Hari Paila – PhD Thesis Defense 04/25/2016 at 1:00 P.M.

JSNN – Hari Paila Ph.D. Thesis Defense

Candidate: Hari Paila

Advisor: Dr. Will Taylor

Department: Nanoscience

Date: Monday, April 25, 2016

Time: 1:00 P.M. – 3:00 P.M.

Location: JSNN Auditorium

2907 E. Gate City Blvd., Greensboro, NC 27401

Title: “Studies of The Human CCR3 Chemokine Receptor: Development of a Cell Line Stably Expressing CCR3, Receptor Purification and Characterization by Mass Spectrometry, and Phosphoproteomic Analysis of the CCR3 GPCR Signaling Pathway.”

Abstract:
Chemokine Receptors are a class of G Protein coupled receptors (GPCRs) or transmembrane
serpentine receptors present on cell membranes and act as gate keepers for the cells. Cellular
homeostasis is maintained by GPCRs by controlling the movement of various signals and
molecules from exterior to interior of the cell. Chemokine receptor subtype 3 has critical role in
homeostasis in organ systems in human body, a novel target in age related macular
degeneration disease progression, and act as co‐receptor for HIV entry into cells. Their role
ranges in mediating allergic and inflammatory reactions, host cell defenses and related
physiological roles. Little is known about the structure ‐ function properties at the receptor
level and the downstream signaling events after receptor stimulation coocurs. My Dissertation
focuses on Chemokine receptor subtype 3 (CCR3). CCR3 expressing cell line available to date
are mortal cell line, meant for single use assay purpose with limited/transient CCR3 receptor
expression. These cell lines are not viable option for CCR3 receptor expression‐purification and
stabilization for biophysical and related structural studies. My work focused on developing a
human endothelial kidney (HEK) cell line stably expressing human CCR3 using a tetracycline
inducible mammalian protein expression vector. This cell line is immortal and can be
propagated for cell culture scale up as of use’s requirement. The HEK ‐ CCR3 cell line was used
specifically for two purposes, work elaborated in the dissertation. First, to overexpress human
CCR3 receptor for purification and characterization, by establish a standard membrane protein
purification method for CCR3 membrane protein. Cellular membrane protein expressing in
human cells is one of the touch challenges in protein biochemistry. The reason behind, protein
loses its functional attributes once they are removed from their lipid bilayer environment of cell
membranes; membrane protein stabilizing environment. As a fundamental pre‐requisite factor
of maintaining the near native environment around the membrane protein molecule during
membrane protein extraction and purification, one have to maintained the biophysical integrity
and to preserve the structural and functional feature of the protein during the overall
extraction and purification processes.
The purified human CCR3 has several utilities such as in protein sequencing to identify any
possible sequence variants of the protein; data useful in receptor modeling and drug discovery
efforts, structure ‐ function relationship studies can be performed, Biophysical characterization
of the receptor at a single molecule level and related dimer and oligomeric state of the
receptor, immobilizing GPCRs on surfaces for ligand/drug screening with SPRI based methods.
All the above mentioned utilities require CCR3 receptor purified in significant quantities;
micrograms. Here we successfully characterized some of the amino acid sequence of the GPCR
by mass spectrometry methods.
In the second goal, we were able to successfully adapt the laboratory developed CCR3
expressing HEK cell line to stable isotope coded amino acid enriched DMEM & 10 % dialyzed
FBS cell culture media. C13 and N15 labelled Arginine (+10 Da) and Lysine (+8 Da) isotopic
enrichment of the cell line was greater than ninety five percent. This cell line was used to study
CCR3 receptor downstream signaling studies by phosphoproteomics studies. Temporal
phosphorylation of signaling protein in cell is the rationale behind global cellular
phosphorylation analysis. Phosphorylation is a reversible post translational modification and is
critical for cellular signaling events. The overall phosphopeptide enrichment from complex whole
cell lysate digests with Titanium dioxide nanoparticles (TiO2) was developed for the developed
HEK CCR3 cell line developed. In efforts to study global cellular level phosphorylation after
receptor stimulation, and application of TiO2 nanoparticles for the above mentioned specific
application. In addition to the above phosphoproteome study, a specific phosphorylation of
tyrosine residues of signaling protein kinases. Tyrosine phosphorylation (pTy) of protein kinases
prevalence of less than one percent of the phosphoproteome of cells, and hard to identify this
pTy phosphorylations, but these are critical phosphorylations of which signaling protein is
phosphorylated. To study the levels of phosphorylation in protein kinases at a cellular level, we
employed sepharose conjugated polyclonal Anti‐phosphotyrosine antibody beads to enrich
tyrosine phosphorylated peptides from whole cell lysate digests. Tyrosine phosphorylation in
kinases is crucial in receptor signaling events in cells. We did identify some significant results
with these methods in protein phosphorylation in HEK CCR3 cell line developed in our
laboratory.
Finally results emphasizes, overall stable cell line development of CCR3 expression in HEKs,
methods in phosphopeptide enrichment and tyrosine phosphorylated peptides from complex
protein samples such as HEK cell lines.