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Optimization of ADRB2 overexpression and reagent characterization for cyclic AMP measurement
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Optimization of ADRB2 overexpression and reagent characterization for cyclic AMP measurement
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Content
OPTIMIZATION OF ADRB2 OVEREXPRESSION AND REAGENT
CHARACTERIZATION FOR CYCLIC AMP MEASUREMENT
By
Josephine Susanto
A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
MOLECULAR PHARMACOLOGY AND TOXICOLOGY
August 2022
Copyright 2022 Josephine Susanto
ii
ACKNOWLEDGEMENTS
I want to acknowledge and express my gratitude to Dr. Curtis Okamoto for his guidance
and reassurance during my year in his lab. Dr. Okamoto welcomed me as part of his lab despite
my lack of experience, believed in my passion in the subject and have given me the freedom to do
research, which have tremendously shaped my understanding of science. He patiently helped me
through the difficulties that I encountered during my research and provided constructive feedbacks
not only in terms of lab skills, but also on how to think as a scientist. In addition, I also thank Dr.
Ian Haworth and Dr. Jennica Zaro for being my committee members and lending me their expertise
in the subject.
I also would like to thank Dr. Anh Tan Truong and Dr. Taojian (Michael) Tu for being the
greatest mentors a student could ever have. My numerous (and often repetitive) questions have
always been answered and explained patiently, which have greatly enhanced my understanding of
biology. Thank you to Dr. Anh Tan Truong, for helping me get onboard in the lab and giving me
a crash course in laboratory skills including on the weekends, despite also working on your
dissertation and defense. Thank you to Dr. Taojian Tu, for always giving me a different perspective
to solve a scientific problem as well as providing reassurance when I encountered an unexpected
problem. It is thanks to these three mentors and role models that solidified my choice to pursue
biology.
I thank everyone in the masters class of 2022, for being the best support system throughout
these past two years. We started in different continents with different time zones during the
COVID-19 pandemic, yet we persevered and the journey with these people have made my masters
experience joyful. I know I could always count of these people I proudly call friends for not only
scientific discussions, but also regular non-scientific conversations. I also want to thank everyone
iii
in Stiles lab, Alachkar lab, Zaro lab and Mackay lab for always keeping the door open when I
needed to ask questions or ask for help.
Finally, I would like to thank my family and my boyfriend (and my two cats) for their
unconditional love and support throughout my scientific journey.
iv
TABLE OF CONTENTS
Acknowledgements ......................................................................................................................... ii
List Of Tables ................................................................................................................................ vi
List Of Figures .............................................................................................................................. vii
Abbreviations ............................................................................................................................... viii
Abstract .......................................................................................................................................... ix
Chapter One: Introduction .............................................................................................................. 1
1.1 G Protein Coupled Receptors (GPCRs) .................................................................... 1
1.2 Structure: Protein ...................................................................................................... 2
1.3 Mechanism: Activation & Inactivation ..................................................................... 7
1.4 Mechanism: Signaling Pathway ................................................................................ 9
1.5 Mechanism: Desensitization ................................................................................... 10
1.6 Polymorphism: Disease State & Drug Response .................................................... 12
1.7: Polymorphism: Receptor Function ........................................................................ 15
Chapter Two: Materials And Methods ......................................................................................... 17
2.1 Cell Culture And Maintenance ............................................................................... 17
2.2 Plasmid Expansion And Isolation ........................................................................... 17
2.3 Site-Directed Mutagenesis ...................................................................................... 20
2.4 Stable Monoclonal Cell Line Generation ............................................................... 21
2.5 Immunoblotting....................................................................................................... 22
2.6 Immunofluorescent Staining ................................................................................... 23
2.7 Total Cyclic Amp Measurement ............................................................................. 24
Chapter Three: Results .................................................................................................................. 26
3.1 ADRB2 Stable Cell Line Generation ...................................................................... 26
3.2 Antibody Verification ............................................................................................. 27
3.3 Immunofluorescent Staining ................................................................................... 29
v
3.4 Effect Of Overexpression On Cyclic Amp Production........................................... 31
Chapter Four: Discussion .............................................................................................................. 32
Conclusions ................................................................................................................................... 37
Bibliography ................................................................................................................................. 39
vi
LIST OF TABLES
Table 1. List of purified cDNAs used for transfection. 22
Table 2. List of primary antibodies used. 23
vii
LIST OF FIGURES
FIGURE 1. PROPOSED ENERGY LANDSCAPE FOR GPCRS. .................................................................... 3
FIGURE 2. CRYSTAL STRUCTURE OF CARAZOLOL BOUND ADRB2-T4L FUSION PROTEIN ..................... 4
FIGURE 3. SIDE VIEW ON LIGAND BINDING SITE. ............................................................................... 4
FIGURE 4. COMPARISON OF AGONIST BOUND TO INVERSE AGONIST BOUND ADRB2 STRUCTURE ....... 6
FIGURE 5. SCHEMATIC OF ALLOSTERIC STATES OF GPCRS. ................................................................ 6
FIGURE 6. SCHEMATIC OF THE GPCR CYCLE. ..................................................................................... 8
FIGURE 7. DOWNSTREAM SIGNALING OF GPCR WHEN COUPLED TO DIFFERENT G𝛼 SUBUNITS. ........ 10
FIGURE 8. ADRB2 DESENSITIZATION AND RECYCLING..................................................................... 11
FIGURE 9. LOCATION OF COMMONLY FOUND ADRB2 POLYMORPHISMS. .......................................... 13
FIGURE 10. PLASMID MAPS ENCODING ADRB2.. .............................................................................. 19
FIGURE 11. PRIMER SEQUENCES FOR MUTAGENESIS REACTIONS.. ................................................... 20
FIGURE 12. SDS-PAGE OF CELL LINES TRANSFECTED WITH FLAG-ADRB2 FUSION GENE. .................. 26
FIGURE 13. SDS-PAGE OF CELL COLONIES TRANSFECTED WITH VECTOR BUILDER PLASMID.. .......... 27
FIGURE 14. SDS-PAGE OF VARIOUS CELL LINES.. ............................................................................. 28
FIGURE 15. SDS-PAGE OF NOVUS BIO CONTROL LYSATES. ............................................................... 29
FIGURE 16. IMMUNOFLUORESCENT STAINING ON HEK293 TRANSFECTED WITH G16E27T164.. ....... 30
FIGURE 17. IMMUNOFLUORESCENT STAINING ON NON-TRANSFECTED HEK293. .............................. 30
FIGURE 18. EFFECT OF ADRB2 OVEREXPRESSION ON CAMP AND ADRB2 EXPRESSION.. .................... 31
viii
ABBREVIATIONS
GPCR: G protein-coupled receptor
ADRB2: Adrenoceptor-β2 / β2-adrenergic receptor
TM: Transmembrane domain
T4L: T4-lysozyme
ECL: Extracellular loops
Nb80: Nanobody 80
GEF: Guanine-nucleotide exchange factor
GDP: Guanosine diphosphate
GTP: Guanosine triphosphate
RGS: Regulators of G protein signaling
GRK: G protein-coupled receptor kinases
ATP: Adenosine triphosphate
cAMP: Cyclic adenosine monophosphate
PKA: Protein kinase A
AMP: Adenosine monophosphate
SNP: Single nucleotide polymorphism
COPD: Chronic obstructive pulmonary disease
ix
ABSTRACT
G protein-coupled receptors (GPCRs) are one of the largest and most important
pharmaceutical targets due to their role in human pathophysiology. Many drugs target β2-
adrenoceptors (ADRB2), a member of GPCRs which are commonly associated with
cardiovascular medicine and asthma. However, pharmacogenetic studies have found that
individuals may differ in treatment responses due to genetic polymorphisms in this gene. Despite
recent population studies, the results have been inconsistent and there is a lack of understanding
of how these polymorphisms can affect agonist binding, which consequently affects the
downstream signaling of these receptors. For this reason, the goal of this thesis is to optimize
experimental procedures and characterize the appropriate reagents for future studies on ADRB2.
While there are many available antibodies to detect ADRB2 on the market, many of these
antibodies have not been verified in peer-reviewed journals. We purchased several different
antibodies on the market to detect the overexpressed ADRB2 level in our cell lines. Unfortunately,
several of them did not seem to be specific for the protein of interest, which hindered our
downstream experiments. In addition, we have optimized the transfection protocol which includes
testing two different plasmids to determine which one would be more experimentally efficient. We
have also optimized the protocols to generate a stable monoclonal cell line as well as the
appropriate condition to maintain the culture. Lastly, we determined that the cyclic adenosine
monophosphate (cAMP) can be measured at basal level through the ELISA kit from Enzo Life
Sciences.
1
CHAPTER ONE: INTRODUCTION
1.1 G PROTEIN COUPLED RECEPTORS (GPCRS)
G protein-coupled receptors (GPCRs) are one of the largest and most diverse groups of
membrane receptors. They function as regulators of intracellular signaling cascades in response to
a variety of ligands, including hormones, neurotransmitters, small molecule drugs as well as
photons (Bockaert & Philippe Pin, 1999). GPCRs are also involved in various physiological
processes, and are not limited to vision, taste, smell, behavioral regulation, immune function,
nervous system, and tumor metastasis.
Currently there have been at least 800 GPCRs encoded by the human genome that are grouped
into six different classes based on amino acid sequence and functional similarities (Josefsson,
1999; Lee, Basith, & Choi, 2018):
Class A - Rhodopsin-like receptors
Class B - Secretin receptor family
Class C - Metabotropic glutamate receptors
Class D - Fungal mating pheromone receptors
Class E - Cyclic AMP (cAMP) receptors
Class F - Frizzled and Smoothened receptors
GPCRs play an essential role in the body, and thus manipulating their signaling pathways
through agonists and inverse agonists have been a continuous interest, including, but not limited
to, cardiovascular and pulmonary diseases, and cancer. In fact, 35% of currently approved drugs
target GPCRs, with approximately 321 agents in clinical trials (Sriram & Insel, 2018). This statistic
shows how important GPCRs are, and therefore studies characterizing these receptors can result
in more possibilities for other therapeutic applications.
2
This thesis will focus on understanding the complexity of β2-adrenergic receptor (ADRB2;
also known as β2-adrenoceptors) (NCBI ID: 154, UniProt ID: P07750), as well as overcoming the
challenges in characterizing its protein expression in vitro.
1.2 STRUCTURE: PROTEIN
Despite having different ligands, all GPCRs are related on amino acid sequence level and are
even proposed to have similar three-dimensional structures. GPCRs are cell surface, seven-
transmembrane receptors that are coupled to G proteins. These receptors pass through the cell
membrane seven times, with an extracellular N-terminus and intracellular C-terminus, giving them
an alternative name, “7 Transmembrane (7TM)-domain receptors”.
Studying the crystal structures of GPCRs has been challenging due to the flexibility in their
structures as well as its properties of being a membrane protein (Weis & Kobilka, 2018). First,
high affinity and long-acting ligands are required as the dissociation of ligands will push the
equilibrium to favor the more stable inactive state (Figure 1). Second, GPCRs are relatively
unstable in the detergents that are often used for crystallization. This however has been overcome
by performing the crystallization in lipids or by mutating key residues to stabilize the receptor
structure (Faham & Bowie, 2002; Magnani et al., 2016; Rasmussen et al., 2007). Third, the
extracellular portion of membrane proteins are essential in forming the crystal structures. In the
case of GPCRs, these are often small and flexible, which makes crystallization more challenging.
Scientists have tried overcoming this issue by fusing a rigid protein such as T4 lysozyme (T4L) to
the extracellular N-terminus or to the intracellular loops (ICLs) and by shortening the flexible
termini to further stabilize the structure (Zou, Weis, & Kobilka, 2012).
3
Figure 1. Proposed energy landscape for GPCRs. (a) Energy states of an unbound GPCR favors the equilibrium onto an inactive
state. (b) Binding of an agonist lowers the energy state of the GPCR, favoring the active state. (c) Binding of an inverse-agonist
lowers the energy state of the inactive conformation, favoring the inactive state. Reprinted from “Conformational complexity of
G-protein-coupled receptors,” by B. Kobilka and X. Deupi, 2007, Trends in Pharmacological Sciences, 28(8), p.397-406.
Copyright 2007 by Elsevier. Reprinted with permission.
Visualization of the inactive state of ADRB2 was first reported in 2007. The structure
discovery was made possible with the binding of carazolol, a high affinity inverse agonist, to an
ADRB2-T4L fusion protein in lipid cubic phase (Figure 2) (Cherezov et al., 2007). The crystal
structure suggests that the ligand binding site is formed mainly by TM3, TM5, TM6, TM7 and
extracellular loops (ECLs), and it is relatively open, enabling a ligand to diffuse easily. Disulfide
bonds within ECL2 and with TM3 also helped maintain the core of the receptor, therefore
preventing the obstruction of the ligand binding site (Figure 3). The flexibility of the structure is
also supported by a 2.4Å resolution structure showing water mediated hydrogen bonds between
the TM residues, suggesting that conformational changes are allowed upon activation (Bang &
Choi, 2015). The flexibility of the ECLs allows the GPCRs to adopt different structures to a
varying degree, which consequently affects its binding kinetics to the ligand.
4
Figure 2. Crystal structure of inverse agonist carazolol bound ADRB2-T4L fusion protein obtained in lipid cubic phase. T4
lysozyme protein (T4L) (grey) is fused to ADRB2 (green) to stabilize the protein structure. (A) Side view of carazolol (space
filling model) bound ADRB2-T4L. (B) Enlarged view of carazolol binding site in ADRB2-T4L fusion protein. (Bang & Choi,
2015).
Figure 3. Side view on ligand binding site. ECL2 (green) is shown to form disulfide bonds (yellow) within itself and with TM3,
thus appearing to maintain the rigidity of the core of the receptor and opening the ligand binding site. In this figure, carazolol acts
as an inverse agonist (blue). Reprinted from “High-Resolution Crystal Structure of an Engineered Human 𝛽 2-Adrenergic G
Protein-Coupled Receptor,” by V. Cherezov, D. Rosenbaum, M. Hanson, S. Rasmussen, et al, 2007, Science, 318(5854), p.1258-
1265. Copyright 2007 by The American Association for the Advancement of Science. Reprinted with permission.
On the other hand, attempts to discover the active conformation of ADRB2 have proven to
be more challenging. In 2011, one study replaced His95 with a Cys residue to make a disulfide
bond with the synthetic agonist FAUC50 to create a more rigid structure (Rosenbaum et al., 2011)
(Figure 4A). The result showed that the binding of the agonist alone was not sufficient to stabilize
the active conformation. It was therefore proposed that the structure will only be stabilized when
the G protein interacts with the receptor. This structural discovery was made possible by fusing
Nb80 (nanobody 80) onto ADRB2-T4L complex, to simulate G protein binding. The binding of
the high-affinity synthetic agonist BI-16707 on ADRB2-T4L/Nb80 complex, elucidated two
5
structures (Figure 4). Through comparison with the previously discovered inactive state, it was
found that there is little conformational change on the extracellular side of the receptor. The major
difference occurs in TM5 which interferes with the hydrophobic interactions, causing TM3, TM5,
TM6 and TM7 re-arrangements (Figure 4A bottom). This shift created a binding pocket in which
the C-terminus of the G protein can insert itself. However, the agonist-bound structures only show
the most thermodynamically stable states of active ADRB2 conformation. It has been suggested
that there is the existence of multiple intermediate states between inactive and active structures in
all GPCRs (Figure 5) (Thal, Glukhova, Sexton, & Christopoulos, 2018). This idea was supported
by a study in 2013 by methyl NMR spectroscopy (
13
CH3ε-Met) that revealed heterogeneity in the
NMR spectrum when BI-167107 was bound, suggesting that the binding of an agonist alone is not
enough to stabilize the active state of ADRB2 (Figure 5) (Nygaard et al., 2013). However, a study
using the endogenous ligand adrenaline indicated that there is not much change in the overall
structure of ADRB2, suggesting that the binding mechanism of ADRB2 is similar for both
synthetic and endogenous ligands (Ring et al., 2013).
6
Figure 4. Comparison of agonist bound ADRB2 structures (A and B) to that of inverse agonist bound ADRB2 structure (C).
Bottom rows show the various cytoplasmic views showing TMs positions with different ligands bound. (A) Covalent binding
interaction between agonist FAUC50 (pink) with ADRB2-T4L (T4 Lysozyme) complex (purple). ADRB2-T4L/Nb80 complex
(orange) is superimposed to indicate movements in TM domains (orange arrows), to simulate the change in receptor structure
upon the binding of G protein. (B) Agonist BI-16707 (green) bound to ADRB2-T4L/Nb80 complex. (C) Carazolol (yellow)
bound to ADRB2-T4L complex (cyan). Reprinted from “Structure and function of an irreversible agonist-𝛽 2 adrenoceptor
complex,” by D. M. Rosenbaum, et al, 2011, Nature, 469(7329), p.236-240. Copyright 2011 by Nature Publishing Group.
Reprinted with permission.
Figure 5. Schematic of allosteric states of GPCRs. Inactive state is represented by inverse agonist carazolol (red sphere) bound
ADRB2 (left) whereas active conformation is represented by agonist BI-16707 (green sphere) bound ADRB2 (right). In between
active and inactive states, there are multiple intermediate states, as exemplified by apo rhodopsin (RHO) and 5-N-ethyl-
carboxamidoadenosine (NECA) bound to the adenosine A2A receptor (A2AR). Reprinted from “Structural insights into G-protein-
coupled receptor allostery,” by D. M. Thal, et al, 2018, Nature, 559, p.45-53. Copyright 2018 by Macmillan Publishers Ltd.
Reprinted with permission.
7
1.3 MECHANISM: ACTIVATION & INACTIVATION
GPCRs are allosteric proteins. The majority of GPCRs are activated by extracellular
ligands, followed by conformational changes such as the shifting of the TM domains. The two
main forms in equilibrium are the active and inactive state, where the inactive, non-ligand-bound
state predominates. As their name suggests, GPCRs are coupled to heterotrimeric G proteins.
Heterotrimeric G proteins, also known as “large G proteins”, consist of G𝛼 , Gβ and Gγ subunits.
Out of these, ADRB2 is known to directly couple with G𝛼 s upon activation. However, it is not
known if it is precoupled to the receptor in the inactive state or before ligand binding (Hilger,
Masureel, & Kobilka, 2018).
When the ADRB2-G protein complex is activated, a conformational change occurs,
revealing the intracellular guanine-nucleotide exchange factor (GEF) activity which is bound to
guanosine diphosphate (GDP) on G𝛼 s in the inactive state (Johnson, 2006) (Figure 6). The GEF
activity induces the dissociation of GDP and recruits guanosine triphosphate (GTP) that is present
in the cytoplasm, now transitioning to the active state. This change dissociates G𝛼 s from the Gβγ
subunits as the GTP-bound-G𝛼 s complex has a lower affinity for the Gβγ subunits. Upon
dissociation, the G𝛼 s-GTP complex and Gβγ subunits bind to adenylyl cyclase and calcium
channels respectively, mediating separate downstream signaling activities. Interestingly, studies
have shown that GPCRs can perform basal GTP exchange activity even without the presence of a
ligand, suggesting that there is an equilibrium favoring the active state in this case (Weis &
Kobilka, 2018). This equilibrium can be manipulated to favor and maintain the active state using
agonists, whereas inverse agonists can be used to favor the inactive, lower-than-basal ligand-free
state (Figure 1).
8
Figure 6. Schematic of the GPCR cycle. Agonist binding induces conformational changes in the GPCR’s TM domain (dark
blue), activating the receptor and allows G proteins (orange), GRKs (red) or arrestins (shades of green) to bind to the intracellular
side of the receptor. Coupling of the G𝛼 s protein to the receptor exchanges GDP for GTP on G𝛼 s, followed by dissociation of the
𝛼 subunit from the β𝛾 subunits. In this case, the GTP-bound-G𝛼 s subunit regulates the activity of adenylyl cyclase, while the
β𝛾 subunit interacts with the potassium channels. The termination is induced by GTP hydrolysis and reassociation of the G𝛼 s
with the β𝛾 subunits to form the inactive heterotrimer. Reprinted from “Structure and dynamics of GPCR signaling complexes,”
by D. Hilger, M. Masureel and B.K. Kobilka, 2018, Nature Structural & Molecular Biology, 2018, 25, p.4-12. Copyright 2018 by
the authors. Reprinted with permission.
On the other hand, the ADRB2 is inactivated when GTP undergoes hydrolysis into GDP
by Regulators of G protein signaling (RGSs), which reduces the affinity of the G𝛼 s to the receptor,
dissociating from the receptor and allowing the receptor to return to its inactivated non-G protein
coupled state (Figure 6) (Hilger et al., 2018). In some instances, the dissociated G𝛼 s subunit can
reassociate with the dissociated Gβγ subunit, thus forming a resting G protein that awaits another
round of receptor activation. However, more studies have revealed that there are multiple forms of
inactivity alone, G𝛼 s-ADRB2 or non-G𝛼 s-bound-ADRB2 on top of the intermediates between
inactive and active states. The definition of active and inactive is not thoroughly discovered yet
and remains an important research question.
9
1.4 MECHANISM: SIGNALING PATHWAY
Studies on the signaling pathway of ADRB2 have been done since the 1960s. One of the
main pathways is through the G𝛼 s proteins, whose activation was described above (Figure 6).
Following the dissociation of the GTP-bound-G𝛼 s protein, the G𝛼 s-GTP complex couples to and
activates adenylyl cyclase (Johnson, 2006). Adenylyl cyclase is an enzyme that catalyzes the
conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP).
cAMP is responsible for activating protein kinase A (PKA), which phosphorylates proteins
responsible for numerous biological functions such as vasodilation in smooth muscles and
stimulation of cardiac muscle contraction. The cAMP level is then decomposed by the activity of
phosphodiesterases which converts it into adenosine monophosphate (AMP).
Some studies have also suggested that ADRB2 can also potentially couple to the G𝛼 i
subunit, which inhibits adenylyl cyclase activity and eventually decreases the production of cAMP
(Daaka, Luttrell, & Lefkowitz, 1997; Sunahara & Taussig, 2002). This alternate signaling pathway
is proposed to occur when PKA phosphorylates ADRB2, uncoupling it from the G𝛼 s proteins, and
with the remaining βγ-subunits, coupling to G𝛼 i. This complex then recruits β-arrestin and
activates the Src, SOS, Ras and MAPK pathway. This mechanism is proposed to act as an alternate
way to terminate the agonist-ADRB2 signaling pathway. In fact, some studies have observed
phosphorylation of the glucocorticoid receptor due to ADRB2-induced-MAPK activation
(Johnson, 2004). In addition, there have been studies that observed altered G𝛼 q response
(responsible for activation of phospholipase C) when ADRB2 is either silenced or overexpressed
(McGraw, Almoosa, Paul, Kobilka, & Liggett, 2003). This indicates that there might be crosstalk
between ADRB2 and other types of G proteins on a cellular level that requires further studies.
10
Figure 7. Downstream signaling of GPCR when coupled to different G𝛼 subunits. The binding of G𝛼 q and G𝛼 s leads to the
activation of phospholipase C (PLC) and adenylyl cyclase (AC) respectively, which results in an increase of intracellular calcium
and cAMP levels, respectively. Stimulation of G𝛼 i upon binding is inhibitory to the elevation of intracellular cAMP while
increasing intracellular potassium level. (Kimura, Pydi, Pham, & Tanaka, 2020).
1.5 MECHANISM: DESENSITIZATION
Like many other regulatory proteins, ADRB2 can be desensitized. This is necessary to
prevent receptor overstimulation when exposed to a large concentration of agonist. Most often,
desensitization occurs when there is an excess of agonists, rather than inverse agonists. The
desensitization of ADRB2 can occur after the inactivation of the receptor, when G𝛼 s is uncoupled
from the receptor (Johnson, 2006). The uncoupled receptors are then internalized and unable to
bind to extracellular ligands, which terminates the signaling pathway. The internalized receptors
can also be phosphorylated to prevent G𝛼 s binding, thus preventing the recruitment of adenylyl
cyclase. However, the extent of desensitization depends on the duration of ADRB2/agonists
response and its binding affinity to the receptor.
The mechanism of reversible, short-term ADRB2 desensitization is through the
phosphorylation of the receptor by PKA or other receptor kinases (Johnson, 1998). The
phosphorylation often occurs in ICL3 and in the cytoplasmic tail, which allows the binding of β-
arrestin and uncoupling of the G𝛼 s protein (Figure 8), thus inhibiting the receptor function. β-
11
arrestin can also recruit phosphodiesterases that metabolize cAMP, terminating further
downstream signaling. Prolonged agonist exposure can also result in desensitization which is
caused by the sequestration of receptors from the cell membrane. The reversal takes longer than
short-term exposure but can often be done within hours. Unfortunately, the mechanism of ADRB2
trafficking has not been fully elucidated. Hours and even days of exposure of agonists can lead to
downregulation of ADRB2. It is speculated that the downregulation mechanism occurs through
the ubiquitination of the receptor by E3 ligase, which induces the degradation process (Shenoy,
McDonald, Kohout, & Lefkowitz, 2001). However, it is important to note that desensitization can
differ in cell and tissue types. One study have found that human lymphocytes desensitize ADRB2
very rapidly, whereas it is persistent in human bronchial smooth muscle cells as observed in a
constant increase of cAMP level in response to isoproterenol exposure (McGraw & Liggett, 1997).
Figure 8. ADRB2 desensitization and recycling. Upon agonist binding, the receptor gets phosphorylated by PKA or GPCR
kinases (GRKs), resulting in receptor desensitization. 𝛽 -arrestin binding is induced, resulting in ADRB2-𝛽 -arrestin complex
sequestration from the cell membrane, and internalization via clathrin-dependent endocytosis. The receptor then becomes
dephosphorylated, is dissociated from 𝛽 -arrestin and recycled back to the cell surface. Reprinted from “Molecular mechanisms of
𝛽 2-adrenergic receptor function, response, and regulation,” by M. Johnson, 2006, Journal of Allergy and Clinical Immunology,
117(1), p.18-24. Copyright 2006 by American Academy of Allergy, Asthma and Immunology. Reprinted with permission.
12
1.6 POLYMORPHISM: DISEASE STATE & DRUG RESPONSE
ADRB2 plays an important role in regulating vascular and bronchial smooth muscle tone
(Guimarães & Moura, 2001). It is also expressed in the heart where it regulates heart contraction
and rate of contraction (Litonjua et al., 2010). However, it has been widely observed that ADRB2
are polymorphic within the human population with more than 80 single nucleotide polymorphisms
(SNPs) identified. Some of the most common nonsynonymous SNPs results in Arg16Gly (R16G;
rs1042713) and Glu27Gln (E27Q; rs1042714) in the N-terminus, as well as Thr164Ile (T164I;
rs1800888) in TM4 (Figure 9) (Reihsaus, Innis, MacIntyre, & Liggett, 1993). Population studies
found that R16G and E27Q have minor allele frequencies of 40-50% and have been well associated
with response variation to asthma medications. On the other hand, T164I is less common with
minor allele frequency of 1-3% and have been found to associate with diminished response to
agonist-induced stimulation (Green, Cole, Jacinto, Innis, & Liggett, 1993). Population studies have
shown that polymorphisms can also vary between ethnicities, which can cause variations in drug
response in different populations (Maxwell et al., 2005).
13
Figure 9. Location of commonly found ADRB2 polymorphisms. Yellow squares indicate silent polymorphisms (no change in
amino acid sequence), whereas red diamonds indicate missense polymorphisms (translate into different amino acid sequence).
Reprinted from “Pharmacogenetics of the human beta-adrenergic receptors,” by M. R. G. Taylor, 2006, The Pharmacogenomics
Journal, 7, p.29-37. Copyright 2006 by Nature Publishing Group. Reprinted with permission.
Activation of ADRB2 in bronchial smooth muscles can induce relaxation of smooth
muscles with consequent bronchodilation. As a result, agonists targeting ADRB2 have been
developed as treatment for asthma and chronic obstructive pulmonary diseases (COPD).
Pharmacogenetics studies on patients have been done to discover the association of asthma
severity, lung function, bronchial hyperreactivity and response to bronchodilators. However, many
observed no major impact of polymorphisms on asthma or COPD progression and severity
(Thomsen, Nordestgaard, Sethi, Tybjærg-Hansen, & Dahl, 2012). Polymorphisms have also been
proposed to cause the varying tolerance to ADRB2 agonists. Population studies using short-term
agonists albuterol and terbutaline showed that patients carrying R16 elicited better response
compared to those with G16 (A. Ahles, Rochais, Frambach, Bünemann, & Engelhardt, 2011).
Unfortunately, studies using long-acting agonists resulted in inconsistent results, suggesting that
the polymorphism at position 16 may only benefit patients for short-term treatments (Andrea Ahles
14
& Engelhardt, 2014). Studies on long-term treatment with ADRB2 agonists have been found to
induce desensitization. In vitro studies found that patients homozygous for G16 developed greater
desensitization to formoterol in the long term (Tan, Hall, Dewar, Dow, & Lipworth, 1997),
whereas studies on short-acting agents such as albuterol found that patients heterozygous or
homozygous for R16 demonstrated larger tolerance than patients homozygous for Gly16 (Israel et
al., 2004; Israel et al., 2000). However, one in vitro study found that endogenous ligands
desensitize ADRB2 in its basal state to a greater extent for G16 compared to R16. Therefore, it is
proposed that the exogenous ligand induced desensitization will have a greater effect on R16, as
G16 is already desensitized by endogenous ligands (Liggett, 2000).
Polymorphisms that alter ADRB2 function and regulation can also be presumed to affect
vasoregulation and blood pressure. However, population studies on the effect of polymorphisms
at positions 16 and 27 on vasodilation, blood pressure regulation and risk for hypertension showed
inconsistent results (Andrea Ahles & Engelhardt, 2014). Some of the largest and smaller studies
(n<1000) showed that there is no association between the SNPs and blood pressure or
hypertension. T164I is a rare SNP and therefore a larger number of eligible participants is still
required. Regarding drug response, there also seems to be variance in response depending on the
mode of application (local or systemic). Studies on healthy individuals found that local infusion
of isoproterenol or terbutaline induced a stronger response when administered locally to
individuals carrying G16 (A. Ahles et al., 2011). However, systemic infusion exhibited a larger
vasodilation effect with patients carrying R16 (Gratze et al., 1999). Response to terbutaline
treatment in healthy volunteers and patients with congestive cardiomyopathy were weaker in
individuals carrying I164 (Barbato et al., 2007; Brodde & Michel, 1999; Bruck et al., 2003). With
regards to ADRB2 antagonists, there seems to be minor effects with polymorphism at position 16
15
and 27, which has been linked to higher blood pressure despite unknown mechanisms (Andrea
Ahles & Engelhardt, 2014).
1.7: POLYMORPHISM: RECEPTOR FUNCTION
Overexpression studies on the impact of SNPs in the extracellular N-terminus, comprising
the R16G and Q27E, have been done on several cell lines. The first study was done in Chinese
hamster fibroblasts that observed no alteration in ligand binding or adenylyl cyclase activity
(Green, Turki, Innis, & Liggett, 1994). In an HEK293 overexpression study with epinephrine
stimulation, the G16 polymorphism displayed increased cAMP formation compared to the normal
allele, R16 (A. Ahles et al., 2011). The result is hypothesized to be due to the faster conformational
changes by phosphorylation by GPCR kinases (GRKs). The same study suggested enhanced
desensitization of R16G polymorphism due to faster interaction with β-arrestin 2. However,
pharmacogenetics studies on isolated, non-transfected human cells showed varying results
following long-term agonist stimulation human cells showed varying results (Aziz, Hall,
McFarlane, & Lipworth, 1998; Bruck et al., 2003; Lipworth, Hall, Tan, Aziz, & Coutie, 1999).
Effects on signaling have been observed in the T164I polymorphism. Amino acid position
164 is located on the upper part of TM4. The change from threonine to isoleucine in the
polymorphism converted a polar residue into a hydrophobic residue. This change is proposed to
affect the interaction between TM4 and TM5, and therefore affect the receptor’s transition to the
activated state. In vitro studies found that I164 exhibited lower affinity for endogenous ligands
isoproterenol and decreased agonist-induced-G𝛼 s interaction (Green et al., 1993). As a result, basal
and agonist-stimulated adenylyl cyclase activity is decreased, leading to a loss of function.
The R16G, Q27E and T164I polymorphisms have been studied using overexpression in
various cell systems decades ago. These studies, however, were mainly done using traditional
16
radioligand and polyclonal antisera that were less optimized and non-specific for ADRB2. Most
of the studies also focused on adenylyl cyclase activity, rather than the impact on cAMP level.
Today, there are not many studies done in vitro regarding the functional effects of ADRB2
polymorphisms, and many reagents for ADRB2 studies have not been optimized. To make things
more complicated, different cell colonies have the possibility of expressing various levels of
endogenous ADRB2 protein, in which epitope antibodies may miss. Therefore, before additional
studies can be conducted, it is necessary to optimize the experimental procedures and characterize
working reagents to obtain experimental results in the future.
17
CHAPTER TWO: MATERIALS AND METHODS
2.1 CELL CULTURE AND MAINTENANCE
Human embryonic kidney 293 wild type cell line (HEK293) was a kind gift from the
MacKay laboratory (University of Southern California, Los Angeles, CA). Cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (cat#11995-065, Thermo Fisher Scientific,
Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (cat#26140-079, Thermo Fisher
Scientific). All cell cultures were maintained at 37°C in a 5% CO2 humidified incubator. Cells
were split into 1:10 ratio at 80-90% confluency. To harvest cells, the culture medium was aspirated,
cells were washed once with DPBS (cat#21-031-CV, Thermo Fisher Scientific) and incubated with
Trypsin EDTA 0.25% (cat#25200-056, Thermo Fisher Scientific) until cells fully detached from
the cell culture plate. The trypsin activity was inhibited by the addition of DMEM added with 10%
FBS and cells were centrifuged for splitting, freezing, or further processing. When necessary, cells
were frozen and stored in DMEM supplemented with 20% FBS and 7% DMSO in -80°C.
2.2 PLASMID EXPANSION AND ISOLATION
Plasmids encoding for human ADRB2 were purchased from GenScript (cat#OHU19730D,
Piscataway, NJ) and VectorBuilder (vector ID #VB900124-0913hym, Chicago, IL) (Figure 10).
Both plasmids harbor an ampicillin resistance gene for selection in E. coli culture and neomycin
resistance gene for selection in mammalian cell culture. Vector from GenScript encodes the fusion
protein ADRB2-DYKDDDDK, which encodes for a FLAG epitope at the end of the ADRB2 C-
terminus. The polymorphism that the ADRB2 gene encodes is R16 Q27 T164. Vector from
VectorBuilder encodes an eGFP marker and the polymorphism G16 E27 T164 corresponding to
Uniprot sequence ID: P07550. Both plasmid DNAs were initially transformed in TOP10
18
competent cells (cat#C404004, Thermo Fisher Scientific), plated on LB agar plates supplemented
with 100 µg/mL carbenicillin (cat#C-103-100, GoldBio, St. Louis, MO), and grown at 37°C
overnight. Bacterial colonies were expanded on a 10 mL scale in Terrific Broth medium
(cat#T15000-2000.0, Research Product International, Mount Prospect, IL) supplemented with 100
µg/mL carbenicillin with constant agitation at 37°C overnight for approximately 16 hours. The
following day, plasmids were isolated using QIAprep Spin Miniprep kit (cat#27106, Qiagen,
Hilden, Germany) following the manufacturer’s protocol. The isolated plasmid sequence was
verified by GENEWIZ. Once sequences were confirmed, the bacterial colonies were further
expanded on a 200 mL scale in Terrific Broth and the plasmids were isolated using QIAGEN
Plasmid Maxi Kit (cat#12162, Qiagen) following the manufacturer’s protocol. The concentration
of the isolated plasmids was determined by measuring the optical density (OD) at 280 nm with a
1 mm light path using Nanodrop ND-1000 spectrophotometer (Bio-Rad, Hercules, CA).
19
Figure 10. Plasmid maps encoding ADRB2. (A) Plasmid purchased from GenScript encoding ADRB2-FLAG fusion protein. (B)
Plasmid purchased from VectorBuilder encoding ADRB2 and eGFP marker.
20
2.3 SITE-DIRECTED MUTAGENESIS
The site-directed mutagenesis of pcDNA3 plasmid from ADDGENE to create mutations
at positions 16 and 164, was performed using the Phusion Site-Directed Mutagenesis Kit
(cat#F541, Thermo Fisher Scientific). The R16G mutation was carried out using a custom PCR
oligonucleotide primer set 5´-CTGGCACCCAATGGAAGCCATGCGC-3´ and 5´-
CAAGAAGGCGCTGCCGTTCCCGG-3´ (Figure 11A). The mutation at position 164 from
threonine to isoleucine was carried out using a custom oligonucleotide primer set 5´-
TGTCAGGCCTTATCTCCTTCTTGCC-3´ and 5´-
CAATCCACACCATCAGAATGATCACCC-3´ (Figure 11B). A double mutation at positions
R16G and T164I were performed by mutating the R16G product with the described primer for
T164I. The PCR product was digested with EcoRI and visualized on a 1% agarose gel using
ethidium bromide to confirm PCR success.
Figure 11. Primer sequences for mutagenesis reactions. (A) Primer pairs used to perform R16G mutation. (B) Primer pairs used
to perform T164I mutation.
21
2.4 STABLE MONOCLONAL CELL LINE GENERATION
At first, transient transfection was performed using Lipofectamine 3000 Transfection
Reagent, (cat#L3000015, Thermo Fisher Scientific) according to the manufacturer’s direction.
However, to obtain a constant measurement of downstream signaling effects, it was decided that
generating a stable cell line would be more appropriate since a more stable and permanent protein
expression would be maintained.
HEK293 cells were transfected with plasmid DNA for stable expression by electroporation.
Cells were grown until 80% confluency, trypsinized and resuspended in Opti-MEM
(cat#11058021, ThermoFisher Scientific). For each electroporation, 400 𝜇 L of 5 x 10
5
cells/mL in
Opti-MEM were combined with 20 𝜇 g of purified plasmid DNA (Table 1) in DEPC-treated water
into a 4 mm gap sterile electroporation cuvette (cat#45-0126, BTX, Hamden, CT). The
electroporation was done at 220 V, 960 𝜇 F with 30 Ω using an electroporation system (Electro Cell
Manipulator 600, BTX). Cells were immediately seeded back into a cell culture dish with DMEM
supplied with 10% FBS and allowed to recover for 48 hours. Transfected clones were selected
with 400 𝜇 g/mL of the geneticin analog G-418 (cat#10131035, Thermo Fisher Scientific) over the
course of 10 days and eGFP expression was monitored under a fluorescence microscope (Diaphot
300 X-Cite 120 LED, Nikon, Melville, NY). Wells demonstrating media color change or eGFP
expression were expanded by splitting cells into 0.5 cells/well concentration in 96-well plates.
Wells containing single cells expressing eGFP were gradually expanded into 100 mm cell culture
dishes. Stable cell line was maintained in full media supplemented with 300 𝜇 g/mL G-418.
22
Table 1. List of purified cDNAs used for transfection.
Source Polymorphisms Marker
GenScript R16 Q27 T164 fused FLAG epitope
Mutagenesis reaction G16 Q27 T164 fused FLAG epitope
Mutagenesis reaction R16 Q27 I164 fused FLAG epitope
Mutagenesis reaction G16 Q27 I164 fused FLAG epitope
VectorBuilder G16 E27 T164 eGFP
2.5 IMMUNOBLOTTING
Positive and negative controls for ADRB2 expression were purchased from Novus
Biologicals (cat#NBP2-11173, Littleton, CO). Cells were plated and allowed to grow to 90%
confluency in 10 cm cell culture dishes with appropriate media (DMEM with 10% FBS or DMEM
with 10% FBS and 300 𝜇 g/mL G-418). Upon confluency, cells were rinsed with DPBS once and
lysed with RIPA buffer (Mackay Lab, University of Southern California). Cell lysates were
collected by scraping into microcentrifuge tubes and kept at -80°C until use. U-937 and Jurkat cell
lysates were kind gifts from Alachkar Lab (University of Southern California). HeLa cell lysates
was a kind gift from Mackay Lab. HepG2 cell lysates was a kind gift from Stiles Lab (University
of Southern California). Before running western blot, the cell lysates were centrifuged at 14,000 x
g for 15 minutes at 4°C and the total protein concentration in the supernatant was determined using
Pierce BCA Protein Assay Kit (cat#23225, ThermoFisher Scientific). Approximately 30 𝜇 g of
total protein from all samples were separated on 10% polyacrylamide gels in Tris-glycine-SDS
running buffer (cat#161-0772, Bio-Rad). Protein bands were transferred onto a pre-activated
PVDF membrane (cat#0307-5L, VWR Life Science, Radnor, PA) in Tris-glycine transfer buffer
(cat#SC-2325, Santa Cruz Biotechnology, Dallas, TX) at 200 mA overnight at 4°C. Membranes
were blocked with 5% non-fat milk (cat#TBST03-03, Bioland Scientifc, Paramount, CA) in TBST
(cat#sc-2325, Santa Cruz Biotechnology) at room temperature for 1 hour. Membranes were then
23
incubated with either DYKDDDDK(FLAG) Rabbit mAb (cat#14793, Cell Signaling Technology,
Danvers, MA), R&D Systems Rabbit anti-ADRB2 mAb antibody (cat#MAB10040, R&D
Systems, Minneapolis, MN) or Abcam Rabbit anti-ADRB2 mAb antibody (cat#ab182136, Abcam,
Cambridge, United Kingdom) at 1:1000 dilution in 1% BSA in TBST supplemented with 0.02%
sodium azide at 4°C overnight. Membranes were washed three times with TBST, followed by
incubation with Rabbit HRP-conjugated secondary antibody (cat#HAF008, R&D Systems) at
1:5000 dilution and GAPDH (HRP Conjugate) Rabbit mAb (cat#3683S, Cell Signaling
Technology) at 1:1000 dilution in 5% milk TBST solution for 1 hour at room temperature.
Incubation was followed by three washes in TBST. Proteins on the membrane were detected with
the addition of chemiluminescent substrates (cat#32106, Thermo Fisher Scientific) and captured
on the ChemiDoc MP imaging system (Bio-Rad).
Table 2. List of primary antibodies used.
Manufacturer Listed Specificity Catalog# Applications
Cell Signaling
Technology
DYKDDDDK (FLAG) 14793
Western Blot,
Immunoprecipitation,
Immunohistochemistry
(Paraffin),
Immunofluorescence
(Immunocytochemistry), Flow
Cytometry, Chromatin IP
R&D Systems Human/Mouse/Rat ADRB2 MAB10040 Western Blot
Abcam Human/Mouse/Rat ADRB2 ab182136
Western Blot,
Immunohistochemistry
(Paraffin, Frozen)
R&D Systems
Human/Mouse/Rat/Monkey/
Bovine/Pig GAPDH
3683S Western Blot
2.6 IMMUNOFLUORESCENT STAINING
Poly-D-lysine (cat#P-1149, Sigma-Aldrich, St. Louis, MO) was dissolved in sterile water
to make a 0.1 mg/mL concentration stock solution. Individual glass coverslips were placed into
24
the bottom of each well in a 6-well plate. Enough Poly-D-lysine solution was poured into the wells
to incubate the coverslips for 15 minutes at room temperature. Poly-D-lysine solution was
removed, and the wells were rinsed with DPBS three times and allowed to dry overnight under
sterile conditions. Approximately 300,000 cells were seeded on top of the Poly-D-Lysine coated
coverslips and allowed to grow at 37°C in 5% CO2 humidified incubator. Upon reaching 70%
confluency, the cultured cells were fixed with 4% paraformaldehyde (PFA) (cat#43368, Alfa
Aesar, Haverhill, MA) for 15 minutes. The PFA was quenched with 50 mM NH4Cl in DPBS for
10 minutes. After washing the wells with DPBS, the cells were permeabilized with 0.1% Triton
X-100 (cat#BP151-500, Fisher Scientific, Waltham, MA) for 5 minutes, washed, and blocked with
1% BSA in DPBS at room temperature for 1 hour. After washing, the fixed cells were incubated
with either R&D Systems Rabbit mAb anti-ADRB2 antibody or Abcam Rabbit mAb anti-ADRB2
antibody at 1:100 dilution at 4°C overnight. The coverslips were washed and incubated with
donkey anti-rabbit rhodamine conjugate (cat#31685, Thermo Fisher Scientific) at 1:100 dilution
at room temperature for 1 hour. After secondary antibody incubation, the coverslips were
incubated with 1:1000 DAPI (cat#D3571, Invitrogen, Paisley, PA) in DPBS for 2 minutes
followed by a DPBS wash, five times. Coverslips were mounted onto slides with Anti-fade
Mounting media (cat#P36961, Invitrogen) and kept in the dark until ready to be imaged. Images
were acquired using fluorescent microscope under rhodamine, DAPI and GFP channels.
2.7 TOTAL CYCLIC AMP MEASUREMENT
Upon reaching confluency, cells were rinsed with DPBS once and incubated with 0.1 M
HCl for 10 minutes. Cell lysates were centrifuged at 14,000 x g for 15 minutes at 4°C, and the
supernatant was collected for measurement by ELISA or kept at -20°C for storage. Dilutions of
samples were made using the 0.1 M HCl supplied in the kit. Cell cAMP levels were measured
25
using the direct cAMP ELISA kit (Enzo Life Sciences, Farmingdale, NY) according to the
manufacturer's instructions.
26
CHAPTER THREE: RESULTS
3.1 ADRB2 STABLE CELL LINE GENERATION
An anti-FLAG antibody was used to verify the expression of GenScript plasmid (R16 Q27
T164) and its mutants (G16 Q27 T164, R16 Q27 I164 and G16 Q27 I164) which encodes the
FLAG-ADRB2 fusion protein. The western blot results showed faint non-specific bands of 28 kDa
(Figure 12A). The same membrane was re-blotted using anti-ADRB2 from Abcam using the same
protocol which revealed bands around 62-70 kDa for the transfected cell lines (Figure 12B). An
antibody against ADRB2 from R&D Systems was used to verify the expression of VectorBuilder
plasmid transfection (G16 E27 T164) which carries an eGFP marker. The Western blot revealed
faint bands for two of the colonies at molecular weight 43 kDa (Figure 13A). Another run was
performed and incubated with the anti-ADRB2 from Abcam, which revealed bands of around 65-
100 kDa (Figure 13B).
Figure 12. SDS-PAGE of polyclonal cell lines transfected with FLAG-ADRB2 fusion gene. (A) Primary antibody specific to
FLAG-epitope was used to blot the membrane. Faint bands were observed for non-transfected HEK293, R16Q27T174 and
G16Q27T164 at molecular weight of 28 kDa. (B) Primary antibody specific to ADRB2 (Abcam) was used to blot the same
membrane. Thick bands were observed for the transfected cell lines with molecular weight of 55-90 kDa.
27
Figure 13. SDS-PAGE of monoclonal cell colonies transfected with Vector Builder (G16E27T164) plasmid. (A) Membrane was
incubated with anti-ADRB2 from R&D Systems. Faint bands were observed for colony 3 and 7 with molecular weight of 43 kDa.
(B) Membrane was incubated with anti-ADRB2 from Abcam. Thick, streaking bands were observed for most of the colonies with
molecular weight 65-100 kDa, with the exception for non-transfected HEK293 and colonies 2-4.
3.2 ANTIBODY VERIFICATION
As listed on Table 2, the primary antibodies used in this experiment were purchased from
three different companies. To verify that the Abcam anti-ADRB2 was specific for ADRB2, we
looked at lysates from various cell lines (HEK293, HepG2, HeLa, U-937 and Jurkat). According
to Protein Atlas database, HepG2, HeLa and Jurkat expressed low concentrations of ADRB2, and
therefore we expected them to act as a negative control (Karlsson et al., 2021). When the blot was
incubated with the anti-ADRB2 from R&D Systems, there were no bands visible (data not shown).
We also ran lysates from U-937 which were supposed to express high concentrations of ADRB2.
However, the blot revealed no bands, and ponceau staining suggested that protein degradation had
occurred as the total protein bands have smeared (data not shown). The membrane was reincubated
with anti-ADRB2 from Abcam, which shows bands for the transfected cell colony 10 at around
70-120 kDa (Figure 14).
To verify the antibody specificity, a positive control was purchased from Novus
Biologicals. The positive control was the whole cell lysate of HEK293T cells overexpressing
28
ADRB2 (G16R variant). The positive and negative controls were blotted with the anti-ADRB2
from R&D Systems, followed by anti-ADRB2 from Abcam. When probed with R&D Systems
antibodies, there are no bands (Figure 15A), whereas the antibody from Abcam revealed a band of
around 47-56 kDa (Figure 15B). No non-specific bands were observed for negative controls
blotted with both antibodies. However, it is important to note that there is a discrepancy in the
molecular weight of the bands of the transfected cells and the purchased positive control (~65-90
kDa compared to ~47-56 kDa).
Figure 14. SDS-PAGE of various cell lines. Membrane was incubated with anti-ADRB2 from Abcam. HeLa, Jurkat and
G16E27T164 colony 2 observed faint non-specific bands. Non-transfected HEK293, U-937 and HepG2 showed no bands.
G16E27T164 colony 10 showed heavy bands at molecular weight 65-100 kDa, consistent with previous blots.
29
Figure 15. SDS-PAGE of Novus Bio control lysates. (A)Membrane was incubated with anti-ADRB2 from R&D Systems. No
bands were visible. (B) Membrane was incubated with anti-ADRB2 from Abcam. Thick bands of molecular weight 45-55 kDa
were observed in overexpressed positive lysate.
3.3 IMMUNOFLUORESCENT STAINING
Adrenergic receptors can be expressed on the cell surface or internalized into endosomes
as part of desensitization. To see where these unbound receptors were expressed,
immunofluorescent staining was performed. Anti-ADRB2 antibody from Abcam was conjugated
to rhodamine (red) to stain for ADRB2 while DAPI was used to stain the nuclei (blue). In addition,
the green fluorescent protein (GFP) expressed as part of the plasmid were also monitored to
confirm transfection efficacy. The transfected HEK293 cells showed high level of ADRB2
(indicated by red color) and GFP staining (indicated by green color), confirming the transfection
of the plasmid (Figure 16). In the non-transfected HEK293 cells, there is low to no GFP signal and
a significantly lower rhodamine signal which stains for ADRB2 (Figure 17).
30
Figure 16. Immunofluorescent staining on HEK293 transfected with VectorBuilder plasmid (G16E27T164). Brightness and
contrast were adjusted to give a better representation of staining. (A) Anti-ADRB2 from Abcam is conjugated onto rhodamine to
stain for ADRB2. (B) eGFP is expressed as part of the plasmid. (C) DAPI was used to stain for nuclei. (D) Merged image of all
three channels to indicate localization for each signal.
Figure 17. Immunofluorescent staining on non-transfected HEK293. Brightness and contrast were adjusted to give a better
representation of staining. (A) Anti-ADRB2 from Abcam is conjugated onto rhodamine to stain for ADRB2. (B) GFP channel
observed no signal as eGFP is not expressed by the non-transfected cells. (C) DAPI was used to stain for nuclei. (D) Merged
image of all three channels to indicate localization for each signal.
31
3.4 EFFECT OF OVEREXPRESSION ON CYCLIC AMP PRODUCTION
cAMP is produced when an agonist binds to ADRB2, as an effect of the activation of
adenylyl cyclase. However, several in vitro studies have revealed that polymorphisms can affect
the degree of activation of adenylyl cyclase, and therefore altering the cAMP level. To see the
effect of overexpression, as well as how different polymorphisms can affect the production of
cAMP in HEK293 cell line, an ELISA was performed to measure the intracellular cAMP
production. The kit is a colorimetric competitive immunoassay, which utilizes alkaline
phosphatase to generate yellow color that can be read on the plate reader. The results show that
HEK293 cells overexpressing G16 E27 T164 contain a high concentration of cAMP (Figure 18A).
However, R16 Q27 T164, G16 Q27 T164, R16 Q27 I164 and G16 Q27 I164 polymorphisms
showed no cAMP production, lower than what was observed with the non-transfected HEK293.
Figure 18. Effect of ADRB2 overexpression on cAMP and ADRB2 expression. Samples from non-transfected HEK293,
R16Q27T164, G16Q27T164, R16Q27I164 and G16Q27I164 were from polyclonal cell lines, whereas G16E27T164 were from a
monoclonal cell colony. (A) Measured total cAMP concentration via Direct cAMP ELISA Kit from ENZO LifeSciences. (B)
Measured relative ADRB2 expression obtained by Western blot, quantified using ImageLab.
32
CHAPTER FOUR: DISCUSSION
ADRB2 is an important pharmaceutical target due to its role in regulating heart and lung
contractions. Recent studies have also started to see associations of ADRB2 in various types of
cancer. Population studies have found that individuals with ADRB2 polymorphisms can react
differently towards β-blockers. However, there is a lack of understanding on the mechanisms of
how these polymorphisms can affect ligand binding, and consequently result in altered
downstream signaling, which this project initially sought to uncover. Unfortunately, several
unexpected problems were encountered, mainly on the lack of antibodies specificity and reliable
controls which shifted our focus onto transfection optimization and reagent characterization for
future experiments.
The first problem encountered was the difficulty in monitoring the transfection efficacy
and stable cell line generation. The FLAG-ADRB2 plasmid purchased from GenScript does not
carry a fluorescent marker that can be monitored through live microscopy. In short, to generate a
monoclonal cell line, it took 7 days post transfection to select for the desired cell line and another
2-3 weeks to grow the single cell into a colony big enough for protein detection via Western blot.
To combat this issue, a plasmid from VectorBuilder, which carries ADRB2 and an eGFP marker,
was tested to monitor the transfection as well as expression level of the protein, eliminating the
possibility of expanding the wrong monoclonal cell colony.
The second problem encountered was the unsuccessful mutagenesis reaction to create G16
E27 I164. G16 Q27 T164, R16 Q27 I164 and G16 Q27 I164 polymorphisms from the FLAG-
ADRB2 fusion plasmid were created using ThermoFisher Site-directed mutagenesis Kit. The
efficacy of the mutagenesis reaction was verified by GeneWiz Sanger Sequencing, resulting in
ADRB2 polymorphisms G16 Q27 T164, R16 Q27 I164 and G16 Q27 I164. However, when the
33
same protocol was performed on VectorBuilder’s plasmid using the same set of T164I primers
(Figure 11B), there were no PCR products, and no E. coli transformation was observed (data not
shown). Based on the plasmid maps, it was unclear why there was no PCR product since the amino
sequence for both plasmids that were covered by the primers were the same. It could be that, due
to the different size of the plasmids (GenScript plasmid is 6632 bp; VectorBuilder plasmid is 7725
bp), different PCR conditions will be required. Through these initial processes, we ended up with
these genotypes: R16 Q27 T164 (GenScript), G16 Q27 T164, R16 Q27 I164, G16 Q27 I164 and
G16 E27 T164 (VectorBuilder).
Another problem encountered was the lack of specific antibodies to ADRB2. Anti-FLAG,
which had been used to detect ADRB2 from the cell lines transfected with FLAG-ADRB2 genes
(Table 1), was tested but no bands of the desired molecular weight (46 kDa) were visible (Figure
12A). We attempted to resolve this situation by purchasing an anti-ADRB2 from R&D Systems.
Further, using anti-ADRB2 from R&D Systems, which also allows for the detection of the basal
level of ADRB2 expression rather than only identifying overexpressed ADBR2, also revealed no
bands (data not shown). Since FLAG-positive or ADRB2-positive controls were not yet available
at this point, it was unclear if the issue lies with the transfection or if there was a fault with the
antibodies. To eliminate the possibility of faulty transfection, anti-ADRB2 from R&D Systems
was used to probe the monoclonal cell line colonies which were successfully transfected with
VectorBuilder plasmid. These colonies have been monitored by their eGFP expression via
fluorescent microscopy, indicating successful transfection, however, no bands of desired
molecular weight were visible with the anti-ADRB2 from R&D Systems (Figure 13A). This result
indicated that both the anti-FLAG from Cell Signaling Technologies and anti-ADRB2 from R&D
Systems were faulty. Therefore, the anti-ADRB2 antibody was purchased from another vendor
34
(Abcam). Incubating the membranes with the new anti-ADRB2 antibody revealed thick bands of
molecular weight 65-100 kDa (Figure 12B and 13B), which were inconsistent with the expected
molecular weight of ADRB2 (46 kDa).
To check if the molecular weight discrepancies were due to non-specific binding, cell lines
with varying levels of ADRB2 expression were compared. However, no bands were visible for the
different cell lines when anti-ADRB2 from Abcam was used (Figure 14). The specificity of the
anti-ADRB2 antibody was then verified by purchasing an ADRB2 control from Novus
Biologicals. The positive control is a whole lysate of transiently overexpressed ADRB2 produced
in HEK293T cells, whereas the negative control is HEK293T transfected with an empty vector.
The controls were analyzed using anti-ADRB2 from R&D Systems (Figure 15A), and the Western
blot result showed no visible bands, which confirms the lack of specificity of the antibody from
R&D Systems. Incubating the membrane with anti-ADRB2 from Abcam (Figure 15B) revealed
bands of 45-55 kDa, which was consistent with the expected molecular weight of ADRB2.
We hypothesized that the discrepancy in molecular weights from our transfected cells (65-
100 kDa) to the Novus Biologicals ADRB2-positive control (45-55 kDa) comes from post
translational modifications or variations in experimental techniques, as mentioned in Abcam’s
product page: “The 50-90 kDa bands detected by ab182136 are consistent with many literatures,
like PMID: 2836733, PMID: 17570158, PMID: 11701618, PMID: 2545714, PMID: 16708760”.
The molecular weight and pattern is similar with the result of various post-translational
modifications reported in other publications such as phosphorylation (Berthouze,
Venkataramanan, Li, & Shenoy, 2009), ubiquitination (Han et al., 2012; Nabhan, Pan, & Lu,
2010), palmitoylation (O'Dowd, Hnatowich, Caron, Lefkowitz, & Bouvier, 1989) or glycosylation
(Li, Zhou, Huang, & Yang, 2017). Based solely on Western blot results, the actual modifications
35
that may have occurred in the transfected cell samples cannot be identified and therefore further
analysis is required.
After determining that the collected lysates are indeed overexpressing ADRB2, the
receptors localization either intracellularly or at the cell surface was investigated. GPCRs can only
bind to ligands when expressed on the cell membrane, and after binding will get internalized into
endosomes and therefore leading to desensitization. Even though studies have shown that ADRB2
can exert basal adenylyl cyclase activity without the presence of ligands, the majority of
downstream signaling relies on agonist binding in which eventually cAMP is produced. The
localization of the ADRB2 was done via immunofluorescent labeling using the anti-ADRB2 from
Abcam which had given results to suggest that this particular antibody was ADRB2-specific. The
G16E27T164 staining results show strong signals for all three channels – rhodamine, GFP and
DAPI, thus, confirming the transfection efficiency (Figure 16). The rhodamine signal seems to
outline the cell boundaries, consistent with cell membrane staining, with some staining within the
cytoplasm. Unfortunately, since the cell membrane was not stained, whether the receptor is
expressed on the cell surface or has been internalized due to endogenous ligands or activation of
signaling pathways could not be determined. On the other hand, staining on revived frozen
monoclonal cells carrying R16 Q27 T164, G16 Q27 T164, R16 Q27 I164, G16 Q27 I164 showed
very low rhodamine signal (data not shown) which could be explained by the loss ADRB2
expression due to being outcompeted by resistant non-transfected cells because of lowered G418
concentration during cell line maintenance. It is also possible that the cells carrying Q27 may have
a growth disadvantage, compared to non-transfected HEK293 cells which resulted in being
outnumbered. On the contrary, the cell lysates ran on the Western blot (Figure 12) were harvested
36
during early transfection period prior to monoclonal cell dilution on Western blots, which explains
the detection of ADRB2 bands.
Total intracellular cAMP measurement showed that HEK293 express basal cAMP
concentration, whereas G16E27T164 expressed a higher cAMP concentration and R16 Q27 T164,
G16 Q27 T164, R16 Q27 I164, G16 Q27 I164 expressed little to no cAMP. The cells carrying
G16E27T164 expressed higher cAMP level compared to non-transfected HEK293 most likely due
to higher levels of ADRB2 overexpression, which was confirmed via Western blot (Figure 13) and
immunofluorescent labeling (Figure 16). We hypothesized that R16 Q27 T164, G16 Q27 T164,
R16 Q27 I164, G16 Q27 I164 expressed low to no cAMP level due to the loss of ADRB2
expression, as explained above. This was confirmed in additional experiment as increasing the
G418 concentration from 300 to 400 𝜇 g/mL in the revived cell lines resulted in complete cell death
over the course of 2 weeks. However, it was unclear why the cAMP concentration is very low,
because if the cell culture has been taken over the non-transfected HEK293 cells, a basal cAMP
activity should still be observed.
37
CONCLUSIONS
Through this study, we have optimized several experimental procedures First, using a
plasmid with a fluorescent marker is more efficient compared to a protein tag, as the fluorescence
can be used as a verification of successful transfection. Second, a stable monoclonal cell generation
through electroporation is more appropriate for future experiments to measure downstream
signaling changes as the protein expression would be more consistent and more permanent. Third,
antibodies purchased from a manufacturer still need to undergo verification, ideally using purified
protein of interest or using an overexpressed cell lysate.
In addition, some interesting observations need to be taken into consideration when
conducting future experiments. The level of ADRB2 expression can vary by colonies, which needs
to be normalized across different polymorphisms when doing comparison studies. Furthermore,
there was a huge discrepancy in molecular weights from cell lines overexpressing ADRB2
compared to one that is from the purchased positive control. Even though we hypothesize that this
could be due to post-translational modifications, we were unable to determine what modifications
have occurred, and how they might affect ligand binding or downstream signaling. Additionally,
receptors localization was not successfully determined, which, can also affect the receptor binding
ability. Lastly, non-transfected HEK293 cells can also express basal level of cAMP in the absence
of catecholamines, which means that it may also bind to other unforeseen compounds.
In conclusion, ADRB2 remains a highly studied receptor due to its role in regulating
cardiovascular and pulmonary functions. Unfortunately, there remains a missing link in
understanding how polymorphisms can affect agonist binding, leading to altered secondary
messenger production. Understanding the molecular mechanisms of ADRB2 downstream
38
signaling coupled with cheminformatics and pharmacogenetic studies will help design β-blockers
that can be personalized to each patient.
39
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Abstract (if available)
Abstract
G protein-coupled receptors (GPCRs) are one of the largest and most important pharmaceutical targets due to their role in human pathophysiology. Many drugs target β2- adrenoceptors (ADRB2), a member of GPCRs which are commonly associated with cardiovascular medicine and asthma. However, pharmacogenetic studies have found that individuals may differ in treatment responses due to genetic polymorphisms in this gene. Despite recent population studies, the results have been inconsistent and there is a lack of understanding of how these polymorphisms can affect agonist binding, which consequently affects the downstream signaling of these receptors. For this reason, the goal of this thesis is to optimize experimental procedures and characterize the appropriate reagents for future studies on ADRB2.
While there are many available antibodies to detect ADRB2 on the market, many of these antibodies have not been verified in peer-reviewed journals. We purchased several different antibodies on the market to detect the overexpressed ADRB2 level in our cell lines. Unfortunately, several of them did not seem to be specific for the protein of interest, which hindered our downstream experiments. In addition, we have optimized the transfection protocol which includes testing two different plasmids to determine which one would be more experimentally efficient. We have also optimized the protocols to generate a stable monoclonal cell line as well as the appropriate condition to maintain the culture. Lastly, we determined that the cyclic adenosine monophosphate (cAMP) can be measured at basal level through the ELISA kit from Enzo Life Sciences.
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Susanto, Josephine
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Optimization of ADRB2 overexpression and reagent characterization for cyclic AMP measurement
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Master of Science
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Molecular Pharmacology and Toxicology
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2022-08
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