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Investigation of Ligand Binding to G-protein Coupled Receptors with the Use of Ellipsometry
Principal Investigator: Anna M. Weber
Advisors: Dr. Jennifer O’Connor and Dr. Maarij Syed
Thesis Research Proposal, Department of Biology and Biomedical Engineering
February 2015

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Introduction
G-protein coupled receptors (GPCRs) are integral cell surface proteins comprised of seven trans-
membrane helices that are responsible for driving many biological processes through the use of
several signal transduction pathways (Kriechbaumer et al., 2012). When a ligand binds the
receptor, a conformational change occurs in which heterotrimeric G proteins are activated.
These proteins are responsible for the transmission of signal molecules to the interior of the cell
(Oldham et al., 2007). Both cellular signaling and mutation of the proteins can potentially result
in detrimental physiological function.
Various diseases such as schizophrenia, cancer, and diabetes as well as others are directly related
to GPCRs (Kriechbaumer et al., 2012). Because GPCRs are both present in high frequencies in
the human genome, with about 800 locations being recognized to date, and play a crucial role in
cell signaling, they are a prime target for drugs and treatment procedures (Giraldo et al., 2011).
Currently, upwards of 60 percent of pharmaceuticals target GPCRs that have been designed to
treat ailments of many physiological systems (Schoneberg et al., 2004). Well known
pharmaceutical companies like GlaxoSmithKline, Eli Lilly, and Pfizer are all investigating in
treatment options targeting these receptors (Kriechbaumer et al., 2012).
Drug discovery is a lengthy process today taking anywhere from 10-12 years from the initial
research and development phase to the creation of a commercially recognized drug (Zang et al.,
2012). The development phase is time consuming due to the extensive testing procedures and
inefficient screening processes currently on the market. More relevant systems of analysis and
high throughput screening are desired in order to expedite the drug discovery.
Literature Review
Though GPCRs represent significant physiological importance, both intensive study and
investigation of therapeutic treatment have prevailed as a challenging task. Structural and
functional analysis of some of the proteins are restricted by the inability to create and maintain
situation comparable to that of nature. The membrane lacks stability when introduced into
certain buffers resulting in fluctuations in data analysis (Kriechbaumer et al., 2012). In some
cases, analysis is typically further limited due to low concentrations in certain tissues rendering
the crystallization technique useless (Zang et al., 2012). Because of these limitations, analysis of
ligand binding is problematic on various facets.
Ligand binding to GPCRs has been previously measured using two main methods: cell based
screen assays and surface plasmon resonance. Cell based screening assays are utilized in over
half of drug screenings to test the efficacy of drug targeting because they are considered to be
relatively simple by the use of cell markers and fluorescence (Zang et al., 2012). Though this is
the case, they take into account several critical assumptions mainly that cell viability is stable
and at a high level, resulting in possible anomalies during data analysis (Azouz, 2014). Cell
based screening assays can take about a lengthy amount of time which is detrimental for cell
viability. Assays may be used as an approach for initial drug screenings but face many
limitations in that the measurements of target goal, cell viability, and medium compositions and
substrate chemistry (Filmore, 2004).

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Surface plasmon resonance is an optical technique that detects substrate enrichment by the
incidence of the light on to the specimen from underneath the cell surface which can determine
the interaction of ligands and receptors. The technique is highly reliant on the molecular weight
of the specimen resulting in inefficient detection of samples of low weights (Ahmed et al., 2010).
Interaction of the light with the surface with which the cells are bound can potentially create
anomalies in data indicating false binding relationships. A General Electric subsidiary, BiaCore,
attempts to use the method to test the effectiveness of drug binding to cell surfaces but have yet
to obtain any prominent results.
Ellipsometry is another optical technique in which the efficacy of ligand bind can be measured
through the analysis of a polarized light spectrum that this refracted off of a specimen. This
method has been utilized by some other researchers to successfully quantify the interaction of
membranes and ligand binding to a GPCR (Kriechbaumer et al., 2012). Ellipsometry is able to
track minute changes in the environment, specifically those at the surface of a membrane making
this method appropriate to investigate ligand and receptor interactions.
As noted, GPCRs are readily seen in the human genome. For a model system, we propose the
use of an endothelial cell line, either KATOIII, HT-29, or A549, said to express the receptors
based upon the findings of previous researchers. The selected cell line will be used in
combination with a chemokine receptor and ligand.
Chemokines are a group of cytokines that act as signaling proteins, functioning primarily as part
of the immune response (Zlotnik et al., 2000). Regulation associated with these proteins is
mainly driven by the interaction of the molecules with GPCRs. In humans, upwards of 40
chemokines have been identified ranging in location from the lymphatic system to the nervous
system (Zlotnik et al., 2000). Chemokine receptors, though representing a small family of
proteins currently, are further subdivided into four subfamilies: CXC, CC, CX3C, and XC. The
nomenclature of the subfamilies is attributed to the arrangement of residues on the side chains of
the proteins (Zlotnik et al., 2000). CXC chemokine receptors (CXCRs) are named for the
separation of the two cysteine residues separated by a one different amino acid.
CXCRs are found on a variety of cells, most notably hematopoietic cells and vascular endothelial
cells. CXCR4 binds naturally with its ligand CXCL12 (Kriechbaumer et al., 2012). CXCR4 has
also been said to be functionally active with the ability pair to certain G-proteins. Many
epithelial cell lines express mRNA for CXCR4 which make them a good target for
experimentation (Murdoch et al., 1999).
Specific Aims
1. To test various substrates such as glass cover slips, slides, and plastic plates both with
and without proteins such as ligands or antibodies, and cells using ellipsometry to collect
a set of baseline data so that signals associated with ligand binding to GPCRs can be
differentiated.
2. To relate ligand binding to ellipsometry data by both the psi delta (method of visual
inspection of spectra) and optical constant (qualitative method with models accounting
for formation of new materials with new properties).

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3. To confirm binding of ligands to receptors in ellipsometry through the use of a well-
establish method, the Enzyme Linked Immunosorbent Assay (ELISA).
Materials and Methods
Cell Culture
Potential cell lines of interest include KATOIII, HT-29, and A549. KATOIII is a stomach cell
line from metastatic sites, HT-29 is an epithelial cell derived from the colon, and A549 is an
epithelial cell derived from the lung. These cell lines are all said to express to CXCR4
molecules. The model system used for experimentation will include one of the cell lines listed
above as well as the chemokine receptor and ligand, CXCR4 and CXCL12, respectively.
Ellipsometry
Ellipsometry is an optical technique used to evaluate the properties of membranes, in this case
receptor and ligand binding. The process begins with the incidence of light through a polarizer
that is introduced to a monolayer of cells secured to some medium in the prism at the base of the
system. The light that reaches the specimens is then refracted back to a detector that produces a
spectrum. The detector analyzes the refracted light data by using two parameters, psi and delta,
which can be related to its amplitude and phase. The machine is able to produce data in about
ten seconds so cell viability is not greatly affected. The machine cancels out stray light that has
entered the system so it does not impact data collection.
Figure 1: The setup of an ellipsometer (Kriechbaumer et al., 2012).
Ellipsometry provides several benefits. The system is very sensitive, allowing for observation of
minute changes to the surface chemistry of the sample. However, because it is very sensitive,
sample preparation must be done properly. The system is able to analyze specimen ranging in
size from several microns up to few millimeters. Biological sample preparation can be difficult,
but with available range, it should not be too problematic. The insrument also utilizes a
multiwavelength approach in which the specimen is scanned picking up signals at various levels.
In addition, ellipsometry when done properly can be used to not only quantify ligand binding but
also provide dynamic information of the timescale behind the binding and protein activation
process.

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Enzyme Linked Immunosorbent Assay
ELISAs are immunological assays used to test for the presence of proteins in a systems. The
tests are typically run in a 96 well plate in which binding and presence of proteins is indicated by
a color change. To confirm data from ellipsometry, ELISAs can be run using a cell line that
grow in the 96 well plate and introduced with CXCR4 and CXCL12 as well as antibodies
correlated to a color indicator like Horse Radish Peroxidase (HRP) to mimic the system.
Data Analysis
Ellipsometry data has the potential to be analyzed on three different levels; each level
incorporates a different approach resulting in information about the sample in varying levels of
significance and sophistication.
Psi and Delta are representative of the raw data from ellipsometry, explaining the amplitude and
phase of the refracted light beam, respectively. They represent change in polarization when light
interacts with the specimen. The psi-delta analysis approach is fairly straightforward. Visual
inspection of spectra is used to describe trends. Because this method is easily attainable and does
not typically involve much calculation and numerical use, it is considered a fairly low level of
analysis. The psi-delta approach is limited to correlating to changes in psi and delta to the
surface chemistry of sample but does not probe the details necessary for obtaining information
about changes in properties.
Figure 2: Example ellipsometry data output (Bizzarri et al., 2005).
The intermediate level of analysis is based upon the use optical constants. This method is more
quantitative, correlating the parameters, psi and delta, to the specific sample by the creation of a
model. Models are used to explain the creation of an optically new material from the binding of
ligands to the original sample. The optically new material displays new characteristics that are
able to be explained by the model and further confirming and explain the analysis from the psi-
delta method.
Finally, the relationship of optical constant to biological properties can be derived from the two
methods of lower level analysis. Though this method is possible, the linkage of optical constants
biological function and structure may include the use of additional techniques like electron

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microscopy may be needed to obtain connection between optical constants and actual biological
significance.
In order to ensure that the same data is being produced and analyzed for both ellipsometry and
ELISA, the experiments will be run under the same conditions. Consistent amounts of receptors
and ligand will be introduced to the cells and the same protocol for washing and blocking can be
followed for both procedures to help eliminate extraneous results.
Timetable
Spring Quarter 2015
Week Task
1 Grow cell cultures
2 Use ellipsometry to test cell line on petri
dish/glass slide.
3 Use ellipsometry to test cell line on petri
dish/glass slide.
4 Psi delta analysis of baseline data
5 Use ellipsometry to test cell and ligand on
petri dish/glass slide.
6 Use ellipsometry to test cell and ligand on
petri dish/glass slide.
7 Psi delta analysis of second layer data
8 Use ellipsometry to test cell, ligand, and
antibody on petri dish/glass slide.
9 Use ellipsometry to test cell, ligand, and
antibody on petri dish/glass slide.
10 Psi delta analysis of third layer data
Quarter Break Further analysis of data
Fall Quarter 2015
Week Task
1 Grow cell cultures
2 Perform previous experiments
3 Perform previous experiments
4 Data analysis
5 Data analysis
6 Development of Models
Quarter Break Determine what further experimentation
needs to be done

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Winter Quarter 2015
Week Task
1 Work week if needed
2 Work week if needed
3 Analyze results
4 Development of figures
5 1st rough draft of thesis
6 Revisions
7 Revisions
8 2nd rough draft of thesis
9 Revisions
10 Turn in final draft
After obtaining the cell line, it will take a about one week to grow the cell culture up to a point in
which the colonies can be moved to another medium such as the glass or plastics plate or a cover
slip. In the interim, ellipsometic data can be collected of the plates to ensure proper calibration
of the machine because spectra can be compared to those already existing in the database. Once
cultures are successfully grown, stage 1 will begin with analysis of cell lines. The process will
continue with the stage 2: the cell line and CXCR4, and then stage 3: the cell line, CXCR4, and
CXCL12. Each stage is designated as a two week period to ensure proper sample preparation
and accurate data collection. A week data analysis period is allotted for each stage so the spectra
can be analyzed.
Once data is collected and analyzed at the psi-delta level, creation of the optical constants model
will commence. Relation of spectra to a model will likely prove to be a substantial task
involving subsequent data analysis and reworking. It is unlikely that the biological properties
portion of data analysis will be reached at this point of the project.
References
Ahmed, F., J. Wiley, D. Weidner, C. Bonnerup, and H. Mota. 2010. Surface Plasmon Resonance
(SPR) Spectrometry as a Tool to Analyze Nucleic Acid–Protein Interactions in Crude Cellular
Extracts. Cancer Genomics and Proteomics. 7(6): 303-309. [Online].
Azouz, H. 2014. Q&A: Brian Kobilka. Nature. 514: S12-S13. [Online].
Bizzarri, A., L. Andolfi, M. Stchakovsky, and S. Cannistraro. 2005. AFM, STM and
Ellipsometry Characterization of a Monolayer of Azurin Molecules Self-Assembled on a Gold
Surface in Air. AZO Nano. [Online].
Filmore, D. 2004. It’s a GPCR World: Cell-based screening assays and structural studies are
fueling G-protein coupled receptors as one of the most popular classes of investigational drug
targets. Modern Drug Discovery. 7(11): 24-28. [Online].
Giraldo, J and J-P. Pin. 2011. G Protein-Coupled Receptors: From Structure to Function.
[Online.] The Royal Society of Chemistry, Cambridge, UK.

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<https://books.google.com/books?id=KMpvQwj1YEQC&pg=PR3&lpg=PR3&dq=ISBN+978-1-
84973-183-6&source=bl&ots=WCWGzxSR3s&sig=jYaF7O-aADoEmwKx6Hp-
V0C7qRk&hl=en&sa=X&ei=fT3YVPv8FYi0yQTqzIC4BA&ved=0CDUQ6AEwBA#v=onepag
e&q=ISBN%20978-1-84973-183-6&f=false>
Kriechbaumer, V., A. Nabok, R. Widdowson, D. P. Smith, and B. M. Abell. 2012. Quantification
of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry. PLoS
One. 7(9): 1-9. [Online.]
Murdoch, C., P. Monk, and A. Finn. 1999. Functional expression of chemokine receptor
CXCR4 on human epithelial cells. Immunology. 98(1): 36-41. [Online].
Oldham, W. M., and H. Hamm. 2007. How do Receptors Activate G Proteins?. Advances in
Protein Chemistry. 74: 67-93. [Online].
Schoneberg, T., A. Schultz, H. Biebermann, T. Hermsdorf, H. Rompler, and K. Sangkul. 2004.
Mutant G-protein-coupled receptors as a cause of human diseases. Pharmacology &
Therapeutics. 104(3): 173-206. [Online].
Zang. R., L. Ding, I-C., Tang, J. Wang, and S-T. Yang. 2012. Cell-Based Assays in High-
Throughput Screening for Drug Discovery. International Journal of Biotechnology for Wellness
Industries. 1(1): 31-51. [Online].
Zlotnik, A. and O. Yoshie. 2000. Chemokines: A New Classification System and Their Role in
Immunity. Cell. 12(2): 121-127. [Online].

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