Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains vi, 200 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains viii, 264 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

published_or_final_version / Surgery / Master / Master of Philosophy

G protein coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins and downregulation. The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension. How GRKs recognize and are activated by GPCRs are not known, in part because the critical N-terminus and the kinase C-terminal extension were not resolved in GRK2 and GRK6 structures. The long-term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1), which represents a model system for studying phosphorylation-dependent desensitization of activated GPCRs. Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide-ligand binding states. One of the (Mg²⁺)₂·ADP·GRK1 structures is the most high-resolution structure (1.85 Å) of a GRK to date. In one (Mg²⁺)₂·ATP·GRK1 structure, almost the entire N-terminal region (residues 5-30) is observed. In addition, different segments of the kinase C-terminal extension are ordered in the various nucleotide-bound structures. Together, these two elements form a putative receptor-docking site adjacent to the hinge of the kinase domain. Based on these structures, a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase. Two novel phosphorylation sites were also identified at the N-terminus. The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS) homology domain of GRK1 was assessed using site-directed mutagenesis. In addition to mediating interaction with activated GPCRs, the N-terminus of GRKs also forms a binding site for calcium sensing proteins. Although its physiological significance is debated, the structures of these complexes could lend further insights into the conformation of the N-terminus of GRKs. The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes. Finally, the RH domain of GRK2 binds G[alpha subscript q], G[alpha]₁₁, and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors. The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆, a member of G[alpha subscript q] family.

published_or_final_version / Zoology / Master / Master of Philosophy

The translational GTPases elongation factor Tu (EF-Tu) and LepA modulate the dynamics of tRNA on the ribosome. EF-Tu facilitates the delivery of aminoacyl-tRNA (aa-tRNA) to the translating ribosome and LepA catalyzes the retro-translocation of tRNA•mRNA from the E- and P-sites of the ribosome back to the P- and A-sites. Although an increasing body of structural and biochemical information is available, little is known about the functional cycle of LepA during retro-translocation, the kinetics of EF-Tu dissociation from the ribosome and the rate of EF-Tu conformational change during aa-tRNA delivery. This thesis reports the successful construction and biochemical characterisation of a mutant form of EF-Tu from Escherichia coli ideal for the specific incorporation of fluorescent labels, enabling measurements pivotal for uncovering the rate of EF-Tu conformational change and dissociation from the ribosome. Furthermore, to determine structural components critical for LepA’s function, mutant versions of the protein were constructed and biochemically characterised. / xii, 127 leaves : ill. (some col.) ; 29 cm

Type 2 Diabetes Mellitus (T2DM) is a metabolic disorder of global concern that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. G- Protein Coupled Receptors (GPCRs), important mediators of cellular signaling responses, have been prime targets of drug discovery efforts in various therapeutic areas. Human G-Protein Receptor 40 (HGPR40) is highly expressed in the pancreas and has been implicated in the regulation of glucose metabolism and pathophysiology of T2DM. For effective control of diabetes, combination therapy is being considered because no single drug can completely control diabetes and its associated complications. This necessitates the identification of novel drug targets including HGPR40 which might permit development of drugs which function differently from existing drugs. This project focused on the construction of two chimeric receptor proteins named HGPR40.1-715_43.709-1013 (Chimera I) and HGPR40.1-431_43.389-1013 (Chimera II) which were made by exchanging domains within trans-membranes regions 6 and 4, of HGPR40 and HGPR43 DNA, homologous receptors which vary in amino acid sequence but which have the same three-dimensional structure. After PCR amplification of sufficient quantities of the desired gene fragments they were ligated together to form the desired recombinant chimeric proteins which were cloned into the expression vector pcDNA3.1 in two successive cloning steps. The vector contains CMV promoter, multiple cloning sites, neomycin resistance gene etc for high-level expression in a wide range of mammalian cells. The two full-length chimeras were designed to be 6452 bp and 6483 bp by exchanging either the first or the first two external domains of HGPR40 with those of HGPR43. The creation of the correct chimeras was verified by both agarose gel electrophoresis and PCR analysis. Then chimeric DNA was transformed into Escherichia coli to obtain sufficient amounts of DNA for sequencing verification of the desired construct. Upon verification, the cloned DNA was to be transfected into cultured mammalian kidney (HEK293) cells for expression of the chimeric proteins. A (FLIPR) Fluorometric Imaging Plate Reader analysis by our collaborators at Eli Lilly would have been used to measure the extent of Ca2+ efflux from the endoplasmic reticulum testing a variety of stimulatory molecules to obtain an indication of which would activate the receptor. Activation would initiate the internal (second messenger cascade) G-protein signaling pathway and result in the secretion of insulin. However, transformation of the chimeric membrane receptors into E. coli resulted in altered sequences which could not be used for the FLIPR analysis. Expression of membrane proteins in E. coli can lead to such gene rearrangements. These experiments will be repeated in the future using another type of competent cell designed to prevent genetic re-arrangements. Construction of stable clones will permit us to test numerous potential ligands to aid in development of novel therapeutic drugs targeting HGPR40 to aid in combating diabetes. / Department of Biology

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 137-156). Also available in electronic version.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 21, 2009) Includes bibliographical references.