Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-191).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 117-153).

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Our group previously reported that caveolin-1 (cav-1) was down-regulated by nitric oxide (NO) during cerebral ischemia and reperfusion (I/R). However, the role of cav-1 in regulating blood-brain barrier (BBB) permeability is unclear yet. This study aims to address whether the loss of cav-1 induced by NO production affects BBB permeability. Data showed that the expression of cav-1 in isolated cortical microvessels was down-regulated in ischemia-reperfused rat brains subjected to middle cerebral artery occlusion (MCAO). Treatment of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, reserved cav-1 expression, inhibited matrix metalloproteinases (MMPs) activity and reduced the BBB permeability. Moreover, cav-1 knockdown remarkably increased MMPs activity in the culture medium of brain microvascular endothelial cells. Cav-1 deficiency mice displayed higher MMPs activity and BBB permeability than that of the wild-type mice. Interestingly, the effects of L-NAME on MMPs activity and BBB permeability were partly reversed in cav-1 deficiency mice. These results suggest that cav-1 plays important roles in regulating MMPs activity and BBB permeability in cerebral I/R injury. After completing the mechanism study, I investigated the potential drug candidate that targets cav-1 for protecting BBB and neuronal damage during cerebral I/R. Results showed that calycosin, an isoflavones from Astragali Radix, up-regulated the expression of cav-1 and inhibited MMPs activity, and decreased the BBB permeability in the MCAO ischemia-reperfused rat brains. I further investigated the neuroprotective effects of isoflavones of Astragali Radix, with in vitro oxygen glucose deprivation (OGD) model and in vivo cerebral ischemia-reperfusion models. In addition to calycosin and formononetin, two major isoflavones in Astragali Radix, daidzein was also included because it is a metabolite of formononetin after absorption. Results showed that all three isoflavones decreased infarction volume and neurological scores in MCAO rats and dose-dependently attenuated neuronal death induced by L-glutamate treatment and oxygen-glucose deprivation plus reoxygenation (OGD/RO). The neuroprotective effects were inhibited by estrogen receptors (ER) antagonist ICI 182,780. Interestingly, combine treatment of isoflavones displayed synergistic effects in both OGD/RO and L-glutamate induced neuronal injury models, as well as in MCAO cerebral ischemia-reperfusion rat brains. Mechanistically, estrogen receptor antagonist partly reduced the synergism in these models. PI3K/Akt activation was synergistically induced by treatment of those isoflavones simultaneously. In summary, cav-1 could be a potential therapeutic target for protecting the BBB in the treatment of cerebral I/R injury. Major findings in this thesis include: 1) Cav-1 plays an important role in maintaining BBB integrity through inhibition of MMPs activity. NO induced MMPs activities and BBB leakage are partially mediated by the down-regulation of cav-1 during cerebral I/R injury. 2) Calycosin treatment reserved cav-1 expression and reduced BBB permeability. 3) Isoflavones synergistically protected neurons against I/R-induced neuronal insults both in vitro and in vivo. The works provide a valuable step towards the clarification of the physiological and pathophysiological functions of cav-1, and a new clue for developing isoflavones as agents targeting cav-1 for the prevention or treatment of ischemic stroke. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

NMR crystallography is a new and developing area. Unlike solution state NMR, solid state NMR has the potential for structural studies of large, motionally restricted biological macromolecules, such as proteins in crystals which may, or may not, diffract. However, finding the best and the most useful sample form and geometry is still a major obstacle to rapid progress. Little has been reported about protein sample preparation for any class of protein for NMR crystallography, mainly since the availability of NMR labelled proteins is still not routine, especially for eukaryotic membrane proteins. The amino acid L-glutamate is the major excitatory neurotransmitter in the brain. Details of glutamate binding to any of its main brain or sensory receptors are not well resolved at the atomic level. In an effort to resolve the glutamate binding mechanism by solid state NMR methods, full-length taste and brain mGluR4 were expressed in E. coli, but proved to be toxic for the cells. The ligand-binding domains (LBD) of mGluR4, with various fusions for the periplasmic expression and with various fusions for expression in the cytoplasm therefore, were used. Solubilisation and then purification of the LBD from inclusion bodies is still under way, no crystals of mGluR4 for NMR were, therefore, grown. Initial NMR spectra of labelled 13C, 15N and 17O glutamate have been recorded to verify sensitivity requirements. Using homology modelling, a model for the truncated version of the ligand binding domain of mGluR4 has been constructed as a basis for designing solid state NMR experiments to probe the ligand binding site in the receptor. Bacteriorhodopsin is a large membrane protein and a model for G-protein coupled receptors (GPCRs). Spectra of bacteriorhodopsin produced in H. salinarium in purple membrane are reported here and compared to spectra of the protein crystallised from bicelles. Optimal conditions for producing spectra suitable for spectral assignment are reported as an initial step towards spectral resolution. Three differently labelled samples of bacteriorhodopsin were prepared to test the applicability of the various assignment strategies and the effects of deuteration on quality of solid state NMR spectra of a large, crystalline membrane protein.

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy