The D1-class receptors (D1R, D5R) each possess distinct signaling characteristics; however, pharmacological selectivity between them remains elusive. The third intracellular loops (IL3) of D1R and D5R harbour divergent residues that may contribute to their individual signalling phenotypes. Here we probe the function of central region of IL3 of D1R and D5R using deletion mutagenesis. Radioligand binding and whole cell cAMP assays suggest that the N-terminal and C-terminal moieties of the central IL3 oppositely contribute to the constitutive and agonist-dependant activity of D1-Class receptors. Whereas the N-terminal deletions ablated constitutive activity and decreased DA-induced activation, C-terminal deletions induced robust increases. These data, interpreted in concert with structural predictions generated from homology modeling implicate the central IL3 as playing an important role in the activation and subtype-specific characteristics of the D1-class receptors. This study may serve as a basis for the development of novel drugs targeting the central IL3 region.

CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.

CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.

<p>Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.</p>

Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPgammaS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation.

Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPgammaS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation.

Current drugs used to treat Congestive Heart Failure target the renin-angiotensin and adrenergic systems. Studies showed increased mortality rates in patients treated with a combination of these medications. Angiotensin-AT1 and ?2-Adrenergic receptors were shown to form receptor heteromers. Blockade of one receptor in the complex can affect the signal transmitted by the other; suggesting that ligand-based therapy is not as selective as we might think. Modulating receptor trafficking after synthesis might prove to be a valid therapeutic strategy. Unfortunately, little is known about receptor assembly and transport from Endoplasmic Reticulum to Plasma Membrane. The objectives of this study are to identify the proteins that participate in the assembly of AT1R-?2AR heteromer and the regulators of the anterograde trafficking of G-Protein Coupled Receptors. This thesis introduces the role of important targets in those poorly understood processes. The identification of such targets could lead to developing better drugs with fewer adverse effects.

An ultra-low dose of a drug is approximately 1000-fold lower than the dose range traditionally used to induce a therapeutic effect. The purpose of the present thesis was to broaden the knowledge of the ultra-low dose effect, that was previously identified in the opioid receptor system, by looking at whether opioids and cannabinoids interact at the ultra-low dose level, whether cannabinoid receptors themselves demonstrate the ultra-low dose antagonist effect, and whether the opioid ultra-low dose effect is maintained in a model of persistent, unavoidable pain. For experiment 1, separate groups of Long Evans rats were tested for antinociception following an injection of vehicle, the cannabinoid agonist WIN 55 212-2 (WIN), the opioid antagonist naltrexone (an ultra-low or a high dose), or a combination of WIN and naltrexone doses. Ultra-low dose naltrexone elevated WIN-induced tail flick thresholds without extending its duration of action. In experiment 2, antinociception was tested in rats following either acute or sub-chronic (7 days) injections of vehicle, WIN, ultra-low doses of the CB1 receptor antagonist rimonabant (SR 141716), or a combination of WIN and ultra-low dose rimonabant. Following the chronic experiment, striatal tissue was rapidly extracted and subjected to co-immunoprecipitation to analyse CB1 receptor coupling to G-protein subtypes. Ultra-low dose rimonabant extended the duration of WIN-induced antinociception, and attenuated the development of WIN-induced tolerance. Animals chronically treated with WIN alone had CB1 receptors predominately coupling to Gs proteins, whereas all other groups had CB1 receptors predominately coupling to Gi proteins. For experiment 3, all animals were subjected to the formalin test following either acute or sub-chronic injections of vehicle, the opiate morphine, ultra-low doses naltrexone, or a combination of morphine and ultra-low dose naltrexone. Ultra-low dose naltrexone had no significant effect on morphine-induced pain ratings in either the acute, or sub-chronic drug treatments. This thesis provides evidence that the ultra-low dose effect, including the agonist-induced G-protein coupling switch, extends to another receptor type. This effect may, therefore, be part of a generalized principle that applies to many G-protein coupled receptors. / Thesis (Ph.D, Psychology) -- Queen's University, 2007-11-05 09:31:30.162 / A portion of this research was supported by a Canadian Institutes of Health Research (CIHR) Proof of Principle Grant to M.C. Olmstead and J.J. Paquette.

Thymoquinone (TQ), a volatile oil component of black seed oil (derived from Nigella sativa), has been shown to have various biological effects including disease treatment and prevention. TQ is believed to share similar properties to the benzoquinones already in use as therapeutic drugs. Based on previous reports on the anti-inflammatory properties of black seed oil and TQ, it was originally hypothesized that TQ would inhibit lipopolysaccharide (LPS)-induced cellular sialidase activity in an anti-inflammatory manner. Sialidase activity was tested on live mouse bone marrow derived primary macrophage cells, BMC-2 macrophage cells, human embryonic kidney epithelial (HEK293) cells and human fibroblast cells using an assay that measures the cleavage of the sialidase specific fluorescent substrate 2’-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (4-MUNANA). The cleavage of 4-MUNANA causes the release of free 4-methylumbelliferone, which fluoresces at 450nm (blue) after excitation at 365nm. Unexpectedly, TQ induced sialidase activation in all three cell lines and wild type primary macrophage cells. TQ was unable to induce sialidase activity in primary macrophage cells isolated from Neu4 knockout mice suggesting that the TQ activates Neu4 sialidase enzyme. TQ-induced sialidase activity in these live cells was found to occur through intermediate GPCR-associated guanine nucleotide Gαi subunit and matrix metalloproteinase 9 (MMP9) by using specific inhibitors. In addition, TQ was found to induce sialidase activity in Toll-like receptor-deficient HEK293 cells. These latter data suggested that TQ may be activating GPCR Gαi and MMP9 signaling associated with Neu4 sialidase independent of TLRs. It is proposed that TQ-induced sialidase activity may activate Toll-like receptors in macrophage cells and the subsequent production of pro-inflammatory cytokines in the absence of LPS. Immunocytochemical staining of BMC-2 cells shows that TQ induced NFκB activation. NFκB activation was confirmed with electrophoretic mobility shift assay (EMSA) and western immunoblotting techniques. Cytokine arrays were used to test the pro-inflammatory cytokine response induced in mice by 5 hour treatment of TQ, compared to LPS. Mice treated with TQ exhibited an increase in IL-1β, IL-6 and TNF-α production, similar to LPS treatment. Taken together, the findings in these studies suggest that TQ is a novel ligand for Neu4 sialidase activation which consequently induces pro-inflammatory cytokine responses. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-04-21 17:38:10.413

Growth factor receptors have significant effects on various normal function of body such as cell proliferation, differentiation and apoptosis. They are also involved in neuronal function and dysfunction, cardiovascular diseases, and malignancies. Recently, multiple G protein-coupled receptors (GPCRs) have been shown to transactivate receptor tyrosine kinases (RTKs). Since both classes of receptors have complicated downstream cascades individually, understanding the signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation is an important challenge. To clarifying this phenomenon we investigated the phosphorylation profile and downstream effectors of ligand-activated vs. transactivated PDGF?? receptors. Dopamine receptors (one of the receptors of the GPCRs family) were used to compare the PDGF?? receptor phosphorylation and activity during direct activation and transactivation. Dose-response and time-course data between these two stimuli were evaluated. Furthermore, the phosphorylation site profiles and the intracellular signaling pathways of PDGF?? receptor after direct activation and transactivation were examined. In addition, possible synergic effects between transactivation and direct activation were explored. The results of this project showed that the phosphorylation profile and downstream effectors of ligand activated receptors versus transactivated receptors are different. Our data indicated that transactivation-induced pathways are more involved in survival and proliferation effects compared to ligand activation. This research answered basic questions about transactivation phenomena and proposes that these transactivation pathways could be exploited as a therapeutic approach for neurodegenerative diseases.