Author: Hari Srinivas Kalyan Paila

Publisher:

Published: 2016

Total Pages: 193

ISBN-13:

"Chemokine Receptors are a class of G Protein coupled receptors (GPCRs) or transmembrane (TM) serpentine receptors present on cell membranes which act as gate keepers and signal transducers 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 a 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 roles range from the mediation of early stage allergic and inflammatory responses, to host cell defenses and related physiological roles. Little is known about their structure - function properties at the receptor level and the downstream signaling events after the receptor is stimulated. My dissertation focuses on Chemokine receptor subtype 3 (CCR3). CCR3 expressing cell lines available to date are mortal cell line, meant for single use assay purpose with limited/transient CCR3 receptor expression. These cell lines are not a 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 293) 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 for semi-preparative scale purification of CCR3. The HEK 293 CCR3 cell line was used specifically for two purposes, as elaborated in this dissertation. The first is to overexpress the human CCR3 receptor for purification and characterization, by establishing a standard membrane protein purification method for human CCR3 membrane protein. Cellular membrane protein expression in human cells is one of the tough challenges in protein biochemistry. Membrane proteins lose their structural and functional integrity once removed from their lipid bilayer environment in cell membranes; amembrane protein is stabilized in its native biophysical environment. As a fundamental pre-requisite for maintaining the near-native conditions around the membrane protein molecule during its extraction and purification, one has to maintain its biophysical integrity and to preserve the structural and functional features of the protein during the overall extraction and purification processes. The purified human CCR3 has several uses, such as 1) in protein sequencing to identify any possible sequence variants and post translational modifications in the protein, 2) to obtain data useful in receptor modeling and structure-based drug discovery efforts of CCR3, 3) biophysical characterization of the receptor at a single molecule level and its dimer and oligomeric states, and 4) immobilizing GPCRs on surfaces for ligand/drug screening with SPRI-based methods. All the above said uses require human CCR3 receptor purified in significant quantities; micrograms to milligrams. Here we successfully characterized a limited amino acid sequence of the purified CCR3 GPCR by mass spectrometry based methods. In the second objective, we were able to successfully adapt the laboratory developed CCR3 expressing HEK 293 cell line to stable isotope amino acid enriched DMEM supplemented with 10 % dialyzed FBS cell culture media. The C13 and N15 labeled Arginine (+10 Dalton) and Lysine (+8 Dalton) isotopic enrichment of the cell line was greater than ninety-five percent. This cell line was intended for the study of CCR3 receptor downstream signaling events by phosphoproteomics studies. Temporal phosphorylation of the signaling protein in the cell is the rationale behind global cellular phosphorylation analysis. Protein phosphorylation is the second most common post-translation modification (PTM) after acetylation. Reversible phosphorylation is critical in the functional aspects of cellular proteome and the signaling events involving biochemical pathways. The overall phosphopeptide enrichment for complex SILAC HEK 293 whole cell lysate protein digested peptide samples is developedwith titanium dioxide nanoparticles (TiO2) as a metal ion affinity resin. The developed SILAC workflow can be applied to study temporal phosphorylation dynamics for different cellular physiological states, following CCR3 receptor stimulation. In summary, the results emphasize overall stable cell line development of CCR3 receptor expression in HEK 293, analytical methods in CCR3 characterization, and methods inphosphopeptide enrichment from complex protein samples digests such as HEK 293 cell line."--Abstract from author supplied metadata.