Browsing by Subject "Receptors, Cell Surface"
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Item Delineating the Role of CD244 in NK Cell Cytotoxicity and the Contribution of Ly108 to Thymocyte Development(2010-11-02) Westcott, Jill Michelle; Schatzle, JohnGenetic analysis of the a murine model of lupus has implicated polymorphisms in the SLAM family of receptors (CD244 (2B4), CD229 (Ly9), CS-1 (CRACC), CD48 CD150 (SLAM), CD84, and Ly108 (NTB-A)) as causative for a breach in tolerance of both T and B cells leading to the production of self reactive antibodies. Analysis of common strains of laboratory mice revealed the existence of two stable haplotypes (b and z) of the SLAM family gene cluster. Given recent studies identifying polymorphisms in the SLAM family in humans, we have determined how polymorphisms in the SLAM family can affect lymphocyte function in mice to model how similar mechanisms may be involved in human pathologies. For these studies, we used mice congenic at the SLAM family locus (B6 –b haplotype and B6.Sle1b–z haplotype) to study two major questions: 1.) how do polymorphisms in CD244 affect NK function? And 2.) how do polymorphisms in Ly108 affect T cell tolerance? In the studies presented herein we demonstrate that CD244 functions largely as an inhibitory receptor in NK cells from B6 mice and as an activating receptor in B6.Sle1b mice. We demonstrate that allelic polymorphisms contribute to this differential function by altering receptor isoform usage, cell surface densities, baseline phosphorylation levels and subsequent adaptor association and receptor downmodulation. We also demonstrate that differential isoform usage of the receptor, Ly108 in B6 and B6.Sle1b mice alters thymocyte differentiation and negative selection events leading to a break in tolerance of T cells in B6.Sle1b mice. This is due to differential affects of the isoforms of Ly108 on thymocyte cell cycle progression and sensitivity to apoptosis. These studies highlight how naturally occurring polymorphisms in SLAM family genes can profoundly affect receptor function and potentially result in pathologic outcomes in certain genetic contexts.Item Dissecting the Function of FARP1 and Rho GTPases in Semaphorin-Plexin Signaling: Structural Perspective(2017-11-30) Kuo, Yi-Chun; Albanesi, Joseph P.; Huang, Jun-Shen; Rizo-Rey, José; Sternweis, Paul C.The Semaphorin-Plexin signaling is important for regulating axon guidance. Binding of Semaphorin to the Plexin receptor induces the dimerization of Plexin and stimulates its cytoplasmic GAP activity towards Rap, resulting in cytoskeleton re-organization. The growth cone of an axon then turns around, which ultimately leads to the repulsive guidance of the axon. Structural studies by our laboratory and others have revealed how the RapGAP activity of Plexin cytoplasmic region is stimulated by dimerization, how the Semaphorin ligand interacts with Plexin ectodomain, and how extracellular domains prevent Plexin from premature activation. Several downstream effectors interacting with Plexin cytoplasmic domain were found; however, the molecular mechanisms by which these effectors regulate Plexin signaling remain largely unknown. Our laboratory was interested in one of the effectors named FARP. Previous studies in our laboratory determined the crystal structures of the functional units of FARP1 and FARP2 but failed to detect any GEF activity in vitro. A recent screening with yeast two-hybrid identified a RhoGTPase, Rif, as a novel interacting protein for FARP1. To investigate how Rif/FARP1 interaction is involved in Plexin signaling, I determined the crystal structure of Rif-bound FARP1. This complex structure explains the functional roles that FARP1 and Rif might exert in dendritic spine formation and neurite outgrowth. Also, the N-terminal FERM domain of FARPs is known to interact with the cytoplasmic domain of Plexin. To interrogate how FERM interacts with Plexin and how this interaction might regulate Plexin signaling, I characterized the interaction of FERM with Plexin both biophysically and biochemically. The last question that I attempted to address in this dissertation is how a RhoGTPase, RhoD, inhibits activation of Plexin. RhoD was known for its remarkable inhibitory effect on Plexin signaling presumably through binding to the defined RhoGTPase binding domain of Plexin. The structure of Rac1 or Rnd1 in complex with Plexin did not explain how this binding regulates Plexin activity. I solved the crystal structure of the RhoD/Plexin complex. Modeling of this structure with that of Plexin active dimer in the context of the plasma membrane reveals a mechanism by which RhoD inhibits Plexin activation. Structure studies in this dissertation added another layer of comprehension on how the Semaphorin-Plexin signaling is regulated.Item Part 1: Isolation of Orexin Receptor Regulators via a Microarray-Based,Two-Color Cell Binding Screen. Part II: Targeted Inactivation of Proteins triggered by Visible Light.(2009-09-04) Lee, Jiyong; Kodadek, Thomas J.Part I. Isolation of orexin receptor regulators via a microarray-based, two-color, cell-binding screen. We have developed a novel two-color, cell-binding peptoid microarray screening approach with which we discovered new orexin receptor ligands. We found that peptoids on microarray, which showed preferential binding to receptor-expressing cells, indeed regulate the function of the receptor in living cells. Although cell-adhesion peptide microarrays have been used to isolate peptides that bind to cell surface receptors, this is the first time that a non-peptide, small molecule microarray has been used to do so. We also demonstrated that the pharmacophore of a hit peptoid can be rapidly identified through sarcosine scanning. Subsequent modifications of the pharmacophore yielded a potent antagonist (IC50 = 1.7 ? and an allosteric potentiator (EC50 = 120 nM) of the orexin receptor. Part II. Targeted Inactivation of Proteins Triggered by Visible Light Advances in genomics and proteomics have helped to provide thousands of potential drug targets and thus target validation strategies are more important than ever. Among target validation technologies, we are interested in chromophore-assisted light inactivation of proteins (CALI) since it allows for time-resolved protein knock-out in living cells. However, the practical use of this technology is limited, partially because of the low CALI efficiency of chromophores that are currently in use. To solve this problem, we developed a convenient system to compare different chromophores for their CALI efficiency, from which we found that Ru(II) complex is a photo-stable and unusually efficient CALI ?rhead?This finding led us to develop photo-chemical protein knock-out reagents?n which Ru(II) complex was conjugated to small molecule ligands targeting VEGFR2 or the 26S proteasome. When irradiated with visible light, these reagents showed significantly increased potencies in inhibiting VEGF-induced VEGFR2 activation or proteolytic activity of the 26S proteasome.Item Phase Transitions of Multivalent Adaptor Proteins(2015-05-27) Banjade, Sudeep; Rice, Luke M.; Rosen, Michael K.; Liou, Jen; Ross, Elliott M.Eukaryotic cells efficiently organize their activities to achieve their functional capabilities. This organization of biochemical reactions is a direct result of the cells' ability to compartmentalize their molecules. For example, within a eukaryotic cell, compartments like the nucleus, the endoplasmic reticulum and the vacuoles exist, which are relatively well known for their specific functions. These aforementioned compartments are surrounded by membranes. However, for the past hundred years, we have also known about assemblies of biomolecules that are not bound by membranes. After the initial discovery of nuages, other structures such as Cajal bodies, the nucleolus, promyelocytic leukemia (PML) bodies, paraspeckles, etc., were also described as membraneless organelles. Furthermore, membranes themselves are self-assembled entities of lipids, proteins and carbohydrates. Additionally, within and on surface of membranes, molecules cluster into signaling compartments in many different biological pathways. Interactions between individual biomolecules have been studied comprehensively in biology. One of our goals as biophysicists is to attempt to propose physical properties that allow these interactions at the subnanometer scale to give rise to formation of cellular structures, the compartments that are listed above. This thesis proposes a hypothesis based on polymerization of multivalent proteins that causes these complexes to phase separate in solution. The behavior of multivalent proteins and their ligands to phase separate may be a general property that allows cells to regulate their activities in certain localized compartments. To study this larger goal, I used a specific example of proteins involved in creating the slit-diaphragm, which is the filtration barrier of our kidneys. Nephrin, an integral membrane protein at the slit-diaphragm, interacts with its partners Nck and N-WASP in a multivalent fashion. I show here that these interactions create large assemblies that phase separate into liquid droplets, both in solution and on membranes. I also find that the creation of these assemblies affects the downstream biochemical activity of N-WASP toward the Arp2/3 complex and actin. The widespread existence of multivalent molecules suggests that these findings may have broad corollaries in different biological systems.