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 / Pathology / Master / Master of Medical Sciences

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor 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

No description available.

In Leishmania glycosomes compartmentalize a variety of essential biochemical and metabolic pathways required for parasite viability. Biogenesis and maintenance of glycosomes involves a family of proteins called peroxins, however the molecular mechanisms governing the early events of this pathway have not been fully established. / A structural-functional analysis of the receptor LdPEX5 revealed the formation of a tetrameric structure stabilized by coiled-coil motifs. A biophysical approach showed that the tetrameric structure of LdPEX5 dissociates to dimers upon binding to the PTS1 ligand. However, the tetrameric LdPEX5 is more thermodynamically favorable to bind. Lastly, LdPEX14 modulates the LdPEX5-PTS1 interaction as the presence of LdPEX14 decreases the Kd of LdPEX5-PTS1 by ∼10 folds. / PTS1-loaded LdPEX5 docks onto the glycosomal membrane via the membrane-associated LdPEX14, an interaction that was characterized by molecular mapping and biophysical approaches. In higher eukaryotes this PEX5-PEX14 interaction involves conserved WXXXF/Y pentapeptide motifs found on PEX5 and a signature sequence found on PEX14. These three repeats in LdPEX5 do not appear to be crucial for interaction with LdPEX14 thus suggesting a unique molecular mechanism mediating the docking event. On the other hand, the conserved signature motif is crucial for interaction with LdPEX5. / The topology and nature of the interaction of LdPEX14 with the glycosomal membrane is not clear. In vivo expression of FLAG-LdPEX14-HA together with proteinase digestion confirmed that both N- and C-termini are cytosolic facing. Mapping analysis revealed that the first 63 N-terminal residues of LdPEX14 are critical for anchoring LdPEX14 to the glycosomal membrane. Interestingly, expression of the first 74 amino acids of LdPEX14 is toxic to the parasites. / Finally, the structure of LdPEX14 on the glycosomal membrane was addressed by molecular mapping and biophysical techniques. Partial trypsin digestion of recombinant LdPEX14 and molecular mapping suggested that the first 250 residues of LdPEX14 were involved in the formation of the complex. Biophysical approaches and cross-linking assays suggest that this complex may consist of ∼12-100 LdPEX14 subunits. Interestingly, the structure of LdPEX14 appears to be modulated by LdPEX5. / Considering all the results, these findings have established important molecular information concerning early events in the targeting and import of PTS1 proteins into the glycosome.

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