GPA1G@12(5) can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste18g6-3841 ste3*1156 tbt1-1 trp1 ura CY14639 FUS1p-HIS3 GPA1p-GPA1-G@13(5) can1 cyh2 far1*1442 his3 leu2 lys2 ste14::trp1::LYS2 ste18g6-3841 ste3*1156 tbt1-1 trp1 ura3 CY15074 FUS1p-HIS3 GPA1G@13(5) STE18g6-3841 can1 far1*1442 his3 leu2 lys2 CY15349 FUS1p-HIS3 GPA1G@13(5) can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 Y4070 FUS1p-HIS3 GPA1-G@12(5) can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 tbt1-1 trp1 ura3 HTB2-::mCherry-HygMx CY11718 FUS1p-HIS3 GPA1-3907 can1 far1*1442 his3 leu2 lys2 sst2*2 ste14::trp1::LYS2 ste3*1156 stp22::TRP1 tbt1-1 trp1 ura3
Table B-1. Genotype of strains used throughout yeast coupling and luciferase assays.
85 transformants on SC-His plates (Amberg, c2005.) in the presence but not the absence of
S1P. Yeast strains used can be seen in Table 1-B.
Wildtype and variant receptor genes were cloned in pCMV-Myc (Clonetech) for transient transfections and into pLV[Exp]-Puro-TRE (Cyagen) for lentiviral transduction.
Plasmid pLV[Exp]-mCherry/Neo-UBC>rtTA_M2 (Cyagen) was used to activate transcription of transduced receptors.
Luciferase Assay and Analysis
Yeast transformants carrying an S1P receptor construct and the luciferase reporter plasmid were grown overnight in SC-Leu-Ura at 30o C on a rotating roller drum.
Cultures were diluted in the same medium to an OD660 of 0.05 and allowed to grow four
o hours at 30 C to an OD660 of 0.2. S1P (Avanti Polar Lipids) was solubilized at 1 mM in methanol by sonication and 100 ul of the solution was evaporated with filtered air. The residual 100 nmol film was reconstituted to 400 uM in a solution of 10 mM Na2CO3 and
2% β-cyclodextrin (Sigma-Aldrich and MP Biomedicals, respectively). Costar 96 well white opaque assay plates were prepared with 5 ul of 3x serial dilutions of S1P prepared in a SC–Leu–Ura and 0.25 % Pluronic F-127 pH 6.8 (Thermo Fisher Scientific). Cells were harvested by centrifugation and resuspended in an equal volume of SC–Leu–Ura, pH 6.8. Aliquots (45 ul) of cell suspension were added to appropriate wells and the plates covered with breathable film and incubated at 30 oC for five hours. Wells were then acidified by addition of 4 ul of 0.1 M HCl (Sigma-Aldrich) prior to addition of with 50 ul of 1 mM D-luciferin (Gold Biotechnology) in 0.1 M sodium citrate pH 4.5. Plates were read using a GloMax luminometer (Promega). Luminescence measurements were imported to Excel and normalized to the average luminescence of untreated control wells. Emax values of WT S1PRs in chapter 2 were calculated as fold stimulation over no treatment controls. EC50 values were calculated using GraphPad Prism and nonlinear fit
86 of the log10 transformed dose response curve using variable slope with four parameters and automatic outlier elimination.
Coupling Assay
Saccharomyces cerevisiae strains expressing an S1P receptor were grown overnight in SC–Leu–Ura medium and then diluted to an OD660 of 0.05 in the same medium. After four hours growth at 30ºC cells (5 ml) were harvested by centrifugation, washed twice with water and resuspended in 1 ml water. Cell suspension (200 ul) was spread onto SC–Leu–Ura–His plates containing 1 mM aminotrizole and adjusted to pH
6.8. After surface liquid was absorbed, 10 ul of 0.0394, 0.394, and 3.94 mM S1P, dissolved in 10% ammonium hydroxide in methanol, were spotted onto the plates and allowed to dry. Plates are incubated at 30oC for 72 hours to allow for growth of cells around spots.
Cell Culture and Cell lines
Human 293T cells were grown in DMEM (Lonza) supplemented with 15% Fetal
Calf Serum. Transient plasmid transfections were performed using Lipofectamine LTX
(Invitrogen). For stable transduction, viral particles were produced using Trans-Lentiviral
ORF Packaging Kit (Dharmacon). Cells were transduced with the receptor expressing virus and selected with 1.5 g/ml puromycin. Cells were also transduced with virus expressing the tetracycline-controlled transactivator rtTA, and mCherry-positive cells were sorted using a FACSAria III cell sorter (BD Biosciences). To induce cDNA expression, cells were treated with 3 ug/ml doxycycline for 24h.
F actin microscopy and analysis
Transiently transfected (S1PR5 WT and L318Q) as well as mock transfected cells were grown on cover slips according to manufacturer’s protocol for F-Actin
87 Visualization Biochem Kit (Cystoskeleton). Cells were treated with 100 nM S1P or buffer for 1 hour. After staining and mounting, cells were imaged using a Leica SP8 scanning confocal microscope with a white light laser. F-actin volume was calculated using Imaris
Imaging software. Volumes were calculated from the bottom 25% of the cell and F-actin volume were averaged over the number of cells per frame. Statistical analysis was performed used GraphPad Prism 6.0 and Two-Way Anova with multiple comparisons comparing cell means regardless of row and column using Bonferroni correction.
F/G actin protocol and analysis
Stably transformed HEK293T cell lines were seeded in a 6-well plate and allowed to grow in DMEM supplemented with 15% Fetal Calf Serum for 24 hours. Cells were then washed, switched to serum free media and treated with doxycycline. Cells were processed at 0, 30, and 60 minutes following addition of 100 nM S1P by removal of medium and addition of 250 ul of warmed F-actin stabilization and lysis buffer
(Cytoskeleton Inc). Cells were harvested and homogenized by repeat pipetting with a pipette tip. Following centrifugation at 350 x g for five minutes at room temperature, supernatants 100 ul were transferred to ultracentrifuge tubes and spun at 100,000 x g at
37oC for one hour. Supernatants were removed and retained and the F-actin pellet was resuspended in 100 ul of F-actin destabilization buffer (Cytoskeleton Inc) on ice.
Samples of supernatants and resuspended pellets were fractionated on a 4-20% SDS-
PAGE gel, transferred to PVDF membrane, probed sequentially with anti-actin rabbit polyclonal antibody (Cytoskeleton Inc) and anti-rabbit IgG antibody and visualized by chemiluminescence (Azure Biosystems) on a UVP Biospectrum Imaging System.
Multiple t-tests of data were performed using GraphPad Prism 6.0.
88 pAkt Analysis
Stably transformed HEK 293T cells were grown as described above and treated with 100 nM S1P for 0, 20, and 40 minutes. Cells were removed by scraping and harvested by centrifugation at 500 x g for five minutes at room temperature. Cell pellets were resuspended in lysis buffer (1% Nonidet P-40 Substitute, 50 mM Tris-HCl ph 7.5,
150 mM NaCl 10% glycerol), lysed by sonication for 15 seconds on ice and nutated at
4oC for one hour. Lysate was cleared by centrifugation at 16000 x g for 25 minutes at
4oC and 50 l supernatant mixed with 50 ul 2x Laemmli sample buffer with 700 mM 2- mercaptoethanol and incubated at 95oC for 10 minutes. Lysates were fractionated by
SDS-PAGE, transferred to PVDF membranes and probed with Anti-pAkt S473 9Cell
Signaling, #4060). After visualization, membranes were stripped using Restore Western
Blot Stripping Buffer (ThermoScientific) and then reprobed with anti-pan Akt (Cell
Signaling, #4685). Two-Way ANOVA with comparing the rows within each column using
Fisher’s LSD test was performed using GraphPad Prism 6.0.
Acyl-biotin exchange
Stably transformed HEK293 cells were seeded in 10 cm plates allowed to grow in
DMEM + 15% FCS to 80% confluency then depleted of serum for 24 hours. Cells were harvested by centrifugation and suspended using ABE LB pH 7.4 (1% Nonidet-P40, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% Glycerol, 75 mM NEM) with protease inhibitors
(G Biosciences) and PMSF. Cells were lysed by sonication for 15 seconds on ice and nutation at 4oC for 1 hour. Cell debris was removed by centrifugation at 4oC for 25 minutes at 16,000 x g. Supernatants were transferred to a cold microcentrifuge tube and precleared by incubation with 30 ul Protein G dynabeads (Thermo Fisher) for one hour at 4oC. A aliquot (50 ul) of precleared supernatant was retained as input control and the remaining supernatant (450 uL) was incubated with 2 ug of Anti-c-Myc Antibody (A7470
89 Sigma Aldrich) by nutation overnight at 4oC. The supernatants were then incubated with
30 ul dynabeads for three hours at 4oC. Beads were collected and washed in ABE LB ph
7.4 without NEM three times. Ten percent of the beads were removed as a negative control for palmitoylation cleavage. ABE LB pH 7.4 containing 1 M hydroxylamine was added to the remaining beads and the suspension nutated at room temperature for one hour. Beads were washed three times with ice cold ABE LB pH 7.4 and then incubated with 2 mM EZ-Link BMCC-Biotin (Thermo Fisher) in ABE LB pH 7.4 by nutation at room temperature for two hours. Beads were washed three times with ABE LB pH 7.4 and eluted into 1x loading buffer (Bio Rad) at 85oC for 15 minutes. Western blot analysis was performed first for biotin signal using Streptavidin-HRP (Cell Signaling) and then using
Anti-c-Myc antibody of mouse origin (Sigma Aldrich). Both biotin and myc-tag signal were statistically analyzed using ordinary one-way ANOVA comparing the WT and
L318Q S1PR5 to the control HEK293T for both signals. Performed using GraphPad
Prism 6.0.
RRMS Drugs
All drugs were obtained from Celgene, except S1P (Avanti Polar Lipids). S1P was solubilized and stored as previously described. Siponimod, ponesimod, MT1303,
MT1303-p, and RPC1074 were solubilized in DMSO to 10 mM while fingolimod was solubilized in DMSO to 0.5 mM. Experimental concentrations were 10x and then diluted
1:10 with mid-log phase growing cells.
EC50 and Emax Calculations of RRMS Drug Assay
All dosages were fold change normalized to no drug control. Using GraphPad
Prism 6.0 fold change normalized dose response curves of WT receptors were then normalized to a 100% scale. This scale was then used to transform variant receptor curves to 100% scale by comparing wild type to variant curves and adjusting the wild
90 type percent by the percent increase or decrease for the same dosage response in the variant. Emax values of RRMS drug treatment of WT and variant S1PRs were calculated as a percent change from the S1P fold stimulation of equal treatment dosage. Emax was calculated from this curve as well. EC50 values were calculated using GraphPad Prism
6.0 and nonlinear fit of the log10 transformed dose response curve using variable slope with four parameters and automatic outlier elimination.
Spectrum2000 Drug Collection Screening
The 2560 compounds in the SPECTRUM Collection were selected by medicinal chemists and biologists to provide a wide range of biological activities and structural diversity for screening. Each compound has a minimum of 95% purity and is reconstituted in DMSO to 10 μM for 1 μM working concentration. The collection is 60% approved drugs from USA, Europe, and Japan, 25% natural products including 640 pure products with unknown biological function selected on the basis of chemical class ensuring structural diversity, and 10% “Other” bioactive compounds including non – drug enzyme inhibitors, competitive inhibitors of receptors, active membrane components, and some known toxins. The screen was performed much in the way previously described except to adjust for assessing competitive inhibition, 150 nM S1P was co – treated with the 1 μM in an empty vector CY12946 strain and the S1PR5 – CY12946 strain. Analysis was performed comparing S1P alone luciferase signal to compound treatments.
References
Allende, M.L., Bektas, M., Lee, B.G., Bonifacino, E., Kang, J., Tuymetova, G., Chen, W., Saba, J.D., and Proia, R.L. (2011). Sphingosine-1-phosphate lyase deficiency produces a pro-inflammatory response while impairing neutrophil trafficking. J. Biol. Chem. 286, 7348–7358.
Amberg, D.C. (c2005.). Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual / (Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press,).
Andrieu, G., Ledoux, A., Branka, S., Bocquet, M., Gilhodes, J., Walzer, T., Kasahara, K., Inagaki, M., Sabbadini, R.A., Cuvillier, O., et al. (2017). Sphingosine 1-phosphate signaling through its receptor S1P5 promotes chromosome segregation and mitotic progression. Sci. Signal. 10.
Antman, K.H. (2001). Introduction: The History of Arsenic Trioxide in Cancer Therapy. The Oncologist 6, 1–2.
Ashley, E.A. (2016). Towards precision medicine. Nat. Rev. Genet. 17, 507–522.
Balatoni, B., Storch, M.K., Swoboda, E.-M., Schönborn, V., Koziel, A., Lambrou, G.N., Hiestand, P.C., Weissert, R., and Foster, C.A. (2007). FTY720 sustains and restores neuronal function in the DA rat model of MOG-induced experimental autoimmune encephalomyelitis. Brain Res. Bull. 74, 307–316.
Baran, Y., Salas, A., Senkal, C.E., Gunduz, U., Bielawski, J., Obeid, L.M., and Ogretmen, B. (2007). Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells. J. Biol. Chem. 282, 10922–10934.
Baud, V., Chissoe, S.L., Viegas-Péquignot, E., Diriong, S., N’Guyen, V.C., Roe, B.A., and Lipinski, M. (1995). EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments. Genomics 26, 334–344.
Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.C., Wang, Y., Macke, J.P., Andrew, D., Nathans, J., and Nusse, R. (1996). A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230.
Bjarnadóttir, T.K., Fredriksson, R., Höglund, P.J., Gloriam, D.E., Lagerström, M.C., and Schiöth, H.B. (2004). The human and mouse repertoire of the adhesion family of G- protein-coupled receptors. Genomics 84, 23–33.
Bode, C., Sensken, S.-C., Peest, U., Beutel, G., Thol, F., Levkau, B., Li, Z., Bittman, R., Huang, T., Tölle, M., et al. (2010). Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1-phosphate. J. Cell. Biochem. 109, 1232–1243.
Bolli, M.H., Abele, S., Binkert, C., Bravo, R., Buchmann, S., Bur, D., Gatfield, J., Hess, P., Kohl, C., Mangold, C., et al. (2010a). 2-Imino-thiazolidin-4-one Derivatives as Potent, Orally Active S1P1 Receptor Agonists. J. Med. Chem. 53, 4198–4211.
92
Bolli, M.H., Abele, S., Binkert, C., Bravo, R., Buchmann, S., Bur, D., Gatfield, J., Hess, P., Kohl, C., Mangold, C., et al. (2010b). 2-Imino-thiazolidin-4-one Derivatives as Potent, Orally Active S1P1 Receptor Agonists. J. Med. Chem. 53, 4198–4211.
Brinkmann, V., Davis, M.D., Heise, C.E., Albert, R., Cottens, S., Hof, R., Bruns, C., Prieschl, E., Baumruker, T., Hiestand, P., et al. (2002). The immune modulator FTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. 277, 21453–21457.
Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010). Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897.
Broach, J.R., and Thorner, J. (1996). High-throughput screening for drug discovery. Nature 384, 14–16.
Campbell, R.M., Cartwright, C., Chen, W., Chen, Y., Duzic, E., Fu, J.M., Loveland, M., Manning, R., McKibben, B., Pleiman, C.M., et al. (1999). Selective A1-adenosine receptor antagonists identified using yeast Saccharomyces cerevisiae functional assays. Bioorg. Med. Chem. Lett. 9, 2413–2418.
Cannavo, A., Rengo, G., Liccardo, D., Pagano, G., Zincarelli, C., De Angelis, M.C., Puglia, R., Di Pietro, E., Rabinowitz, J.E., Barone, M.V., et al. (2013). β1-Adrenergic Receptor and Sphingosine-1-Phosphate Receptor 1 Reciprocal Down-Regulation Influences Cardiac Hypertrophic Response and Progression Toward Heart Failure: Protective Role of S1PR1 Cardiac Gene Therapy. Circulation 128, 1612–1622.
CDC.gov (2018). FastStats.
Charest, P.G., and Bouvier, M. (2003). Palmitoylation of the V2 Vasopressin Receptor Carboxyl Tail Enhances β-Arrestin Recruitment Leading to Efficient Receptor Endocytosis and ERK1/2 Activation. J. Biol. Chem. 278, 41541–41551.
CHARGE Consortium Hematology Working Group (2016). Meta-analysis of rare and common exome chip variants identifies S1PR4 and other loci influencing blood cell traits. Nat. Genet. 48, 867–876.
Chaudhry, B.Z., Cohen, J.A., and Conway, D.S. (2017). Sphingosine 1-Phosphate Receptor Modulators for the Treatment of Multiple Sclerosis. Neurotherapeutics 1–15.
Choi, J.W., and Chun, J. (2013). Lysophospholipids and their receptors in the central nervous system. Biochim. Biophys. Acta 1831, 20–32.
Christoffersen, C., Obinata, H., Kumaraswamy, S.B., Galvani, S., Ahnström, J., Sevvana, M., Egerer-Sieber, C., Muller, Y.A., Hla, T., Nielsen, L.B., et al. (2011). Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M. Proc. Natl. Acad. Sci. U. S. A. 108, 9613–9618.
93
Clemens, J.J., Davis, M.D., Lynch, K.R., and Macdonald, T.L. (2005). Synthesis of 4(5)- phenylimidazole-based analogues of sphingosine-1-phosphate and FTY720: Discovery of potent S1P1 receptor agonists. Bioorg. Med. Chem. Lett. 15, 3568–3572.
D’Ambrosio, D., Freedman, M.S., and Prinz, J. (2016). Ponesimod, a selective S1P1 receptor modulator: a potential treatment for multiple sclerosis and other immune- mediated diseases. Ther. Adv. Chronic Dis. 7, 18–33.
Debien, E., Mayol, K., Biajoux, V., Daussy, C., De Aguero, M.G., Taillardet, M., Dagany, N., Brinza, L., Henry, T., Dubois, B., et al. (2013). S1PR5 is pivotal for the homeostasis of patrolling monocytes. Eur. J. Immunol. 43, 1667–1675.
Deng, H., Bernier, S.G., Doyle, E., Lorusso, J., Morgan, B.A., Westlin, W.F., and Evindar, G. (2013). Discovery of Clinical Candidate GSK1842799 As a Selective S1P1 Receptor Agonist (Prodrug) for Multiple Sclerosis. ACS Med. Chem. Lett. 4, 942–947.
Di Pardo, A., Castaldo, S., Amico, E., Pepe, G., Marracino, F., Capocci, L., Giovannelli, A., Madonna, M., van Bergeijk, J., Buttari, F., et al. (2018). Stimulation of S1PR5 with A- 971432, a selective agonist, preserves blood-brain barrier integrity and exerts therapeutic effect in an animal model of Huntington’s disease. Hum. Mol. Genet.
Dilokthornsakul, P., Valuck, R.J., Nair, K.V., Corboy, J.R., Allen, R.R., and Campbell, J.D. (2016). Multiple sclerosis prevalence in the United States commercially insured population. Neurology 86, 1014–1021.
Dinkel, B.A., Kremer, K.N., Rollins, M.R., Medlyn, M.J., and Hedin, K.E. (2018). GRK2 mediates TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that drives PI3Kγ/PREX1 signaling and T cell cytokine secretion. J. Biol. Chem. 293, 14022–14039.
Doorn, R. van, Pinheiro, M.A.L., Kooij, G., Lakeman, K., Hof, B. van het, Pol, S.M. van der, Geerts, D., Horssen, J. van, Valk, P. van der, Kam, E. van der, et al. (2012). Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier. J. Neuroinflammation 9, 133.
Dowell, S.J., and Brown, A.J. (2009). Yeast assays for G protein-coupled receptors. Methods Mol. Biol. Clifton NJ 552, 213–229.
Drouillard, A., Mathieu, A.-L., Marçais, A., Belot, A., Viel, S., Mingueneau, M., Guckian, K., and Walzer, T. (2017). S1PR5 is essential for human NK cell migration towards sphingosine-1 phosphate. J. Allergy Clin. Immunol.
Escribá, P.V., Wedegaertner, P.B., Goñi, F.M., and Vögler, O. (2007). Lipid–protein interactions in GPCR-associated signaling. Biochim. Biophys. Acta BBA - Biomembr. 1768, 836–852.
Evindar, G., Deng, H., Bernier, S.G., Doyle, E., Lorusso, J., Morgan, B.A., and Westlin, W.F. (2013). Exploring amino acids derivatives as potent, selective, and direct agonists of sphingosine-1-phosphate receptor subtype-1. Bioorg. Med. Chem. Lett. 23, 472–475.
94
Finney, C.A., Hawkes, C.A., Kain, D.C., Dhabangi, A., Musoke, C., Cserti-Gazdewich, C., Oravecz, T., Liles, W.C., and Kain, K.C. (2011). S1P is associated with protection in human and experimental cerebral malaria. Mol. Med. Camb. Mass 17, 717–725.
Foord, S.M., Bonner, T.I., Neubig, R.R., Rosser, E.M., Pin, J.-P., Davenport, A.P., Spedding, M., and Harmar, A.J. (2005). International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol. Rev. 57, 279–288.
Fredriksson, R., Lagerström, M.C., Lundin, L.-G., and Schiöth, H.B. (2003). The G- protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272.
Fritze, O., Filipek, S., Kuksa, V., Palczewski, K., Hofmann, K.P., and Ernst, O.P. (2003). Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation. Proc. Natl. Acad. Sci. U. S. A. 100, 2290–2295.
Fryer, R.M., Muthukumarana, A., Harrison, P.C., Mazurek, S.N., Chen, R.R., Harrington, K.E., Dinallo, R.M., Horan, J.C., Patnaude, L., Modis, L.K., et al. (2012). The Clinically- tested S1P Receptor Agonists, FTY720 and BAF312, Demonstrate Subtype-Specific Bradycardia (S1P1) and Hypertension (S1P3) in Rat. PLOS ONE 7, e52985.
Fujino, M., Funeshima, N., Kitazawa, Y., Kimura, H., Amemiya, H., Suzuki, S., and Li, X.-K. (2003). Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720 treatment. J. Pharmacol. Exp. Ther. 305, 70–77.
Gacasan, S.B., Baker, D.L., and Parrill, A.L. (2017). G protein-coupled receptors: the evolution of structural insight. Biophys. 2017 Vol 4 Pages 491-527.
Garris, C.S., Wu, L., Acharya, S., Arac, A., Blaho, V.A., Huang, Y., Moon, B.S., Axtell, R.C., Ho, P.P., Steinberg, G.K., et al. (2013). Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation. Nat. Immunol. 14, 1166–1172.
Gentile, A., Musella, A., Bullitta, S., Fresegna, D., De Vito, F., Fantozzi, R., Piras, E., Gargano, F., Borsellino, G., Battistini, L., et al. (2016). Siponimod (BAF312) prevents synaptic neurodegeneration in experimental multiple sclerosis. J. Neuroinflammation 13, 207.
Gergely, P., Nuesslein‐Hildesheim, B., Guerini, D., Brinkmann, V., Traebert, M., Bruns, C., Pan, S., Gray, N.S., Hinterding, K., Cooke, N.G., et al. (2012). The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate. Br. J. Pharmacol. 167, 1035–1047.
Gloriam, D.E., Fredriksson, R., and Schiöth, H.B. (2007). The G protein-coupled receptor subset of the rat genome. BMC Genomics 8, 338.
Goth, C.K., Tuhkanen, H.E., Khan, H., Lackman, J.J., Wang, S., Narimatsu, Y., Hansen, L.H., Overall, C.M., Clausen, H., Schjoldager, K.T., et al. (2017). Site-specific O- Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase
95
T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage. J. Biol. Chem. 292, 4714–4726.
Gräler, M.H., Bernhardt, G., and Lipp, M. (1998). EDG6, a novel G-protein-coupled receptor related to receptors for bioactive lysophospholipids, is specifically expressed in lymphoid tissue. Genomics 53, 164–169.
Guo, Y.-X., Ma, Y.-J., Han, L., Wang, Y.-J., Han, J.-A., and Zhu, Y. (2015). Role of sphingosine 1-phosphate in human pancreatic cancer cells proliferation and migration. Int. J. Clin. Exp. Med. 8, 20349–20354.
Hakalahti, A.E., Vierimaa, M.M., Lilja, M.K., Kumpula, E.-P., Tuusa, J.T., and Petäjä- Repo, U.E. (2010). Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage. J. Biol. Chem. 285, 28850–28861.
Hänel, P., Andréani, P., and Gräler, M.H. (2007). Erythrocytes store and release sphingosine 1-phosphate in blood. FASEB J. 21, 1202–1209.
Hannun, Y.A., and Obeid, L.M. (2008). Principles of bioactive lipid signalling: lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 9, 139–150.
Hansen, M.R., and Okuda, D.T. (2018). Precision medicine for multiple sclerosis promotes preventative medicine. Ann. N. Y. Acad. Sci. 1420, 62–71.
Hanson, M.A., Roth, C.B., Jo, E., Griffith, M.T., Scott, F.L., Reinhart, G., Desale, H., Clemons, B., Cahalan, S.M., Schuerer, S.C., et al. (2012). Crystal Structure of a Lipid G Protein–Coupled Receptor. Science 335, 851–855.
Harmar, A.J. (2001). Family-B G-protein-coupled receptors. Genome Biol. 2, reviews3013.1-reviews3013.10.
Hauser, A.S., Chavali, S., Masuho, I., Jahn, L.J., Martemyanov, K.A., Gloriam, D.E., and Babu, M.M. (2018). Pharmacogenomics of GPCR Drug Targets. Cell 172, 41-54.e19.
Hecht, S.M. (2000). Bleomycin: new perspectives on the mechanism of action. J. Nat. Prod. 63, 158–168.
Herr, D.R., Grillet, N., Schwander, M., Rivera, R., Müller, U., and Chun, J. (2007). Sphingosine 1-phosphate (S1P) signaling is required for maintenance of hair cells mainly via activation of S1P2. J. Neurosci. Off. J. Soc. Neurosci. 27, 1474–1478.
Hill, S.J. (2006). G-protein-coupled receptors: past, present and future. Br. J. Pharmacol. 147, S27–S37.
Hla, T., Venkataraman, K., and Michaud, J. (2008). The vascular S1P gradient—Cellular sources and biological significance. Biochim. Biophys. Acta BBA - Mol. Cell Biol. Lipids 1781, 477–482.
96
Horan, J.C., Kuzmich, D., Liu, P., DiSalvo, D., Lord, J., Mao, C., Hopkins, T.D., Yu, H., Harcken, C., Betageri, R., et al. (2016). Late-stage optimization of a tercyclic class of S1P3-sparing, S1P1 receptor agonists. Bioorg. Med. Chem. Lett. 26, 466–471.
Horstmeyer, A., Cramer, H., Sauer, T., Müller-Esterl, W., and Schroeder, C. (1996). Palmitoylation of Endothelin Receptor A DIFFERENTIAL MODULATION OF SIGNAL TRANSDUCTION ACTIVITY BY POST-TRANSLATIONAL MODIFICATION. J. Biol. Chem. 271, 20811–20819.
Hou, J., Chen, Q., Wu, X., Zhao, D., Reuveni, H., Licht, T., Xu, M., Hu, H., Hoeft, A., Ben-Sasson, S.A., et al. (2017). S1PR3 Signaling Drives Bacterial Killing and Is Required for Survival in Bacterial Sepsis. Am. J. Respir. Crit. Care Med. 196, 1559– 1570.
Huang, Y.-L., Huang, W.-P., and Lee, H. (2011). Roles of sphingosine 1-phosphate on tumorigenesis. World J. Biol. Chem. 2, 25–34.
Huwiler, A., and Zangemeister-Wittke, U. (2018). The sphingosine 1-phosphate receptor modulator fingolimod as a therapeutic agent: Recent findings and new perspectives. Pharmacol. Ther. 185, 34–49.
Im, D.S., Clemens, J., Macdonald, T.L., and Lynch, K.R. (2001). Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors. Biochemistry 40, 14053–14060.
Ingham, N.J., Carlisle, F., Pearson, S., Lewis, M.A., Buniello, A., Chen, J., Isaacson, R.L., Pass, J., White, J.K., Dawson, S.J., et al. (2016). S1PR2 variants associated with auditory function in humans and endocochlear potential decline in mouse. Sci. Rep. 6, 28964.
Jaillard, C., Harrison, S., Stankoff, B., Aigrot, M.S., Calver, A.R., Duddy, G., Walsh, F.S., Pangalos, M.N., Arimura, N., Kaibuchi, K., et al. (2005). Edg8/S1P5: An Oligodendroglial Receptor with Dual Function on Process Retraction and Cell Survival. J. Neurosci. 25, 1459–1469.
Jiang, L.I., Collins, J., Davis, R., Lin, K.-M., DeCamp, D., Roach, T., Hsueh, R., Rebres, R.A., Ross, E.M., Taussig, R., et al. (2007). Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway. J. Biol. Chem. 282, 10576–10584.
Jin, X., Liu, Q., Jia, L., Li, M., and Wang, X. (2015). Pinocembrin Attenuates 6-OHDA- induced Neuronal Cell Death Through Nrf2/ARE Pathway in SH-SY5Y Cells. Cell. Mol. Neurobiol. 35, 323–333.
Justin R. Sysol, Jiwang Chen, Panfeng Fu, Joe G.N. Garcia, Viswanathan Natarajan, and Roberto F. Machado (2015). Sphingosine-1-Phosphate (S1P) Induces Hypoxia- Inducible Factor-1 Alpha (HIF1-α) Activation in Human Pulmonary Artery Smooth Muscle Cells. In D43. CELL FATE, (American Thoracic Society), pp. A5957–A5957.
97
Kang, Y., Zhou, X.E., Gao, X., He, Y., Liu, W., Ishchenko, A., Barty, A., White, T.A., Yefanov, O., Han, G.W., et al. (2015). Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567.
Kappos, L., Bar-Or, A., Cree, B.A.C., Fox, R.J., Giovannoni, G., Gold, R., Vermersch, P., Arnold, D.L., Arnould, S., Scherz, T., et al. (2018). Siponimod versus placebo in secondary progressive multiple sclerosis (EXPAND): a double-blind, randomised, phase 3 study. Lancet Lond. Engl. 391, 1263–1273.
Keller, C.D., Rivera Gil, P., Tölle, M., van der Giet, M., Chun, J., Radeke, H.H., Schäfer- Korting, M., and Kleuser, B. (2007). Immunomodulator FTY720 Induces Myofibroblast Differentiation via the Lysophospholipid Receptor S1P3 and Smad3 Signaling. Am. J. Pathol. 170, 281–292.
Kim, H.R., Duc, N.M., and Chung, K.Y. (2018). Comprehensive Analysis of Non- Synonymous Natural Variants of G Protein-Coupled Receptors. Biomol. Ther. 26, 101– 108.
Klein, C., Paul, J.I., Sauvé, K., Schmidt, M.M., Arcangeli, L., Ransom, J., Trueheart, J., Manfredi, J.P., Broach, J.R., and Murphy, A.J. (1998). Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat. Biotechnol. 16, 1334–1337.
Kleinau, G., Jaeschke, H., Worth, C.L., Mueller, S., Gonzalez, J., Paschke, R., and Krause, G. (2010). Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin Receptor. PLOS ONE 5, e9745.
Kohno, T., and Igarashi, Y. (2003). Truncation of the N-terminal ectodomain has implications in the N-glycosylation and transport to the cell surface of Edg-1/S1P1 receptor. J. Biochem. (Tokyo) 134, 667–673.
Kothapalli, R., Kusmartseva, I., and Loughran, T.P. (2002). Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia. Biochim. Biophys. Acta 1579, 117–123.
Kozian, D., Evers, A., Schafer, M., Marz, W., Bohm, B., Winkelmann, B., Mace, S., and Deleuze, J.-F. (2010). A Novel Val286Ala Polymorphism in the NPXXY Motif of the Sphingosine-1-Phosphate Receptor S1PR2 Associates with the Incidence and Age of Onset of Diabetes. J. Diabetes Metab. 1.
Lagerström, M.C., and Schiöth, H.B. (2008). Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357.
Lamy, T., Moignet, A., and Loughran, T.P. (2017). LGL leukemia: from pathogenesis to treatment. Blood blood-2016-08-692590.
LeBlanc, F.R., Wang, H.-G., Feith, D.J., and Loughran, T.P. (2015). FTY720 (Fingolimod) Targets the Sphingolipid Pathway and Induces Autophagy-Related
98
Apoptosis in Human Natural Killer Large Granular Lymphocyte Leukemia. Blood 126, 1288–1288.
Li, B., He, X., Jia, W., and Li, H. (2017a). Novel Applications of Metabolomics in Personalized Medicine: A Mini-Review. Mol. Basel Switz. 22.
Li, X., Zhou, M., Huang, W., and Yang, H. (2017b). N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization. FEBS J. 284, 2004– 2018.
Liao, A., Broeg, K., Fox, T., Tan, S.-F., Watters, R., Shah, M.V., Zhang, L.Q., Li, Y., Ryland, L., Yang, J., et al. (2011). Therapeutic efficacy of FTY720 in a rat model of NK- cell leukemia. Blood 118, 2793–2800.
Lima, M. (2013). Aggressive mature natural killer cell neoplasms: from epidemiology todiagnosis. Orphanet J. Rare Dis. 8, 95.
Liu, R., Wong, W., and IJzerman, A.P. (2016). Human G protein-coupled receptor studies in Saccharomyces cerevisiae. Biochem. Pharmacol.
Liu, Y., Wada, R., Yamashita, T., Mi, Y., Deng, C.X., Hobson, J.P., Rosenfeldt, H.M., Nava, V.E., Chae, S.S., Lee, M.J., et al. (2000). Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J. Clin. Invest. 106, 951–961.
Liu, Y., Deng, J., Wang, L., Lee, H., Armstrong, B., Scuto, A., Kowolik, C., Weiss, L.M., Forman, S., and Yu, H. (2012). S1PR1 is an effective target to block STAT3 signaling in activated B cell-like diffuse large B-cell lymphoma. Blood 120, 1458–1465.
Lo-Coco, F., Avvisati, G., Vignetti, M., Thiede, C., Orlando, S.M., Iacobelli, S., Ferrara, F., Fazi, P., Cicconi, L., Di Bona, E., et al. (2013). Retinoic Acid and Arsenic Trioxide for Acute Promyelocytic Leukemia. N. Engl. J. Med. 369, 111–121.
Lucchinetti, C., Brück, W., Parisi, J., Scheithauer, B., Rodriguez, M., and Lassmann, H. (2000). Heterogeneity of multiple sclerosis lesions: Implications for the pathogenesis of demyelination. Ann. Neurol. 47, 707–717.
Lyamani, G., Rahmoune, I., Filali, H., and Hakkou, F. (2017). Treatments of Multiple Sclerosis.
Malnic, B., Hirono, J., Sato, T., and Buck, L.B. (1999). Combinatorial receptor codes for odors. Cell 96, 713–723.
Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J., Thornton, R., Shei, G.-J., Card, D., Keohane, C., et al. (2002). Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346–349.
99
Massé, K., Kyuno, J., Bhamra, S., and Jones, E.A. (2010). The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus laevis. Int. J. Dev. Biol. 54, 1361–1374.
Matloubian, M., Lo, C.G., Cinamon, G., Lesneski, M.J., Xu, Y., Brinkmann, V., Allende, M.L., Proia, R.L., and Cyster, J.G. (2004). Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427, 355–360.
Mayol, K., Biajoux, V., Marvel, J., Balabanian, K., and Walzer, T. (2011). Sequential desensitization of CXCR4 and S1P5 controls natural killer cell trafficking. Blood 118, 4863–4871.
Medjakovic, S., Mueller, M., and Jungbauer, A. (2010). Potential Health-modulating Effects of Isoflavones and Metabolites via Activation of PPAR and AhR. Nutrients 2, 241–279.
Mendelson, K., Zygmunt, T., Torres-Vázquez, J., Evans, T., and Hla, T. (2013). Sphingosine 1-Phosphate Receptor Signaling Regulates Proper Embryonic Vascular Patterning. J. Biol. Chem. 288, 2143–2156.
Mitchell, L.A., Wang, A., Stracquadanio, G., Kuang, Z., Wang, X., Yang, K., Richardson, S., Martin, J.A., Zhao, Y., Walker, R., et al. (2017). Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science 355, eaaf4831.
Mizugishi, K., Yamashita, T., Olivera, A., Miller, G.F., Spiegel, S., and Proia, R.L. (2005). Essential role for sphingosine kinases in neural and vascular development. Mol. Cell. Biol. 25, 11113–11121.
Moore, B.S., and Mirshahi, T. (2018). Genetic variants help define the role of the MC4R C-terminus in signaling and cell surface stability. Sci. Rep. 8, 10397.
Moore, C.A.C., Milano, S.K., and Benovic, J.L. (2007). Regulation of receptor trafficking by GRKs and arrestins. Annu. Rev. Physiol. 69, 451–482.
Murone, M., Rosenthal, A., and de Sauvage, F.J. (1999). Sonic hedgehog signaling by the patched-smoothened receptor complex. Curr. Biol. CB 9, 76–84.
Nagahashi, M., Abe, M., Sakimura, K., Takabe, K., and Wakai, T. (2018). The role of sphingosine-1-phosphate in inflammation and cancer progression. Cancer Sci. 109, 3671–3678.
Nagana Gowda, G.A., Zhang, S., Gu, H., Asiago, V., Shanaiah, N., and Raftery, D. (2008). Metabolomics-Based Methods for Early Disease Diagnostics: A Review. Expert Rev. Mol. Diagn. 8, 617–633.
Nazarullah, A., Don, M., Linhares, Y., Alkan, S., and Huang, Q. (2016). Aggressive NK- cell leukemia: A rare entity with diagnostic and therapeutic challenge. Hum. Pathol. Case Rep. 4, 32–37.
100
Oakley, R.H., Laporte, S.A., Holt, J.A., Caron, M.G., and Barak, L.S. (2000). Differential Affinities of Visual Arrestin, βArrestin1, and βArrestin2 for G Protein-coupled Receptors Delineate Two Major Classes of Receptors. J. Biol. Chem. 275, 17201–17210.
Obinata, H., Gutkind, S., Stitham, J., Okuno, T., Yokomizo, T., Hwa, J., and Hla, T. (2014). Individual variation of human S1P1 coding sequence leads to heterogeneity in receptor function and drug interactions. J. Lipid Res. 55, 2665–2675.
O’Brien, P.J., and Zatz, M. (1984). Acylation of bovine rhodopsin by [3H]palmitic acid. J. Biol. Chem. 259, 5054–5057.
Oggungwan, K., Glaharn, S., Ampawong, S., Krudsood, S., and Viriyavejakul, P. (2018). FTY720 restores endothelial cell permeability induced by malaria sera. Sci. Rep. 8, 10959.
Ohashi, H., Maruyama, T., Higashi-Matsumoto, H., Nomoto, T., Nishimura, S., and Takeuchi, Y. (2002). A novel binding assay for metabotropic glutamate receptors using [3H] L-quisqualic acid and recombinant receptors. Z. Naturforschung C J. Biosci. 57, 348–355.
Ohno, Y., Ito, A., Ogata, R., Hiraga, Y., Igarashi, Y., and Kihara, A. (2009). Palmitoylation of the sphingosine 1-phosphate receptor S1P1 is involved in its signaling functions and internalization. Genes Cells 14, 911–923.
Okada, T., Le Trong, I., Fox, B.A., Behnke, C.A., Stenkamp, R.E., and Palczewski, K. (2000). X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles. J. Struct. Biol. 130, 73–80.
Oo, M.L., Thangada, S., Wu, M.-T., Liu, C.H., Macdonald, T.L., Lynch, K.R., Lin, C.-Y., and Hla, T. (2007). Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor. J. Biol. Chem. 282, 9082–9089.
Oprea, T.I., Bologa, C.G., Brunak, S., Campbell, A., Gan, G.N., Gaulton, A., Gomez, S.M., Guha, R., Hersey, A., Holmes, J., et al. (2018). Unexplored therapeutic opportunities in the human genome. Nat. Rev. Drug Discov. 17, 317–332.
O’Sullivan, S., and Dev, K.K. Sphingosine-1-phosphate receptor therapies: Advances in clinical trials for CNS-related diseases. Neuropharmacology.
Ovchinnikov, Y.A. (1987). Structure of rhodopsin and bacteriorhodopsin. Photochem. Photobiol. 45, 909–914.
Pan, S., Gray, N.S., Gao, W., Mi, Y., Fan, Y., Wang, X., Tuntland, T., Che, J., Lefebvre, S., Chen, Y., et al. (2013). Discovery of BAF312 (Siponimod), a Potent and Selective S1P Receptor Modulator. ACS Med. Chem. Lett. 4, 333–337.
101
Patmanathan, S.N., Wang, W., Yap, L.F., Herr, D.R., and Paterson, I.C. (2017). Mechanisms of sphingosine 1-phosphate receptor signalling in cancer. Cell. Signal. 34, 66–75.
Pelletier, D., and Hafler, D.A. (2012). Fingolimod for multiple sclerosis. N. Engl. J. Med. 366, 339–347.
Pfitscher, A., Reiter, E., and Jungbauer, A. (2008). Receptor binding and transactivation activities of red clover isoflavones and their metabolites. J. Steroid Biochem. Mol. Biol. 112, 87–94.
Powell, E., Shanle, E., Brinkman, A., Li, J., Keles, S., Wisinski, K.B., Huang, W., and Xu, W. (2012). Identification of estrogen receptor dimer selective ligands reveals growth- inhibitory effects on cells that co-express ERα and ERβ. PloS One 7, e30993.
Punsawad, C., and Viriyavejakul, P. (2017). Reduction in serum sphingosine 1- phosphate concentration in malaria. PloS One 12, e0180631.
Quint, K., Stiel, N., Neureiter, D., Schlicker, H.U., Nimsky, C., Ocker, M., Strik, H., and Kolodziej, M.A. (2014). The role of sphingosine kinase isoforms and receptors S1P1, S1P2, S1P3, and S1P5 in primary, secondary, and recurrent glioblastomas. Tumor Biol. 35, 8979–8989.
Rivera, R., and Chun, J. (2008). Biological effects of lysophospholipids. Rev. Physiol. Biochem. Pharmacol. 160, 25–46.
Rosen, H., Gonzalez-Cabrera, P.J., Sanna, M.G., and Brown, S. (2009). Sphingosine 1- Phosphate Receptor Signaling. Annu. Rev. Biochem. 78, 743–768.
Rosen, H., Stevens, R.C., Hanson, M., Roberts, E., and Oldstone, M.B.A. (2013). Sphingosine-1-phosphate and its receptors: structure, signaling, and influence. Annu. Rev. Biochem. 82, 637–662.
Sanchez, T., and Hla, T. (2004). Structural and functional characteristics of S1P receptors. J. Cell. Biochem. 92, 913–922.
Santos, R., Ursu, O., Gaulton, A., Bento, A.P., Donadi, R.S., Bologa, C.G., Karlsson, A., Al-Lazikani, B., Hersey, A., Oprea, T.I., et al. (2017). A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34.
Santos-Cortez, R.L.P., Faridi, R., Rehman, A.U., Lee, K., Ansar, M., Wang, X., Morell, R.J., Isaacson, R., Belyantseva, I.A., Dai, H., et al. (2016). Autosomal-Recessive Hearing Impairment Due to Rare Missense Variants within S1PR2. Am. J. Hum. Genet. 98, 331–338.
Sauer, B., Gonska, H., Manggau, M., Kim, D.S., Schraut, C., Schäfer-Korting, M., and Kleuser, B. (2005). Sphingosine 1-phosphate is involved in cytoprotective actions of calcitriol in human fibroblasts and enhances the intracellular Bcl-2/Bax rheostat. Pharm. 60, 298–304.
102
Scott, F.L., Clemons, B., Brooks, J., Brahmachary, E., Powell, R., Dedman, H., Desale, H.G., Timony, G.A., Martinborough, E., Rosen, H., et al. (2016). Ozanimod (RPC1063) is a potent sphingosine‐1‐phosphate receptor‐1 (S1P1) and receptor‐5 (S1P5) agonist with autoimmune disease‐modifying activity. Br. J. Pharmacol. 173, 1778–1792.
Shah, M.V., Zhang, R., Irby, R., Kothapalli, R., Liu, X., Arrington, T., Frank, B., Lee, N.H., and Loughran, T.P. (2008). Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes. Blood 112, 770–781.
Shao, Y., Lu, N., Wu, Z., Cai, C., Wang, S., Zhang, L.-L., Zhou, F., Xiao, S., Liu, L., Zeng, X., et al. (2018). Creating a functional single-chromosome yeast. Nature 560, 331.
Sivasubramanian, M., Kanagaraj, N., Dheen, S.T., and Tay, S.S.W. (2015). Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson’s disease and in MPP+ -treated MN9D cells in vitro. Neuroscience 290, 636–648.
Smith, J.S., and Rajagopal, S. (2016). The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.
Sobel, K., Menyhart, K., Killer, N., Renault, B., Bauer, Y., Studer, R., Steiner, B., Bolli, M.H., Nayler, O., and Gatfield, J. (2013a). Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling. J. Biol. Chem. 288, 14839– 14851.
Sobel, K., Menyhart, K., Killer, N., Renault, B., Bauer, Y., Studer, R., Steiner, B., Bolli, M.H., Nayler, O., and Gatfield, J. (2013b). Sphingosine 1-phosphate (S1P) receptor agonists mediate pro-fibrotic responses in normal human lung fibroblasts via S1P2 and S1P3 receptors and Smad-independent signaling. J. Biol. Chem. 288, 14839–14851.
Sokol, L., and Loughran, T.P. (2006). Large Granular Lymphocyte Leukemia. The Oncologist 11, 263–273.
Soto, A.G., Smith, T.H., Chen, B., Bhattacharya, S., Cordova, I.C., Kenakin, T., Vaidehi, N., and Trejo, J. (2015). N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias. Proc. Natl. Acad. Sci. U. S. A. 112, E3600-3608.
Sugahara, K., Maeda, Y., Shimano, K., Mogami, A., Kataoka, H., Ogawa, K., Hikida, K., Kumagai, H., Asayama, M., Yamamoto, T., et al. (2017a). Amiselimod, a novel sphingosine 1-phosphate receptor-1 modulator, has potent therapeutic efficacy for autoimmune diseases, with low bradycardia risk. Br. J. Pharmacol. 174, 15–27.
Sugahara, K., Maeda, Y., Shimano, K., Mogami, A., Kataoka, H., Ogawa, K., Hikida, K., Kumagai, H., Asayama, M., Yamamoto, T., et al. (2017b). Amiselimod, a novel sphingosine 1‐phosphate receptor‐1 modulator, has potent therapeutic efficacy for autoimmune diseases, with low bradycardia risk. Br. J. Pharmacol. 174, 15–27.
103
Sun, X., Singleton, P.A., Letsiou, E., Zhao, J., Belvitch, P., Sammani, S., Chiang, E.T., Moreno-Vinasco, L., Wade, M.S., Zhou, T., et al. (2012). Sphingosine-1-phosphate receptor-3 is a novel biomarker in acute lung injury. Am. J. Respir. Cell Mol. Biol. 47, 628–636.
Sun, Y., Cheng, Z., Ma, L., and Pei, G. (2002). Beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J. Biol. Chem. 277, 49212–49219.
Takabe, K., Paugh, S.W., Milstien, S., and Spiegel, S. (2008). “Inside-out” signaling of sphingosine-1-phosphate: therapeutic targets. Pharmacol. Rev. 60, 181–195.
Tu, Z., Argmann, C., Wong, K.K., Mitnaul, L.J., Edwards, S., Sach, I.C., Zhu, J., and Schadt, E.E. (2009). Integrating siRNA and protein-protein interaction data to identify an expanded insulin signaling network. Genome Res. 19, 1057–1067.
Turner, T.N., Yi, Q., Krumm, N., Huddleston, J., Hoekzema, K., F Stessman, H.A., Doebley, A.-L., Bernier, R.A., Nickerson, D.A., and Eichler, E.E. (2017). denovo-db: a compendium of human de novo variants. Nucleic Acids Res. 45, D804–D811.
Van Brocklyn, J.R., and Williams, J.B. (2012). The control of the balance between ceramide and sphingosine-1-phosphate by sphingosine kinase: Oxidative stress and the seesaw of cell survival and death. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 163, 26–36.
Vinson, C.R., Conover, S., and Adler, P.N. (1989). A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains. Nature 338, 263–264.
Walzer, T., Chiossone, L., Chaix, J., Calver, A., Carozzo, C., Garrigue-Antar, L., Jacques, Y., Baratin, M., Tomasello, E., and Vivier, E. (2007). Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat. Immunol. 8, 1337–1344.
Wishart, D.S., Tzur, D., Knox, C., Eisner, R., Guo, A.C., Young, N., Cheng, D., Jewell, K., Arndt, D., Sawhney, S., et al. (2007). HMDB: the Human Metabolome Database. Nucleic Acids Res. 35, D521-526.
Wishart, D.S., Feunang, Y.D., Marcu, A., Guo, A.C., Liang, K., Vázquez-Fresno, R., Sajed, T., Johnson, D., Li, C., Karu, N., et al. (2018). HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res. 46, D608–D617.
Wisler, J.W., DeWire, S.M., Whalen, E.J., Violin, J.D., Drake, M.T., Ahn, S., Shenoy, S.K., and Lefkowitz, R.J. (2007). A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling. Proc. Natl. Acad. Sci. U. S. A. 104, 16657–16662.
Wu, Y., Li, B.-Z., Zhao, M., Mitchell, L.A., Xie, Z.-X., Lin, Q.-H., Wang, X., Xiao, W.-H., Wang, Y., Zhou, X., et al. (2017). Bug mapping and fitness testing of chemically synthesized chromosome X. Science 355, eaaf4706.
104
Wüller, S., Wiesner, B., Löffler, A., Furkert, J., Krause, G., Hermosilla, R., Schaefer, M., Schülein, R., Rosenthal, W., and Oksche, A. (2004). Pharmacochaperones post- translationally enhance cell surface expression by increasing conformational stability of wild-type and mutant vasopressin V2 receptors. J. Biol. Chem. 279, 47254–47263.
Yanagida, K., Liu, C.H., Faraco, G., Galvani, S., Smith, H.K., Burg, N., Anrather, J., Sanchez, T., Iadecola, C., and Hla, T. (2017). Size-selective opening of the blood–brain barrier by targeting endothelial sphingosine 1–phosphate receptor 1. Proc. Natl. Acad. Sci. 201618659.
Ye, D., and Lin, F. (2013). S1pr2/Gα13 signaling controls myocardial migration by regulating endoderm convergence. Dev. Camb. Engl. 140, 789–799.
Yoshida, Y., Tsuji, T., Watanabe, S., Matsushima, A., Matsushima, Y., Banno, R., Fujita, T., and Kohno, T. (2013). Efficacy of combination treatment with fingolimod (FTY720) plus pathogenic autoantigen in a glucose-6-phosphate isomerase peptide (GPI325-339)- induced arthritis mouse model. Biol. Pharm. Bull. 36, 1739–1746.
Zhang, D., and Loughran, T.P. (2012). Large granular lymphocytic leukemia: molecular pathogenesis, clinical manifestations, and treatment. Hematol. Am. Soc. Hematol. Educ. Program 2012, 652–659.
Zhang, J., Barak, L.S., Anborgh, P.H., Laporte, S.A., Caron, M.G., and Ferguson, S.S.G. (1999). Cellular Trafficking of G Protein-coupled Receptor/β-Arrestin Endocytic Complexes. J. Biol. Chem. 274, 10999–11006.
Zheng, H., Pearsall, E.A., Hurst, D.P., Zhang, Y., Chu, J., Zhou, Y., Reggio, P.H., Loh, H.H., and Law, P.-Y. (2012). Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling. BMC Cell Biol. 13, 6.
VITA
Jacob Hornick
EDUCATION
Pennsylvania State University College of Medicine 2010-2019 (expected) Ph.D.-Biochemistry and Molecular Biology
Washington and Jefferson College, Washington, Pennsylvania, 2006-2010 B.A.-Biochemistry
TEAMWORK/LEADERSHIP/PROFESSIONAL EXPERIENCES
Adjunct Instructor of Brewing Science, 2016-2019 Co-Chair and Mentor, PSU College of Medicine Junior Mentor Program, 2012- 2017 Treasurer, PSU College of Medicine Graduate Student Association, 2012-2014 Co-Chair, PSU College of Medicine Career Day, 2012-2014 Assistant Wrestling Coach, Lower Dauphin High School, 2015-2019
AWARDS and HONORS
Graduate Alumni Endowed Scholarship, PSU College of Medicine, 2014 Multi-time Award Winning Homebrewer, 2016-2018 Mixed Fermentation and Belgian Ales